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the word pilonidal is derived from the latin words of pilos, meaning hair, and nidus, meaning nest. a pilonidal sinus is an acquired disease most commonly affecting the sacrococcygeal region when broken or cut hairs penetrate the skin. they can occur in any hair - bearing area, primarily where there is an anatomical cleft. rarely, pilonidal sinuses have been found in non - hair bearing areas such as the web spaces of hands and feet where the cause is likely to be the individual s occupation. here it has mainly been documented as a disease of barbers but has been reported infrequently in animal groomers, milkers and abattoir workers.(1 - 5) although a common aetiology is believed to be sheep shearing, in reality, it is quite rare. over 50 cases of inter - digital pilonidal sinus have been described but there have only been two cases in male sheep shearers with the most recent in 1966.(4,6,7) a 31-year - old right - handed male sheep shearer presented with a longstanding sinus, for 3 years, in the 3 web space of his left hand. the patient had been shearing for 4 months of the year for the past 11 years working all over the world with different varieties of sheep. he often noticed that burrs, thorns or grass seeds would puncture the skin, especially of his non - dominant hand web spaces, and would be left with small wounds that would fill with tufts of wool (fig. he first presented to medical attention when experiencing an episode of infection with a painful, inflamed middle finger and nodule in the web space. he was treated with erythromycin to clear the infection and referred to our care for surgical excision that was performed with primary closure with a local skin flap. the wound healed satisfactorily and the patient was back at work and used fingerless gloves for a short period. at two months post - surgery, however, we reviewed him at his workplace and found that he was not wearing gloves and that the sinus had recurred (fig 2). the patient was reluctant to regular follow - up and felt that daily clearing of the wool from the sinus was a satisfactory method of management. sharp material caught in the wool is the likely cause of puncture to the skin. the wool is then forced in when the sheep is handled. recurrence at the original site. in the non - dominant hand, 3rd web space (arrow). histology results showed that microscopically the dermis was found to contain an inflamed pilonidal sinus (fig. the sinus revealed sheep hair of different colour and maturity (fig 4.). this was explained by the large varieties of sheep with which the patient came into contact. a low power cross section image (h&e x10) of the pilonidal sinus imaged with a digital macro lens. a high power image (h&e x100) of the sinus displaying epithelial lining of the sinus (white arrow), ulceration (black arrow) and multiple different coloured wool fragments (red arrows). inter - digital pilonidal sinus is an acquired disease caused by the penetration of hair through the thin epidermis of the inter - digital region.(8) this provokes an inflammatory response and the initiation of a foreign body granulomatous reaction. the constant friction between the fingers and negative pressure within the sinus is thought to facilitate the penetration and accumulation of hairs.(9) in hairdressing the aetiology is the penetration of short, sharp hair clippings whilst in sheep shearing it is thought to be due to an initial puncture from grass seeds, thorns and burrs, found in the sheep s fleece, followed by wool fibres entering at the puncture site.(1,6,8) it is a benign condition and many individuals are asymptomatic. however, the concern arises due to the susceptibility to recurrent infection that may result in a chronic draining sinus. infection is a concern with any pilonidal sinus although it is suggested that this is a more common occurrence in sheep shearers with up to six exacerbations in one year.(6) in the case of our patient only one episode of infection was noted which was at the time he first presented to his doctor. a previously reported case in 1966 was managed conservatively by removing visible fibres from the sinus and placing protective strips of plaster over the web space to prevent further infiltration of wool.(6) our patient, however, chose surgical excision over a conservative watch - and - wait policy. in symptomatic cases antibiotic therapy is also considered to be unsuccessful unless used to treat the infection in combination with surgery to excise the sinus. the preferred surgical procedure is excision and primary closure either directly or with the help of a local flap. this method produces a good cosmetic result but has been suggested to have a higher risk of recurrence when compared with healing by secondary intention, although the disadvantage of the latter is a prolonged recovery.(10) surgical excision is largely believed to be curative although some recurrences have been reported.(3,8,9) a late recurrence may be due to a return to the same occupation without any alterations to their practice that may act as preventative measures.(3,9) prevention is the most effective method of treating this disease.(4) it is important that those at risk adhere to a good hygiene routine ; meticulous cleaning and drying of the area, wearing gloves and ensuring the removal of any hairs that may have penetrated the skin during their work.(6,9) often in the instance of inter - digital sinus, multiple hairs of different colour are visible either protruding from the sinus itself or on further inspection intra - operatively.(4,5,9) in the example of one abattoir worker the sinus was found to be filled with animal hairs.(1)figure 4 shows the microscopic findings within the excised sinus of our patient, displaying fibres of different colour and this may reflect the different varieties of sheep with which the patient works.	occupationally acquired inter - digital pilonidal sinuses are seldom seen and typically found on the hands of hair dressers. sheep shearing has traditionally been believed to be a cause of inter - digital pilonidal sinuses ; although in reality it is infrequently so with only two previously reported cases in 1951 and 1966.we present a rare case of a shearer who worked with a variety of sheep and presented with a chronic pilonidal sinus in the 3rd web space of his hand. this work demonstrates for the first time a histological confirmation of the presence of wool, and in this case different varieties and coloured fibres within the sinus.the report presents our investigations into the aetiology and the mechanical theories supporting it. the clinical presentation and surgical management of this condition are discussed alongside occupational realities and expectations.
over the last century, the surgical management of ureteropelvic junction obstruction (upjo) has dramatically evolved. various open surgical techniques have been described based on the cause, location, and length of the upjo. the most popular repair is the anderson - hynes dismembered pyeloplasty, which has universal application and is accepted as the gold standard of treatment. with the development of endoscopic techniques and equipment, minimally invasive approaches have come into favor in the treatment of patients with primary and secondary upjo. more recently, with advancing laparoscopic skills and the introduction of robotic - assisted surgery, many centers have moved to laparoscopic pyeloplasty (lp) as first - line therapy. improved suturing skills and the use of robotic assistance have greatly facilitated laparoscopic dismembered pyeloplasty for primary and secondary repairs. the use of ureteral stents following pyeloplasty ensures adequate drainage, particularly in the presence of postoperative edema. for similar reasons, stents are commonly used after ureteroscopy although some controversy still exists regarding its necessity after uncomplicated ureteroscopic stone removal. in the pediatric population the advantages of stent placement following pyeloplasty include lowering the risk of urinoma formation following upj repair and providing support and alignment of the fresh suture line. the importance of the stent is highlighted when the anastamosis is not watertight, or after endopyelotomy, allowing healing of the defect while urine is diverted by the stent. however, ureteral stents are not free from risk, and potential problems include migration, encrustation, retained or forgotten fragments, irritative urinary symptoms, exposure of the upper urinary tract to high pressure during urination, flank pain, and increased risk of infection. in a porcine model, soria evaluated whether it is possible to reduce the duration of ureteral stenting following endopyelotomy, and thus reduce side - effects. ureteral stent placement for 1 week was found to be insufficient to assure correct healing and evolution of the upj following endopyelotomy. stenting for 3 weeks was shown to be effective, and it was therefore not necessary to extend stenting time to 6 weeks. in the senior surgeon 's experience (als), 17 stented lps have been performed to date. excellent outcomes have been previously reported with the use of the lapraty clip during collecting system reconstruction. although the role of stents has been well described after endopyelotomy, its role after laparoscopic pyeloplasty, where watertight closure can be achieved, is unclear. this is an institutional review board approved, prospective collection and retrospective analysis of data obtained from patients specifically undergoing lsp. between november 2004 and february 2006, 6 consecutive patients were treated without the placement of a postoperative stent, all of whom were operated on by the same surgeon (als). in addition, all patients had undergone retrograde ureteral stenting by their local urologist before being seen at our center. briefly, after cystoscopy and ureteral stent removal followed by repeat retrograde urography, patients were placed in a lateral decubitous position. after port placement, the colon was reflected medially followed by identification of the ureter either at the level of the lower pole or near the level of the renal hilum. the collecting system was divided just cranial to the narrowest point, and the ureter was then spatulated 2 cm along its lateral aspect. the capacious pelvis was minimally spatulated along its medial aspect and reduced appropriately if excessive tissue was present. the anastomosis was performed using 4 0 absorbable vicryl suture on an sh - needle beginning with the posterior layer with a free hand - tied knot and proceeding with a continuous suture in a lateral - to - medial direction. the suture line was performed by placing the needle from outside - to - in on the renal pelvis and inside - to - out on the ureter side. a lapra - ty clip (ethicon endosurgery, cincinnati, oh) was used to complete the suture line at the medial aspect of the renal pelvis. the anterior layer was completed in a similar fashion by using a second 4 0 vicryl suture secured with a lapra - ty clip. forced diuresis along with intravenous indigo - carmine was provided by the anesthesia team to visually inspect the anastamotic closure. on the morning of postoperative day (pod) 1, the bladder catheter was removed, and the drain was removed 12 hours later before hospital discharge, after the patient had voided without any increase in drain output. all patients had a follow - up tc-99 m mag3 renal scintigraphy at 4 weeks and 6 months following surgery. total analgesia was reported in equivalent milligrams of injectable morphine and was calculated for duration of in - patient stay only. data were maintained and analyzed with file maker pro (file maker inc, santa clara, ca). five patients (3 female, 2 male) underwent lsp with a mean follow - up of 15.7 months (range, 7 to 24). average age of the patients was 42.8 years (range, 33 to 64) with an average body mass index of 29.3 kg / m (range, 19.3 to 54.9). mean split renal function for the obstructed kidney by tc-99 m mag3 scan was 36% (range, 15 to 52) (table 1). three patients were noted to have a crossing vessel, while the other 2 patients had dense, fibromuscular adhesions surrounding the upj. mean operative time and estimated blood loss were 196 minutes (range, 145 to 284) and 50 ml (range, 10 to 150), respectively. average time to regular diet was 13.3 hours (range, 5 to 35). mean durations of postoperative urinary catheter and abdominal drain were 0.9 days (range, 0.5 to 1.5) and 1.6 days (range, 1 to 2.5), respectively. one patient was kept in the hospital one extra day due to difficulty voiding after catheter removal. mean analgesia consumption was 42.1 mg (range, 21 to 65) of equivalent morphine. summary of preoperative and postoperative tc-99 m mag3 renal scans upjo = ureteropelvic junction obstruction ; cv = crossing vessel ; da = dense adhesion. this involved patient # 5 in our series who was a 42-year - old male with a left - sided upjo. the night of the surgery, the patient complained of colic - type flank pain with intractable nausea and vomiting. a retrograde pyelogram was performed on the morning of pod 1, which demonstrated a normal caliber ureter with mild hydronephrosis. although there was no obvious evidence of obstruction, a 6f ureteral stent was placed without difficulty. multiplanar fluoroscopic views ensured proper placement of the guide - wire and stent in the collecting system. the patient noted complete resolution of symptoms, however, did note moderate irritatitve voiding symptoms requiring anticholinergic medications and flank discomfort while voiding during his recovery period. follow - up split function with mag-3 renal scans at both 4 weeks and 6 months following lsp were not significantly different from preoperative values (table 1). otherwise, all patients remained asymptomatic throughout their follow - up period with no evidence of obstruction. abdominal ultrasound at that time revealed no hydronephrosis, however, did demonstrate several gallbladder stones. follow - up scan at that time noted drainage with t- of 26 minutes, which had improved from the patient 's preoperative state of complete obstruction. a more recent renal scan 18 months postoperatively demonstrated a further improvement in t of 16 minutes and a stable split renal function, with no recurrence of flank discomfort. in addition, this patient, the only one with preoperative baseline renal insufficiency, had an improvement in serum creatinine from 2.5 mg / dl to 2.0 mg / dl. ureteral stenting in children typically requires a second procedure with the patient under anesthesia for stent removal. the case for nonstented pyeloplasty has been addressed in a review study of the pediatric literature where it has been shown to be safe and accepted as a standard of care. in general, the indications for ureteral stenting are many, and their use has become common in a urologic practice, especially after a pyeloplasty where a ureteral stent typically will be placed for 4 weeks to 6 weeks postop. the most recent and largest published series to date, from johns hopkins, documents 4-week routine stenting following laparoscopic pyeloplasty. unfortunately, stent use is not without potential complication. in one study evaluating the morbidity of stents, a complication rate of 94% (103/110 patients) was reported, which included infection, flank pain with voiding, stent migration, and stent fragmentation. similarly, using validated questionnaires, joshi reported that 78% of the 62 respondents noted bothersome urinary symptoms that encompassed urinary urgency, frequency, incontinence, and hematuria. in addition, 80% of respondents experienced stent - related pain affecting daily activities, as well as some respondents noting sexual dysfunction (32%) and reduced work capacity (58%). in the senior surgeon 's prior experience with 17 stented laparoscopic pyeloplasties, nine (53%) of these individuals required anticholinergic medication to treat these symptoms and 2 (12%) required early stent removal 3 weeks postoperatively (unpublished data). in our initial lsp experience, we found no increased morbidity from performing this technique. the risks of stent migration, encrustation, stent syndrome (defined as dysuria, frequency, flank pain, and hematuria commonly seen with short - term placement), increased risk of infection, and stent encrustation were replaced by the risk of possible postop obstruction and/or urinoma formation that would require retrograde stent placement. such a maneuver is potentially dangerous in that wire placement may injure the anastamosis or be mistakenly placed outside the collecting system. proper endoscopic equipment (eg, hydrophilic coated guidewire) and skill are required to ensure safe placement. in our study, only one patient experienced symptoms suggestive of postoperative obstruction. nevertheless, a ureteral stent was placed for 4 weeks, during which time, the patient complained of irritative voiding symptoms and renal pain during voiding. furthermore, the oldest patient in this cohort (patient # 2) with a bmi of 54.9 kg / m who had recurrent bouts of pyelonephritis and flank pain at the time of presentation had dense peri - ureteral adhesions noted at the time of surgery. this patient with a preoperative split function of 35% on the right side, and complete upjo was noted to have a partial obstruction on follow - up renal scan. at 10-months postoperatively, the concept of cholecystitis was considered by the presence of gallbladder stones and sludge coupled with moderate gallbladder wall thickening. this patient has otherwise remained asymptomatic with improved serum creatinine and no recurrent bouts of pyelonephritis despite moderate objective improvement. no infectious or obstructive complications related to the use of lapra - ty clips were encountered. although postoperative ct - abdominal scans were not routinely obtained, none of the patients exhibited signs or symptoms consistent with urinoma formation, which compares favorably with a previous report that noted a 5% prolonged urinary leak and 5% rate of urinoma formation in a nonstented pediatric cohort. the benefits of a stentless procedure, if proven as efficacious and safe as a stented pyeloplasty, include reducing the risk for infection, eliminating the risk of developing stent syndrome and the need for follow - up cystoscopy 4 weeks to 6 weeks postoperatively for stent removal. the technical advantage of using the lsp technique is the avoidance of stent interference during suture - related reconstruction. lsp allows for optimal visualization and direct access to the spatulated edges for anastamosis, eliminating the risk of entangling the suture around the stent and stent migration. as such, these factors help optimize the likelihood of a watertight anastamosis. comparative results were noted to our previously published stented laparoscopic pyeloplasty series. comparing our operative times to times of previously published series shows that our mean time of 196 minutes is comparable to that of other contemporary large series, which range between 123 minutes and 252 minutes. when assessing hospitalization, we noted a shorter mean stay of approximately 1.6 days versus that of other series, which are reported to be between 2.6 days to 4.5 days. overall, there was 100% subjective success in our 5 patients with a mean follow - up of 15.7 months. with regards to objective success, all patients demonstrated stabilized or improved split renal function on mag-3 renal scan and improved drainage t- times. several limitations of this study warrant discussion. the small patient number and short follow - up limit the power to perform statistical analysis. although some critics may argue that extended follow - up beyond 24 months would be optimal, inagaki from johns hopkins evaluated 147 lps with a mean follow - up of 24 months and demonstrated that most failures occur within 6 months. due to small numbers in a retrospective review, we were not able to assess the benefit of lsp for primary versus secondary upjo. a prospective, randomized study comparing stented with nonstented methods is needed to address the dogma of ureteral stent placement and evaluate the postoperative complication parameters. as such, it will take a significantly larger cohort of patients with a longer follow - up to assess whether adult lsp is comparable to published success rates of stented laparoscopic pyeloplasty of 81 to 99. we still advocate the use of a stent in all patients with a solitary kidney, patients with a difficult ureteral anastomosis, significant bleeding during upjo repair, and those individuals with a thick, noncompliant ureter due to chronic inflammation. utilizing a stentless technique has proved to be efficacious in a small cohort of patients with limited follow - up. our minimal incidence of postoperative complications is likely related to meticulous surgical technique and ability to minimize laxity on our suture line with the aid of additional lapra - ty clips as needed. certainly, a larger cohort with longer follow - up will be required to prove the durability and safety of a stentless laparoscopic pyeloplasty, which may ultimately revolutionize previous dogmatic assertions.	background and objectives : pyeloplasty, whether open or laparoscopic, has been the mainstay of treatment for ureteropelvic junction obstruction (upjo). a nonstented pyeloplasty has only been reported in the pediatric literature. herein, to the best of our knowledge, we report the first published experience with laparoscopic stentless pyeloplasty (lsp) in the adult population.methods:patients with a normal contralateral kidney who underwent a laparoscopic pyeloplasty were included in this study. a dismembered pyeloplasty was performed without the placement of a ureteral stent. functional tc-99 m mag3 renal - scan data were compared with results at 4 weeks and 6 months postoperatively. perioperative complications and long - term follow - up were prospectively gathered.results:to date, 5 patients have undergone lsp with a mean follow - up of 15.7 months. mean age and body mass index of this group were 42.8 years and 29.3 kg / m2, respectively. mean operative time, estimated blood loss, and hospital stay were 196 minutes, 58 ml, 1.6 days, respectively. three patients had right - sided upjo, and 2 patients had left upjo. no patient had undergone previous surgery for upjo. all patients had a ureteral stent in place at the time of surgery. no intraoperative complications occurred. only one patient complained of flank pain on pod1. no obstruction or urinary extravasation was seen on retrograde pyelography, but a ureteral stent was placed. during our follow - up, all patients had complete resolution of their symptoms. postoperative renal scans demonstrated improved urinary drainage in all patients.conclusion:our initial experience suggests that in experienced hands, lsp may be an effective method for treating upjo.
as mentioned before, the increase in phase measurement sensitivity in the nci is not due to noise reduction but due to signal enhancement. in order to demonstrate this, we experimentally compare it with a ci, which works at the snl. as the snl is related to the phase - sensing photon number. the schematic diagrams of an su(1,1) nci and a conventional mz interferometer (ci) are shown in fig. beam splitters are replaced by parametric amplifiers (pa1, pa2), which amplify the incoming idler input field is labelled as and is in vacuum. in the present realization of nci, the parametric amplifier is based on non - degenerate four - wave mixing in hot rb-85 atomic vapour cells29 (see methods for the details of the experimental arrangement). output relation for a parametric amplifier is given by : where g is the amplitude gain and g = g1. with an overall phase shift in the idler arm, we obtain the output fields of the interferometer : with ()=ge+g, ()=gg(1+e) as the phase - sensitive gains. for a coherent state \| injected in the signal input and vacuum in the idler input, we obtain the output intensity in the idler port or one of the outputs of the nci as where g(+1) is the intensity of the phase - sensing field (idler field in fig. 1a) with \|\| as the input intensity. for > > 1, g. a conventional mz interferometer shown in fig. 1b, on the other hand, has a fringe given by with 50:50 beam splitters and a coherent state \| input. since the sensitivity of an interferometer is related to the photon number inside the interferometer, for a fair comparison between nci and ci, we set =, which can be achieved by adjusting, independently. therefore, we see from equations (3) and (4) that, under the condition of the same phase - sensing intensity, the output of the nci is 2 g times that of the ci. however, as is well known, for any amplifier, the amplification of signal is also accompanied by the amplification of the noise. so, the signal - to - noise ratio at best does not change in the process31. fortunately, it is not the case for the nci discussed here. from equation (2), at dark fringe when =(2n+1), we have. however, since is in vacuum, the output of the nci is at vacuum noise level, just like the ci at dark fringe. thus, the signal in the nci is increased while the noise level is not and we should expect an enhancement of 2 g in signal - to - noise ratio of the nci over the ci. what happens here for the nci is that although each amplifier increases signal and noise together, the first parametric amplifier (pa1) produces two fields (two arms of the nci) that are quantum mechanically entangled323334 so that their noises are also correlated, and a destructive interference occurs at the second parametric amplifier (pa2) when the interferometer is operated at dark fringe so that most of the amplified noise from pa1 are cancelled and only the vacuum noise is left35. therefore, the nci increases the signal while it keeps the noise unchanged, leading to an enhancement in signal - to - noise ratio for phase measurement as compared with the ci. figure 1c shows the interference fringes observed for both nci and ci with the same phase - sensing intensity =. the fringe size of the nci is bigger than that of the ci because of the amplification at pa2, which leads to an enhancement in the phase signal. when operated at dark fringe with a small phase change : =(2n+1)+ (> 1, g. a conventional mz interferometer shown in fig. 1b, on the other hand, has a fringe given by with 50:50 beam splitters and a coherent state \| input. since the sensitivity of an interferometer is related to the photon number inside the interferometer, for a fair comparison between nci and ci, we set =, which can be achieved by adjusting, independently. therefore, we see from equations (3) and (4) that, under the condition of the same phase - sensing intensity, the output of the nci is 2 g times that of the ci. however, as is well known, for any amplifier, the amplification of signal is also accompanied by the amplification of the noise. so, the signal - to - noise ratio at best does not change in the process31. fortunately, it is not the case for the nci discussed here. from equation (2), at dark fringe when =(2n+1), we have. however, since is in vacuum, the output of the nci is at vacuum noise level, just like the ci at dark fringe. thus, the signal in the nci is increased while the noise level is not and we should expect an enhancement of 2 g in signal - to - noise ratio of the nci over the ci. what happens here for the nci is that although each amplifier increases signal and noise together, the first parametric amplifier (pa1) produces two fields (two arms of the nci) that are quantum mechanically entangled323334 so that their noises are also correlated, and a destructive interference occurs at the second parametric amplifier (pa2) when the interferometer is operated at dark fringe so that most of the amplified noise from pa1 are cancelled and only the vacuum noise is left35. therefore, the nci increases the signal while it keeps the noise unchanged, leading to an enhancement in signal - to - noise ratio for phase measurement as compared with the ci. figure 1c shows the interference fringes observed for both nci and ci with the same phase - sensing intensity =. the fringe size of the nci is bigger than that of the ci because of the amplification at pa2, which leads to an enhancement in the phase signal. this is the key difference between an nci and a ci. when operated at dark fringe with a small phase change : =(2n+1)+ (<<1, n = integer), we obtain from equations (3) and (4) the change in the output intensities of the two interferometers due to as : so, the signal is enhanced by bsinci/ici=2 g for the same and =. the fringe enhancement in fig. the noise performance of the nci is measured by homodyne detection (hd) method at the idler output port, as shown in fig. 2c shows the noise level at the output of the nci when the phase is scanned. the minimum noise occurs at dark fringe when there is a destructive quantum interference at pa2. this is in contrast to the trace in yellow when one arm of the nci is blocked and there is no interference effect. the trace in yellow corresponds to two uncorrelated amplifiers in series, as depicted in fig. respectively show the noise levels for individual amplifiers when the other one is not active. notice that at some phases, the output noise level (the trace in dark blue) of the nci is even lower than the amplified noise levels of individual amplifiers (the traces in green and dark brown) as a result of noise cancelation. 2c respectively show the measured noise levels at dark fringe setting for the nci and the ci under the same condition when the fringes in fig. the observed noise level for the nci is 3.30.3 db higher than that of the ci. this is not what is predicted in previous paragraphs, that is, the noise level of the nci is the same as that of the ci at the vacuum noise level. the higher measured output noise of the nci is due to internal losses of the interferometer (more on this later). 30 that the signal due to a small phase shift in hd of an interferometer has the same form as that in direct intensity detection), we still obtain an enhancement of 4.10.3 db in signal - to - noise ratio for the nci over the ci, that is, snrnci / snrci=4.1 db=2.5. from equation (5), we know that the signal is proportional to, whereas the noise measured in the experiment is the noise power. so, we have the phase measurement sensitivity as in rms value. as the ci has a phase measurement sensitivity at snl3, our nci beats the shot noise level by a factor of = 1.6 in rms value. the simple theory in the previous part does not include losses in the system and predicts the continuous increase of the snr with the gain of the amplifiers. losses, however, will introduce extra vacuum noise, which is also amplified by pa1 and pa2 but is not correlated and can not be cancelled, thus leading to higher noise level for the nci than the ci but at a manageable level, as observed in fig. 2c. the extra amplified vacuum noise will increase with the gain of the amplifiers in the same way as the phase signal. 3 where we plot in blue diamond shape the ratio of the output noise level of the interferometer at dark fringe (the light blue trace in fig. 2c) as a function of the average noise gain of individual amplifiers (the traces in green and dark brown in fig. since the vacuum noise level is also the output noise level of the ci at dark fringe, this is also the noise level ratio of the nci to the ci. shown also in fig. 3 in black square shape are the ratio of the signal level of the nci to that of the ci, or the signal enhancement factor bs extracted from the fringes similar to fig. 1c. therefore, the ratio of the black square to the blue diamond gives the enhancement of the signal - to - noise ratio of the nci over the ci, which is shown as red triangle in fig. 3. as seen in the figure, both the signal enhancement factor (black) and the noise level (blue) at the output increase with the amplifier gain in about the same rate at high gain. so, the signal - to - noise improvement factor (red) levels out at a gain of ~4 db, in qualitative agreement with ref. 30. next, we characterize the effect of losses on the performance of the nci by introducing some extra losses. first, we place an attenuator with loss l in front of the homodyne detector and vary the attenuation. the signal enhancement factor bs and noise n above vacuum noise level after the attenuator will change with the loss l by where bs0 and n0 are the respective values before the attenuator. so, the signal - to - noise ratio after the attenuator becomes the solid lines in fig. 4a are fitted to equations (6) and (7) with bs0=7.5 db and n0=3.2 db. so, the signal - to - noise improvement factor does not change much at small loss for large n0. this is well known for amplification process : the amplified noise is above the vacuum level so that the noise will decrease under loss just like the signal, making snr less sensitive to external losses. this is quite different from the squeezed state scheme56 in which the improvement will suffer under loss. for large loss, however, snr will drop with the increase of the loss because the noise level is mostly vacuum noise already and wo n't decrease further. for the effect of internal loss, we place a variable attenuator in the paths of both arms of the nci. we need to readjust the gain of pa2 to re - balance the interferometer so that it produces fringe visibility close to 100%. in the meantime, gain balancing also leads to optimized output noise levels shown fig. 4b, the output noise level (blue diamond) increases as the loss increases more uncorrelated vacuum noise enters the interferometer and gets amplified. therefore, the improvement factor in signal - to - noise ratio decreases more quickly with internal loss (fig. the effect of internal loss on the nci is complicated so we can not fit the data to a theoretical prediction we have demonstrated an su(1,1) nci with 4 db improvement in signal - to - noise ratio in phase measurement over a ci operated under the same condition. the improvement is mostly limited by the loss inside the interferometer and is less sensitive to external loss than the squeezed state method. the improvement in signal - to - noise ratio comes from the increase in signal rather than the reduction of noise. furthermore, if we inject squeezed states into the unused idler input port of pa1 (see fig. 1a), reduction of the noise of the interferometer is possible, leading to further improvement of signal - to - noise ratio. therefore, the current scheme and the squeezed state scheme are complementary to each other. notice that our nci involves amplification of the input field whereas a conventional mz interferometer (ci) does not. however, according to refs 21, 25 it is the photon number of the phase probing field that counts for the limit of sensitivity. therefore, when we compare the performance of our nci with the ci, we make sure that the light fields probing the phase change have the same intensity in both nci and ci. when we reduce the intensity of the injected coherent state, this interferometer will approach to the originally proposed su(1,1) interferometer by yurke.19, which can have a phase measurement sensitivity reaching the heisenberg limit. 3, the internal loss of the interferometer is the main limiting factor in achieving higher sensitivity at higher gain. 30 and demkowicz - dobrzanski.36 then, it is natural to ask what kind of phase measurement sensitivity limit we can reach in our new interferometer. to answer that unfortunately, our measurement technique, that is, the hd method, does not allow us to perform an analysis with fisher information method, which requires photon counting technique. phase estimate with fisher information method for this new type of interferometer will be the topic in future researches. what we did in current work is to compare the performance of our new interferometer with a conventional mz interferometer, which has the well - known snl (1/-dependence for phase measurement uncertainty), and show a 4-db improvement in signal - to - noise ratio. the use of a nonlinear process to mix the signal and idler fields means that the two fields may have different frequencies. indeed, in our experiment, the frequency difference between the signal and the idler fields is ~6 ghz. this opens up a new way to construct hybrid interferometers involving different waves and thus broadens the scope of precision measurement involving interferometry. 5. the two parametric amplifiers (pa1, pa2) employed for the nci as shown in fig. 1a are based on non - degenerate four - wave mixing process in two hot rb-85 atomic vapour cells temperature - stabilized at 117 c (ref. 29) ; see inset of fig. 5 for energy diagram of relevant components). they are respectively pumped by a vertically polarized beam (p1, p3) at a maximum power of 400 mw with a waist of 500 m. the pump beams are from a ti : sapphire laser frequency - stabilized to a stable reference cavity. the pump beams are detuned about 0.8 ghz above the transition line of rb-85 f=2f at 795 nm. the pump power can be adjusted with a combination of polarization beam splitters (pbs) and half - wave plates. a horizontally polarized seed beam (also known as signal) with waist of 200 m is injected with an angle of 0.4 relative to the pump. signal beam is 3.04 ghz red shifted from the pump beam by an acousto - optic modulator in double - pass configuration. the pump and signal beams are combined with a glan - laser pbs before being sent into the atomic cell. at the output of the atomic cell, the seed signal beam is amplified with a gain that depends on the power and frequency detuning of the pump beam. the amplification of the signal beam is accompanied by a conjugate beam (called idler) with a frequency of 3.04 ghz blue shift from the pump at the other side of the pump beam. the second cell is identical to the first one except that both the signal and the idler fields have inputs that are from the first cell. a 4-f imaging system (l1, l2) is used to mode match the fields of the two cells. a ci of mz type is formed in the path of the idler field with a pair of flipping pbs (pbs1, pbs2) and a beam - redirecting mirror. in this way we block the signal arm and the pump to cell 2 (p3) when we operate the ci. a pzt - mounted mirror (pzt) is placed in both interferometers as the phase modulator so that we can compare the performance of both interferometers under the same phase change. since the pbs splits the idler beam into two equal parts in the ci and reduces the phase - sensing intensity to half, we need to increase the injected signal at cell 1 to bring the phase - sensing intensity of the ci to the same level as that of the nci (ips=60 w with iseed=16 w). a regular silicon photo - detector (d) is placed in the idler output port of the nci, which is also the output port for the ci. this detector records the fringes of the nci and the ci in a digital scope, as shown in fig. the noise performance of the nci is measured by hd (with a 98% homodyne efficiency) at the output of the idler side of the nci under different circumstances. the local oscillator of 726 w for hd is obtained from cell 1 with another seed (196 w) and pump (p2=300 mw)34. the local oscillator goes through the same imaging system to match its mode with those in the interferometer. 5. the two parametric amplifiers (pa1, pa2) employed for the nci as shown in fig. 1a are based on non - degenerate four - wave mixing process in two hot rb-85 atomic vapour cells temperature - stabilized at 117 c (ref. 29) ; see inset of fig. 5 for energy diagram of relevant components). they are respectively pumped by a vertically polarized beam (p1, p3) at a maximum power of 400 mw with a waist of 500 m. the pump beams are from a ti : sapphire laser frequency - stabilized to a stable reference cavity. the pump beams are detuned about 0.8 ghz above the transition line of rb-85 f=2f at 795 nm. the pump power can be adjusted with a combination of polarization beam splitters (pbs) and half - wave plates. a horizontally polarized seed beam (also known as signal) with waist of 200 m is injected with an angle of 0.4 relative to the pump. signal beam is 3.04 ghz red shifted from the pump beam by an acousto - optic modulator in double - pass configuration. the pump and signal beams are combined with a glan - laser pbs before being sent into the atomic cell. at the output of the atomic cell, the seed signal beam is amplified with a gain that depends on the power and frequency detuning of the pump beam. the amplification of the signal beam is accompanied by a conjugate beam (called idler) with a frequency of 3.04 ghz blue shift from the pump at the other side of the pump beam. the second cell is identical to the first one except that both the signal and the idler fields have inputs that are from the first cell. a 4-f imaging system (l1, l2) is used to mode match the fields of the two cells. a ci of mz type is formed in the path of the idler field with a pair of flipping pbs (pbs1, pbs2) and a beam - redirecting mirror. in this way we block the signal arm and the pump to cell 2 (p3) when we operate the ci. a pzt - mounted mirror (pzt) is placed in both interferometers as the phase modulator so that we can compare the performance of both interferometers under the same phase change. since the pbs splits the idler beam into two equal parts in the ci and reduces the phase - sensing intensity to half, we need to increase the injected signal at cell 1 to bring the phase - sensing intensity of the ci to the same level as that of the nci (ips=60 w with iseed=16 w). a regular silicon photo - detector (d) is placed in the idler output port of the nci, which is also the output port for the ci. this detector records the fringes of the nci and the ci in a digital scope, as shown in fig. the noise performance of the nci is measured by hd (with a 98% homodyne efficiency) at the output of the idler side of the nci under different circumstances. the local oscillator of 726 w for hd is obtained from cell 1 with another seed (196 w) and pump (p2=300 mw)34. the local oscillator goes through the same imaging system to match its mode with those in the interferometer.	conventional interferometers usually utilize beam splitters for wave splitting and recombination. these interferometers are widely used for precision measurement. their sensitivity for phase measurement is limited by the shot noise, which can be suppressed with squeezed states of light. here we study a new type of interferometer in which the beam splitting and recombination elements are parametric amplifiers. we observe an improvement of 4.10.3 db in signal - to - noise ratio compared with a conventional interferometer under the same operating condition, which is a 1.6-fold enhancement in rms phase measurement sensitivity beyond the shot noise limit. the improvement is due to signal enhancement. combined with the squeezed state technique for shot noise suppression, this interferometer promises further improvement in sensitivity. furthermore, because nonlinear processes are involved in this interferometer, we can couple a variety of different waves and form new types of hybrid interferometers, opening a door for many applications in metrology.
intracellular ca regulates many distinct aspects of neuronal function over a very wide range of timescales. the transduction of changes in ca concentration into a specific physiological outcome involves the action of ca - binding proteins that act as sensors and regulate the activity of various target proteins. many ca sensors bind ca via so - called ef - hand domains and the best known of these is the ubiquitous protein calmodulin. during vertebrate evolution, however, there has been an expansion in the number of ef - hand proteins, particularly in protein families expressed in neurons. amongst these are the neuronal calcium sensor (ncs) proteins and the calcium - binding proteins (cabps). the cabps are more similar to calmodulin than the ncs proteins are but they all have in common a high affinity for ca, allowing them to respond with high sensitivity to small increases in neuronal cytosolic ca concentration. the ncs family consists of 14 genes in mammals, and some of these genes are expressed as multiple splice variants. these include ncs-1, hippocalcin, neurocalcin, visinin - like protein (vilip) 1 - 3, recoverin, guanylyl cyclase - activating proteins (gcaps) 1 - 3, and k channel - interacting protein (kchip) 1 - 4. ncs-1 was discovered in flies and named frequenin due to its ability to enhance neurotransmitter release in a frequency - dependent manner and was thought to be a neuronal - specific protein. ncs-1 has, however, an orthologue in yeast and other fungi whereas other ncs proteins appeared at later stages of evolution and in some cases have a more restricted neuronal expression. the ncs proteins have varied patterns of intracellular localisation, and some of these proteins are cytosolic whereas others are associated with membrane organelles due to the lipid modifications of myristoylation or palmitoylation. it was originally shown for recoverin and later for some other ncs proteins such as hippocalcin that they can reversibly associate with membranes on ca binding and extrusion of their myristoyl tail (the ca / myristoyl switch). other ncs proteins such as ncs-1 have their myristoyl tail exposed even in the absence of ca and therefore can associate with membranes even at resting intracellular ca concentrations, allowing them to respond to short - duration and local ca signals. the cabps are expressed in fish onwards and are encoded by seven genes with alternate splice forms of cabp1 (including caldendrin) and cabp2. again, these proteins can be found to be either cytosolic or membrane - associated although none shows the ca / myristoyl switch mechanism that appears to be unique to members of the ncs family. recoverin and the gcaps have defined roles in regulating signalling pathways specifically in the retina. recent work has begun to reveal the physiological functions of other ncs and cabp proteins : for example, roles in the regulation of plasma membrane ca channels [14 - 18 ]. they have also been suggested to regulate intracellular ion channels such as the inositol 1,4,5-trisphosphate receptor [19 - 21 ], although the effect of cabps initially reported was opposite to that seen in later studies. the molecular basis for the functions of these proteins has been revealed in part by identification of some of their target proteins. below, we will highlight recent advances in our understanding of the roles of these ca sensors in membrane traffic and in forms of synaptic plasticity. it has become increasingly apparent that ncs and cabp proteins have roles in regulating intracellular traffic in cells either through interaction with specific cargo molecules or through general effects on membrane traffic processes. the first example to be characterised was the ability of kchips to stimulate the traffic of the kv4 potassium channels to the cell surface. this requires a direct physical interaction of the kchips with the kv4 channel subunits. for kchip1, traffic with kv4 channels occurs via a novel non - conventional pathway, from the endoplasmic reticulum to the golgi apparatus, that is dependent on functional ef - hands in kchip1 before subsequent traffic to the plasma membrane. this pathway may be related to the local satellite traffic pathways present within neuronal dendrites. other specific effects of ncs proteins include stimulation by vilip-1 of the cell surface expression of 42 nicotinic acetylcholine receptors and inhibition by ncs-1 of dopamine d2 receptor internalisation, which is further discussed below. a more general effect of ncs-1 and cabp7/8 (calneurons 2/1) on membrane traffic is exerted through regulation of a key enzyme in phosphoinositide metabolism, phosphatidylinositol-4-kinase (pi4k) iii. a role for ncs-1 in stimulating this enzyme was first shown in yeast and subsequently the mammalian ncs-1 protein was shown to stimulate pi4kiii and also to interact with both the enzyme and the small gtpase adp - ribosylation factor 1 (arf1) on the golgi complex. these interactions regulate general traffic from the trans - golgi network (tgn) to the plasma membrane, with the effects of ncs-1 and arf1 being antagonistic. the importance of these interactions is indicated by findings that ncs-1, arf1 and pi4kiii control inner ear development in zebrafish. recently, cabp7 and 8 were found to interact with pi4kiii but, in contrast to ncs-1, inhibited the enzyme activity at resting ca levels. this inhibition was released on binding of ncs-1 at elevated ca levels, providing a tight ca threshold for control of the traffic from the tgn to the plasma membrane in neurons (figure 1). release of ca from the golgi through channels such as the inositol 1,4,5-trisphosphate receptor may influence trafficking through and from this organelle. under conditions of high local ca concentration (high - ca), ncs-1 binds to and activates phosphatidylinositol-4-kinase iii (pi4kiii) (activated pi4kiii is indicated by an asterisk) to drive the production of phosphatidylinositol 4-phosphate (pi4p) (shown enriched in hatched regions of the membrane), a lipid essential for stimulation of vesicular traffic from the tgn. a further level of complexity in this model stems from a ca - dependent interaction between ncs-1 and the key golgi trafficking gtpase adp - ribosylation factor 1 (arf1). at high ca, ncs-1 and arf1 interact, preventing activation of pi4kiii in regions where the two proteins coexist. since arf1 activates pi4kiii independently of ca, this interplay may represent a mechanism for spatial organisation of the tgn into discrete subdomains tasked with serving either ca - dependent or ca - independent trafficking functions that are segregated by domain boundaries populated by non - productive arf1/ncs-1 complexes. under conditions of low local ca concentration (low - ca), ncs-1 no longer activates pi4kiii and instead cabp7 and cabp8 are able to interact with the kinase to inhibit its activity. lack of pi4p production under such circumstances would prevent vesicular traffic from ca - dependent trafficking domains. it has also become clear that ncs-1 has key roles in aspects of synaptic plasticity underlying learning and memory. there are three ncs-1 homologues in caenorhabditis elegans and knockout of the one most similar to the mammalian protein impaired learning and memory in the worms. increased levels of ncs-1 switched the mode of short - term plasticity in hippocampal synapses in culture from depression to facilitation. further progress was made when it was shown that ncs-1 was required for a form of synaptic plasticity, known as long - term depression (ltd), induced through metabotropic glutamate receptor activation in cortical neurons. in this system, ncs-1 acted as a ca sensor in conjunction with another ca - binding protein, pick1 (protein interacting with c kinase 1). as noted above, ncs-1 interacts with and controls the surface expression of dopamine d2 receptors. most recently, a study extended these findings to directly link ncs-1 regulation of dopamine d2 receptor activity with the processes of learning and memory in adult mice. inducible targeted expression of ncs-1 specifically in the dentate gyrus of transgenic animals enhanced acquisition of spatial memory and had the novel effect of increasing exploratory behaviour. importantly, these effects could be abolished through antagonism of the ncs-1/dopamine d2 receptor interaction by use of a membrane - permeant inhibitory peptide. another ncs protein, hippocalcin, has been of interest in considerations of synaptic plasticity as it is expressed to the highest extent in the hippocampus, which is a site of memory formation. like ncs-1, hippocalcin has been suggested to act as a ca sensor for ltd but in this case in the hippocampus. kchip3 was independently discovered and named the downstream regulatory element antagonistic modulator (dream). dream can act in the nucleus by binding in a ca - dependent manner to a specific dna sequence and may control aspects of synaptic plasticity through changes in gene expression. in fact, one of its major physiological roles revealed in a knockout mouse is in pain modulation. a recent novel role that has been discovered is that of caldendrin in regulating nuclear signalling in neurons by interaction with the protein jacob. this leads to changes in dendritic remodelling following nmda (n - methyl - d - aspartic acid) receptor activation, which could also be important for synaptic plasticity. it has become increasingly apparent that ncs and cabp proteins have roles in regulating intracellular traffic in cells either through interaction with specific cargo molecules or through general effects on membrane traffic processes. the first example to be characterised was the ability of kchips to stimulate the traffic of the kv4 potassium channels to the cell surface. this requires a direct physical interaction of the kchips with the kv4 channel subunits. for kchip1, traffic with kv4 channels occurs via a novel non - conventional pathway, from the endoplasmic reticulum to the golgi apparatus, that is dependent on functional ef - hands in kchip1 before subsequent traffic to the plasma membrane. this pathway may be related to the local satellite traffic pathways present within neuronal dendrites. other specific effects of ncs proteins include stimulation by vilip-1 of the cell surface expression of 42 nicotinic acetylcholine receptors and inhibition by ncs-1 of dopamine d2 receptor internalisation, which is further discussed below. a more general effect of ncs-1 and cabp7/8 (calneurons 2/1) on membrane traffic is exerted through regulation of a key enzyme in phosphoinositide metabolism, phosphatidylinositol-4-kinase (pi4k) iii. a role for ncs-1 in stimulating this enzyme was first shown in yeast and subsequently the mammalian ncs-1 protein was shown to stimulate pi4kiii and also to interact with both the enzyme and the small gtpase adp - ribosylation factor 1 (arf1) on the golgi complex. these interactions regulate general traffic from the trans - golgi network (tgn) to the plasma membrane, with the effects of ncs-1 and arf1 being antagonistic. the importance of these interactions is indicated by findings that ncs-1, arf1 and pi4kiii control inner ear development in zebrafish. recently, cabp7 and 8 were found to interact with pi4kiii but, in contrast to ncs-1, inhibited the enzyme activity at resting ca levels. this inhibition was released on binding of ncs-1 at elevated ca levels, providing a tight ca threshold for control of the traffic from the tgn to the plasma membrane in neurons (figure 1). release of ca from the golgi through channels such as the inositol 1,4,5-trisphosphate receptor may influence trafficking through and from this organelle. under conditions of high local ca concentration (high - ca), ncs-1 binds to and activates phosphatidylinositol-4-kinase iii (pi4kiii) (activated pi4kiii is indicated by an asterisk) to drive the production of phosphatidylinositol 4-phosphate (pi4p) (shown enriched in hatched regions of the membrane), a lipid essential for stimulation of vesicular traffic from the tgn. a further level of complexity in this model stems from a ca - dependent interaction between ncs-1 and the key golgi trafficking gtpase adp - ribosylation factor 1 (arf1). at high ca, ncs-1 and arf1 interact, preventing activation of pi4kiii in regions where the two proteins coexist. since arf1 activates pi4kiii independently of ca, this interplay may represent a mechanism for spatial organisation of the tgn into discrete subdomains tasked with serving either ca - dependent or ca - independent trafficking functions that are segregated by domain boundaries populated by non - productive arf1/ncs-1 complexes. under conditions of low local ca concentration (low - ca), ncs-1 no longer activates pi4kiii and instead cabp7 and cabp8 are able to interact with the kinase to inhibit its activity. lack of pi4p production under such circumstances would prevent vesicular traffic from ca - dependent trafficking domains. it has also become clear that ncs-1 has key roles in aspects of synaptic plasticity underlying learning and memory. there are three ncs-1 homologues in caenorhabditis elegans and knockout of the one most similar to the mammalian protein impaired learning and memory in the worms. increased levels of ncs-1 switched the mode of short - term plasticity in hippocampal synapses in culture from depression to facilitation. further progress was made when it was shown that ncs-1 was required for a form of synaptic plasticity, known as long - term depression (ltd), induced through metabotropic glutamate receptor activation in cortical neurons. in this system, ncs-1 acted as a ca sensor in conjunction with another ca - binding protein, pick1 (protein interacting with c kinase 1). as noted above, ncs-1 interacts with and controls the surface expression of dopamine d2 receptors. most recently, a study extended these findings to directly link ncs-1 regulation of dopamine d2 receptor activity with the processes of learning and memory in adult mice. inducible targeted expression of ncs-1 specifically in the dentate gyrus of transgenic animals enhanced acquisition of spatial memory and had the novel effect of increasing exploratory behaviour. importantly, these effects could be abolished through antagonism of the ncs-1/dopamine d2 receptor interaction by use of a membrane - permeant inhibitory peptide. another ncs protein, hippocalcin, has been of interest in considerations of synaptic plasticity as it is expressed to the highest extent in the hippocampus, which is a site of memory formation. hippocalcin knockout mice have impaired memory formation. like ncs-1, hippocalcin has been suggested to act as a ca sensor for ltd but in this case in the hippocampus. kchip3 was independently discovered and named the downstream regulatory element antagonistic modulator (dream). dream can act in the nucleus by binding in a ca - dependent manner to a specific dna sequence and may control aspects of synaptic plasticity through changes in gene expression. in fact, one of its major physiological roles revealed in a knockout mouse is in pain modulation. a recent novel role that has been discovered is that of caldendrin in regulating nuclear signalling in neurons by interaction with the protein jacob. this leads to changes in dendritic remodelling following nmda (n - methyl - d - aspartic acid) receptor activation, which could also be important for synaptic plasticity. major issues still to be resolved include the physiological roles of the ncs family members that have been less studied (e.g., neurocalcin and most of the cabp family). all of the knockouts of ncs proteins have differing phenotypes, indicating a lack of redundancy of these proteins. to date, the only knockouts in mice for the cabp family are for cabp4 and 5 that may be expressed only in the retina in mice. these mice have abnormalities in retinal function, and indeed mutations in human cabp4 result in congenital stationary night blindness. various studies have made indirect links between ncs proteins and neuronal disease states, and the relationship of these proteins to neurological deficits requires further work as they could potentially be targets for pharmacological treatment of these disease processes. of further interest is the relationship between the described effects of these proteins on membrane traffic and on synaptic plasticity and whether these two aspects are causally related. finally, while the structures of several of the ncs and cabp proteins have been solved, there are still only a few examples of structural analysis of the sensors bound to their target proteins [23,24,47 - 49 ] and so additional structure / function studies would help to reveal more about the molecular basis of their actions as we currently know relatively little about how they regulate their target proteins.	ca2 + plays a crucial role in the regulation of neuronal function. recent work has revealed important functions for two families of neuronally expressed ca2 + sensor proteins. these include roles in membrane traffic and in alterations in synaptic plasticity underlying changes in behaviour.
multiple sclerosis (ms) is a chronic, inflammatory demyelinating disease, which mainly affects the white matter of the central nervous system (cns) but may also affect the cortex. the disease presents itself with a wide range of clinical manifestations, usually beginning with a relapsing remitting (rrms) phase. rrms is characterized by attacks of worsening neurologic function (relapses) that are followed by partial or full recovery (remissions). relapses are directly related to an underlying inflammation of the cns, which affects the myelin of the axons and consequently leads to focal ms lesions. because magnetic resonance imaging (mri) is sensitive to inflammatory and demyelinating changes, it is often used to monitor, identify and quantify ms lesions (fazekas., 1999) that are hyperintense on t2-weighted (t2w) magnetic resonance (mr) images and may become hypointense on t1-weighted (t1w) images. lesion counts are often used to assess the disease burden and track disease progression as new lesions are related to current disease activity. both counts are used to assess the efficacy of new therapies (polman., 2011). for the purpose of this article, we focus on lesions commonly called t2-lesions (those that are hyperintense on t2w images) and do not consider other sub - types of lesions (i.e., gadolinium enhancing active lesions, black holes and cortical lesions). ms lesions in mr images are extremely difficult to identify because of inter - subject anatomical variability, lesion location, size, texture and shape. manual segmentation of ms lesions is still recognized as the gold standard in ms, but it is time consuming and subjects to intra- and inter - expert variability. as an alternative, a multitude of automatic techniques to detect and segment ms lesion has been proposed. however, recent reviews of the literature (llad., 2012 ; garca - lorenzo., 2013) concluded that automatic ms lesion segmentation is still an unsolved topic. for example, many techniques are not robust across imaging centers or differing mri protocols. unsupervised methods do not require manual segmentation of the lesions, but estimate tissue classes or clusters of similar voxels with or without the help of anatomical and mri knowledge. many unsupervised techniques were initially developed to classify healthy brain tissues based on mri intensities into three classes (cerebral spinal fluid (csf), white matter (wm) and grey matter (gm)). this was done by using fuzzy c - mean and gaussian mixture models with expectation maximization (em) (wells., 1996). to detect ms lesions, some groups adapted the gaussian models by adding an extra class for ms lesions (kikinis., 1999 ; 2008) and/or added topological constrains (i.e., the publically available approach lesiontoads (shiee., 2010)). others defined lesions as outliers of the mixture model (van leemput., 2001 ; schmidt., 2012 ; cabezas., 2014) or as outliers when comparing the spatial and intensity information of the images to be segmented and library of healthy subjects (tomas - fernandez and warfield, 2011 ; tomas - fernandez and warfield, 2015). to correct for noise and image artifacts, graph - cut techniques have been used to combine spatial information from the local neighborhoods with the intensity model (garca - lorenzo. unsupervised techniques suffer from both intensity non - uniformity, in the whole image and in the lesion since this variability in intensities can not be captured with a single global model. furthermore, the properties of each image need to be specifically defined which can be difficult when artifacts have properties similar to lesions. the supervised methods use machine - learning techniques to extract relevant features (e.g. intensity or local gradient) and train automatic classifiers from manual or automatic lesion segmentation datasets. then, the features of new images to be segmented are compared with the training sets to estimate the lesions. these methods can use different machine - learning approaches including : artificial neural networks (ann) (zijdenbos., 1994), k - nearest neighbors (k - nn) (vinitski., 1999), decision trees (kamber., 1995), random decision forests (rdf) (geremia., 2011), bayesian frameworks (harmouche., 2006) and logistic regression (sweeney., 2013). the common limitation of both supervised and unsupervised techniques is their sensitivity to image and lesion variability. however, using the appropriate training set and image features, supervised techniques can potentially identify the ms lesion and compensate for variability in image intensities. indeed, it has been shown that many supervised library - based (or multi - atlas) techniques outperform unsupervised model - based segmentation methods (lao., 2008). for example, patch - based methods using non - local means (nlm) for structural segmentation have gained in popularity and shown promising results despite their simplicity (coup., 2011). patch - based approaches have been applied to segment a multitude of anatomical structures including the hippocampus (coup., 2011), brain (eskildsen., 2012), lateral ventricles (fonov., 2012), deep nuclei (xiao., 2014), intracranial cavity (manjn., 2014), brain tissues (cordier., 2013) and other structures of the brain (rousseau., 2011 although nlm has proven to be useful in segmenting anatomically well - defined structures (e.g. hippocampus and lateral ventricles) they have not yet been applied intensively to ms lesion segmentation. given the success of patch - based approaches, we present a library - based nlm approach where voxels with similar surrounding neighborhoods (or patches) are used to estimate the presence of lesions. contrary to the original patch - based segmentation method (coup., 2011), we offer two main contributions in order to efficiently address the problem of ms lesion segmentation : i) a rotationally - invariant similarity metric for patch comparison which better captures lesion shape variability and ii) a multi - contrast framework that takes advantage of information derived from t2w and flair images. indeed, in the context of ms lesion segmentation the dimension, shape, orientation and position of lesions vary greatly (fig. the sum of squared differences (or l2-norm), which originally used as patch - based distance measure (buades. 2005), is sensitive to the orientation of the patch. while this is good for structure segmentation, this could potentially miss lesion labels (fig. 1). similar to the work by manjn. (2012), we replace the l2-norm distance measure with a rotation - invariant distance (ri) where only the intensity of a central voxel and the mean intensity of the patches are considered. furthermore, the existing nlm segmentation algorithms used a single contrast library (e.g. only t1w images), however, a single image contrast does not hold enough information to separate lesions from healthy tissues. most lesion segmentation algorithms use t2w and flair mr images, as most ms lesions appear hyperintense on these modalities indicating inflammation or scar tissue. on t1w images, lesions appear hypointense but present a larger intensity variability which might reflect the different sub - types of lesion such as the so - called black hole associated with irreversible tissue damage (van walderveen. (2013), which introduced multi - contrast nlm for image super - resolution and xiao. (2014) for dual - channel nlm segmentation of deep brain structures, we propose an adaptation of the nlm segmentation algorithm to take advantage of multi - contrast images for ms lesion segmentation. this library - based approach captures the potential global and local variability of the anatomy as well as the intensity variability in lesions. to our knowledge, despite the increasing popularity of patch - based techniques, only a few recent methods have been developed to segment ms lesions. 2013) presented a supervised segmentation technique using sparse coding with patches from a library of healthy subjects to reconstruct ms patient images. the reconstruction estimates an error map, which detects outliers believed to describe ms lesions. their method shows promising preliminary results but was not assessed on a large clinical cohort and might not be specific enough to distinguish ms lesions from artifacts when detecting image outliers. the approach by roy., they estimate a weighted average of the most similar patches of a kd - tree using a nearest neighbor search on a library of pre - segmented multi - contrast (t1w and flair) images. while sparse strategies present the advantage of decreasing the dimensionality of the library, kd - tree removes the 3d spatial knowledge of the training images which may hold additional pertinent information that can help identifying ms lesions. indeed, despite careful pre - processing, the local ms lesion properties in mr images (i.e., intensity, contrast, noise) depend on the local anatomical and/or spatial location of the lesion (meier and guttmann, 2003). thus, by using a 3d volume library, this local information can help capture the spatial layout of different ms lesions. finally, neither weiss. (2013) nor roy. 's (2014) patch - based approaches for lesion segmentation use rotationally invariant features. as will be demonstrated in section 3.1.3, this aspect is crucial in the context of ms lesion detection. we assessed our rotation - invariant multi - contrast non - local means segmentation (rmnms) approach on 108 rrms patients from a multi - site clinical study. our method obtained a dice similarity measure of 60.1 16.4%, a sensitivity 75.4 15.7% and a precision 55.0 20.1% in cross - validation. using the parameters established for our initial evaluation, we compared rmnms to several different state - of - the - art techniques using the datasets from the ms lesion grand challenge (msgc, miccai, 2008 (styner., 2008)), on which we obtained very competitive results holding the first rank at the time of the submission. in the following section we first describe the developed algorithm (section 2.1), then the dataset (section 2.2), and lastly our evaluation techniques (section 2.3). our algorithm adapts the nlm estimator (section 2.1.1) to account for multi - modal images (section 2.1.2) and rotation - invariant distance measure of the patches (section 2.1.3). the nlm estimator, which takes advantage of image redundancy, was initially proposed by buades. the idea of the nlm is to reduce the noise of the image by averaging the voxels that would have a similar intensity in the noise - free image. to achieve the denoising of voxel x(i), the patch p(x(i)) centered on i is compared with all the patches p(x(j)) centered on j of the images in the neighbourhood such that:(1)x^i=jwijljjwijwherewij = epxipxjs22h2where the term w(i, j) is a weight based on the similarity of between the patches p(x(i)) and p(x(j)), and is designed to attribute a smaller weight to the greater l2-norm (\|\|.\|\|2) distance measures. the nlm approach has been used for structural segmentation (coup., 2011) by employing a library of atlases with co - registered anatomical images and manually segmented structures to segment those particular structures on new subjects. for nlm segmentation, the weights are estimated between intensities of the subject patch, p(x(i)), and patches from a subject s from library of n pre - segmented subjects, p(x(js)), such that:(2)x^i=s = njwijsljss = njwijswijs = epxipxjs22h2where in this case the h parameter is set based on the patch minimum distance of the search area (coup., 2011). thus, if similar patches are found in the library the minimum distance h will be low and the weight function will decay quickly such that it is not influenced by other patches. in ms, multi - contrast images for manual and automatic segmentation have shown to improve the identification of ms lesions. inspired by previous work on multi - contrast nlm (mnlm) for denoising (manjn., 2009) and for super - resolution (coup., 2013), we apply the nlm weighting function to allow for various contrasts (m) such that:(3)wijs = empmxipmxjs22hm2 here m represents the different mr contrasts commonly used in ms lesion segmentation : t1w, t2w, pdw, or flair for example. it is important to note that the smoothing parameter hm is estimated for each considered contrast (i.e., the per contrast minimum distance). subjects with similar lesion load and spatial distribution may be more similar with respect to their brain intensity characteristics. therefore, focusing the weight estimation on the most similar subjects should potentially hold more similar patches, and also presents the advantage of reducing computation. in the context of label fusion segmentation methods, aljabar. (2009) proposed a pre - selection for single contrast images of the most similar structures present in the training library. in our multi - contrast method, we seek the most similar training subjects by measuring the multi - contrast l2-norm (ml2-norm) distance of the subject being segmented and the training subjects across their brain mask region, defined as:(4)ml2norm=mimxilmxjs2 the n subjects with smallest ml2 distances are selected as they represent the most similar training subjects and thus provide the most similar set of features. previous nlm segmentation implementations have shown convincing results in segmenting anatomically well defined structures (e.g. hippocampus and lateral ventricles (coup., 2011)). anatomically, these structures present a relatively small variability of shape, contrast and spatial location making the orientation of the structure to be segmented an important constraint when looking for similar patches in the library. however, this strong advantage for structural segmentation could be a drawback in the context of ms lesion segmentation where no structural, orientation nor spatial location can be assumed. (2011) show that ms lesions can be found almost anywhere in the brain. however, there is spatial predilection for lesions to occupy the peri - ventricular area, the cortico - spinal tract and the optic radiations the lesion distribution probability map as can be shown in fig. 3. the lesions themselves do not appear to have a constraint on their size, shape or number (as shown in fig. 10 on three ms cases). in order to increase the ability of the nlm segmentation approach to detect ms lesions, we propose a rotation - invariant distance (ri) metric instead of the l2-norm metric that is used in the multi - contrast nlm framework. (2012) on sparseness and self - similarity for mri denoising, we replaced the l2-norm with a ri distance measure. therefore, only the intensity of a central voxel (x) and the mean of surrounding patch () are considered:(5)wijs = emxmixmjs2+mimjs2hm2 in our experiments, we found that the intensity difference of the central voxels (xm(i)xm(js)), was roughly the same as the intensity difference of the patch average (m(i)m(js)), thus we chose = 1, whereas manjn. the need for a difference in alpha might be due to our pre - processing and in particular the denoising step, which tends to smooth the neighbouring intensity values surrounding the central voxel. the image denoising step used in our pre - processing (coup., 2008) is indeed a crucial step as it removes the variability of the central voxel with respect to its neighborhood and therefore allowing a better identification of similar ri features. in order to be fully invariant to rotation, patches should be spherical, however, we found that cubic patches significantly reduce computational burden while preserving the distance accuracy. 1, where the distance of a cubic patch containing a lesion and the identical patch subject to different rotations is measured. indeed, ri provides identical distance measures for different rotations of the same patch, only varying due to sampling and/or interpolation error, while l2-norm varies greatly and favors a larger distance between patches. another advantage of the ri distance measure is a reduced computational cost owing to considering only the central voxel and the mean of the patch, rather than all voxels in the patch. to further reduce computational cost we used multithread processing. (2013), simulated datasets, e.g. brainweb, enable a good proof of concept validation for image processing methods by providing ground truth images and lesion masks. however, brainweb images present multiple limitations : synthetic images are much easier to segment, only one phantom anatomy exists, and brainweb lacks contrasts such as flair. therefore, in this article we focus on two clinical datasets, an rrms multi - center clinical dataset and the miccai (2008) ms grand challenge dataset (styner., 2008). one clinical multi - center dataset of 108 rrms patients [age = 42.6 10.7, 72 females ] was used to assess the proposed segmentation algorithm. the dataset contains t1w [te = 911 ms, tr = 3040 ms, flip angle = 30, in - plane resolution = 0.977 0.977 mm, slice thickness = 3 mm ], t2w [te = 66100 ms, tr = 35506610 ms, flip angle = 90, in - plane resolution = 0.977 0.977 mm, slice thickness = 3 mm ], pdw [te = 1018 ms, tr = 18673750 ms, flip angle = 90, in - plane resolution = 0.977 0.977 mm, slice thickness = 3 mm ] and flair [te = 5994 ms, tr = 79779630 ms, ti = 19932500 ms, flip angle = 90, in - plane resolution = 0.977 0.977 mm, slice thickness = 3 mm ] for all subjects. the mri data were acquired at 32 sites on 1.5 t scanners from different manufacturers : ge (n = 19), philips (n = 3) and siemens (n = 10). this contains gold standard ms lesion segmentation labels that were first automatically segmented by a multi - spectral bayesian classifier (francis, 2004) with the t1w, t2w and pdw images and manually assessed and corrected by expert raters who underwent extensive training on similar ms patient mri data. in a previous study (caramanos., 2012), seven raters with similar expertise, corrected the automatically generated lesion labels and were evaluated on a set of 10 ms patients with similar mri protocols to those used in this study. thanks to the initial automatic segmentation, this evaluation revealed an excellent inter - rater reliability relative to their trainer 's reference segmentations (dsc = 93.5 1.5%) and intra - class correlations (icc = 99.0 0.5%). this rrms cohort presents a large range of lesion loads (0.548.8 ml) and lesion counts (1156 lesions) which are depicted in fig. only lesions with at least three connected voxels (or a lesion volume of 0.009 ml) in the 3d volume are kept and considered in our experiments. the mri data and the expert lesion masks were used to form the template library of our proposed algorithm, which was tested in a leave - one - out fashion. our proposed rmnms algorithm was further validated using the clinical data provided by the ms lesion segmentation challenge introduced at miccai (2008) (styner., 2008). from the ms challenge website1, 20 training mr datasets with ground truth manual lesion segmentations and 23 testing cases were provided from the boston children 's hospital (chb) and the university of north carolina (unc). while lesions masks for the 23 testing cases are not available for download, an automated system is available to evaluate the output of a given segmentation algorithm. we downloaded the co - registered t1w, t2w, flair images for all 43 datasets as well as the ground truth lesion mask images for the 20 training datasets. we used the msgc training set as a library to segment the msgc t2w and flair images. all the images from both ms datasets (clinical rrms and msgc) were processed using the same pipeline, which does : a)nlm image denoising (coup., 2008).b)intensity non - uniformity correction (n3) (sled., 1998). linear intensity normalization of the image histogram to our in - house ms templates that were created with an unbiased template creation algorithm (fonov., 2011) from the 108 t1w images of the rrms patients (fig. 3).c)linear registration of each t1w image to our ms template which is in the mni152 template space (collins., 1994).d)rigid registration of the t2w and flair to the t1w image, followed by resampling onto a 1 1 1 mm grid in the mni space. note that for the purpose of the validation describe here, we used the t1w as the reference image for registration, but other modalities (t2w, flair) could be chosen if a t1w image is not present or required.e)brain extraction (eskildsen., 2012). intensity non - uniformity correction (n3) (sled., 1998). linear intensity normalization of the image histogram to our in - house ms templates that were created with an unbiased template creation algorithm (fonov., 2011) from the 108 t1w images of the rrms patients (fig. linear registration of each t1w image to our ms template which is in the mni152 template space (collins., 1994). rigid registration of the t2w and flair to the t1w image, followed by resampling onto a 1 1 1 mm grid in the mni space. note that for the purpose of the validation describe here, we used the t1w as the reference image for registration, but other modalities (t2w, flair) could be chosen if a t1w image is not present or required., 2013), we did not apply non - linear registration to segment and create the library. this is due to the fact that non - linear registration and interpolation of ms lesions could alter the anatomical and intensity characteristics of ms lesions. after pre - processing, all of the images and their respective manual segmentation lesion maps are spatially aligned and their intensity distributions are normalized. the denoising step of the pipeline is crucial for the ri distance measure as the central voxel value of a patch is given as much weight as its surrounding patch average. the ms library, used for the segmentation, was built using the output images from the pre - processing stages d, e and f. to double the size of the library and increase the spatial distribution of ms lesions, left we describe the general evaluation strategy (section 2.3.1) and the different metrics (section 2.3.2) used to assess the proposed segmentation method. on the clinical rrms dataset, we performed a leave - one - out cross - validation of the proposed rmnms method. this leave - one - out cross - validation is achieved by first removing the subject and its respective left right - mirrored images from the training library, then the multi - modal pre - selection of the n closest subjects are selected and finally the segmentation is performed. we first evaluated the performance of rmnms with respect to the search area radius of the patches in the library and the number of pre - selected training subjects (as described in section 2.1.2). we also assessed rmnms using i) different contrast combinations (t1w + flair, t2w + t1w + flair and t2w + flair contrasts), ii) the original l2-norm distance measure version of the nlm segmentation algorithm using t2w + flair contrasts (t2w + flair nlm), iii) without the left right mirror addition to the training library, and iv) the lesiontoads2 approach proposed by shiee. lesiontoads is an iterative atlas based segmentation technique that uses a topological and a statistical atlas within the fuzzy c - means algorithm. as it was originally developed to segment healthy brain tissues (bazin and pham, 2008), the algorithm was adapted to use multi - contrast (t1w + flair) and an extra lesion class within the wm class. lesiontoads was chosen as it is publically available and obtained one the best results during the 2008 msgc (styner., 2008). furthermore, we explored the effect of patients ' total lesion - load and lesion - by - lesion detection measures on the rmnms method. first, our algorithm was validated on the training set using leave - one - out cross - validation. second, our segmentation results on the testing msgc dataset were submitted online3 and compared with other published techniques including i) lesiontoads, ii) souplet. (2008), winner of the msgc at miccai 2008, iii) a recent supervised technique by geremia. (2011), who hold the current best score on the msgc website before our submission. for the online msgc evaluation the organizers normalized different metric results between 0 and 100, where 100 is a perfect score and 90 is the typical score of an independent rater as described by styner. (table 1)) were measured by comparing the automatic segmentation to the manual segmentation of two experts (chb and unc). the quantitative evaluation of our method is carried out using different metrics, summarized in table 1 as suggested by styner. (ppv) and sensitivity (tpr) indicate that the automatically segmented lesions correspond well to the manually labeled lesion voxels. a low fall - out (fpr) indicates that the procedure does not identify voxels as lesion when they are not. we measure the absolute volume difference (vold) of the manual versus the automatic segmentation (0% indicates a perfect lesion volume agreement) and the symmetric surface distance (surfd) estimates the euclidean distance between the surfaces of both segmentations at each voxel of their contours (0 mm indicates a perfect match of the surfaces). to estimate the surfd values, we first estimate the distance transform from the binary segmentation using a 3d - euclidean metric (borgefors, 1988) where the surface has a value of 0. then, we look at the value of the binary segmentation and the corresponding transform distance value to estimate the distance to the surface. usually, the true positive (tp), false positive (fp) and false negative (fn) rates are voxel - based ; however, this measure can also be performed in a lesion - wise manner. indeed, in some studies, detecting small lesions we use lppv and ltpr, which are lesion - wise version of the ppv, and tpr metrics where lesion - wise tp (ltp), fp (lfp) and fn (lfn) are measured at each distinct lesion (table 1). in this case, instead of applying the metrics on a voxel - by - voxel basis, we measure the ability of the method to detect the presence of a lesion. following the expert manual segmentation protocol, only lesions with at least 3 voxels (or 0.009 ml in the native image space) and an overlap of at least 1 voxel (or 0.003 ml) were considered (karimaghaloo., 2012). finally, we assess the behaviour of our method with regard to the patient 's lesion load, size and location. the t1w, t2w, and flair rrms average templates created for the pre - processing stages of our method are presented in fig. 3 along with the spatial distribution of t2w lesions. while one can appreciate the anatomical definition of the different contrast templates, we can still visually identify the hypo - intense intensity distribution around the lateral ventricles on the t2w and the corresponding hyper - intense intensity on the flair. as expected, the t2w lesion spatial probability distribution is higher is the peri - ventricular region. however, the presence of lesions is diffuse, and our library of ms patients holds enough spatial variation to capture the spatial distribution of lesions. the results for different search area radii on the different metrics (dsc, vold, tpr, ppv, ltpr and lppv) are presented in fig. 4 for rmnms using t2w + flair. in our experiments, we found that using a patch size of 3 3 3 provides the best compromise between accuracy and computational burden. with a 3 3 3 patch (i.e., radius of 1 voxel) and a pre - selection of 50 subjects, the dsc, tpr, ppv, ltpr and lppv results plateau at their best results with a search area radius of 5 voxels (i.e., 11 11 11 search area). the volume difference (vold) results are not as clear but the best results are achieved for any search area radius bigger than 2 voxels. increasing the search area increases the chance of capturing a patch that is more similar to the considered patch, however, increasing the search area needs to balance against computational cost where for instance, increasing the search area from 5 to 6 voxels increases the computational time by 15%. we found a search area radius of 5 voxels to be a good compromise (median results : dsc = 60.1 16.4%, tpr = 75.4 15.7%, ppv = 55.0 20.1%, vold = 33.5 68.9%, ltpr = 79.8 14.6% and lppv = 85.7 24.2%) and was chosen for the rest of the evaluation. pre - selecting more subjects from the template library can increase segmentation accuracy. in fig. 5, the results for the rmnms method using t2w + flair with different numbers of pre - selected training subjects on the different metrics (dsc, tpr, vold, ppv, fpr and vold) are presented. the experiment was performed with a patch radius of 2 (voxels), and a search area radius of 5 voxels while varying the number of pre - selected training subjects from 10 to 80. as expected, increasing the number of subjects in the library improves the quality of the segmentation. using 50 subjects provides a good comprise between segmentation results and computational cost (median results with 50 pre - selected subjects are the same as in section 3.1.1, as we used the same parameters) and was chosen for the rest of the evaluation. here, we compare rmnms using t1w + flair, t2w + t1w + flair, t2w + flair with and without the mirrored library images as well as the previous mnlm technique using t2w + flair images, and lesiontoads using t1w + flair images. rmnms with t2w + flair was selected as the baseline for comparison and the similarity metric results are summarized in fig. 6 is that rmnms t2w + flair provides a higher ltpr (79.8 14.6%) than t2w + flair mnlm (67.3 18.6%). furthermore, t2w + flair rmnms consistently obtains the highest results (dsc, vold, ppv, tpr, ltpr and lppv) when compared to the different modalities used with rmnms but also when compared with the unsupervised lesiontoads approach. using t2w + flair images provides overall better segmentation results than the other modality combination and the addition of the left right mirrored images to the training set improves consistently the segmentation results of t2 + flair rmnms. the computational time for rmnms using 16 threads on an intel core i7 - 950 processor at 3.06 ghz was around 40 min per subject. our method with these settings is about three times faster (p - value 45) by submitting our segmentation results of the 23 ms test subjects to the msgc website (styner., 2008). while our rmnms approach attained the first position at the time of writing with a score of 86.11, this result must be considered with the limitations described above. we feel that our evaluation with the multi - site clinical dataset is much more representative of quality and robustness of the rmnms technique. we also compared our approach on our rrms dataset to the popular and publicly available lesiontoads approach (shiee., 2010). compared to rmnms, lesiontoads is a topology preserving approach guided by probabilistic and topologic atlases. this approach was developed to segment t1w and flair images and as any unsupervised approach it is less flexible to image variability that is not described by the underlying models. future work will focus on improving segmentation results for smaller lesions, further decrease in the computational time with more advanced patch matching strategy (ta., 2014), investigate the performance and the pre - selection preferences with respect to scanner machine, site, gender and other clinical variables. our method, rmnms, is a multi - contrast and rotation - invariant distance adaptation of the non - local means operator. rmnms presents highly competitive results compared to state - of - the - art supervised and unsupervised methods and provides segmentation quality near inter - rater variability for ms lesion segmentation. rmnms, with multi - contrast and rotation - invariant patch distance, demonstrates that the non - local approach is able to detect structures that vary in size, shape and location such as ms lesions.	multiple sclerosis (ms) lesion segmentation is crucial for evaluating disease burden, determining disease progression and measuring the impact of new clinical treatments. ms lesions can vary in size, location and intensity, making automatic segmentation challenging. in this paper, we propose a new supervised method to segment ms lesions from 3d magnetic resonance (mr) images using non - local means (nlm). the method uses a multi - channel and rotation - invariant distance measure to account for the diversity of ms lesions. the proposed segmentation method, rotation - invariant multi - contrast non - local means segmentation (rmnms), captures the ms lesion spatial distribution and can accurately and robustly identify lesions regardless of their orientation, shape or size.an internal validation on a large clinical magnetic resonance imaging (mri) dataset of ms patients demonstrated a good similarity measure result (dice similarity = 60.1% and sensitivity = 75.4%), a strong correlation between expert and automatic lesion load volumes (r2 = 0.91), and a strong ability to detect lesions of different sizes and in varying spatial locations (lesion detection rate = 79.8%). on the independent ms grand challenge (msgc) dataset validation, our method provided competitive results with state - of - the - art supervised and unsupervised methods. qualitative visual and quantitative voxel- and lesion - wise evaluations demonstrated the accuracy of rmnms method.
. these injuries can occur in a fashion similar to those of the biliary system, resulting in laceration, transection or occlusion of the hepatic arterial. this case report describes a patient who presented with acute bleeding from a pseudoaneurysm of the replaced right hepatic artery and the left main hepatic artery following a laparoscopic cholecystectomy. a 64-year - old male referred to our hospital 4 weeks after having a laparoscopic cholecystectomy complicated by a common bile duct injury. he had undergone a difficult laparoscopic cholecystectomy with intraoperative bile duct injury suspected at the end of the procedure at a peripheral hospital. the surgeon inserted a drain in the surgical bed at the time of the surgery. postoperatively, the patient was started on piperacillin and tazobactam, and underwent an endoscopic retrograde cholangiopancreatography (ercp), which confirmed the presence of a common bile duct injury. a biliary stent was placed. the patient continued to have daily bile drainage ranging from 100 to 350 ml per day. upon presentation to our center, the patient was febrile (38.9c) and complained of a left upper limb pain and swelling. hematology was consulted, and a diagnosis of acute upper limb venous thrombosis was established. owing to the presence of blood in the drain, he was kept on the maximum prophylactic dose of unfractionated heparin. a ct angiography of the abdomen showed two collections, one at the surgical bed near the drain, and the other was subcapsular below the left lateral lobe of the liver, as well as an aneurysm of the replaced right hepatic artery with an active bleeding blush (fig. 1). the patient was immediately referred for an angiography, which confirmed the ct scan findings (fig. an ercp was also performed, which revealed a strasberg class d injury, and a plastic biliary stent was inserted. after the angio - stent insertion and stabilization of the patient, five days later he developed hematemesis and melena with a significant drop in his hb to 2 g / l, and his total bilirubin became 183 mol / l of which 91 mol / l is direct. a gastroscopy was performed and showed hemobilia (bleeding from the ampulla of vater). subsequent angiography demonstrated a leak of contrast just above the arterial stent ; hence, a further stent was placed to cover that area of the aneurysm. similar symptoms reoccurred a week later, and a new angiography showed a new aneurysm from the left proper hepatic artery. a percutaneous thrombin injection of the aneurysm was performed as the bleeding branch was unreached via direct angiography and was filling in retrograde perfusion. during recovery a chest spiral ct figure 1:a ct scan showed replaced right hap inside the collection (straight arrow). figure 2:an angiographic scan showed superior mesenteric artery (straight arrow) and replaced right hap (angulated arrow). an angiographic scan showed superior mesenteric artery (straight arrow) and replaced right hap (angulated arrow). an angiographic scan showed stent in the replaced right hepatic artery. on day 7, the patient bled again from the same aneurysm of the left hepatic artery. a repeated angiography revealed the bleeding with a reduced flow in the stented, replaced right hepatic artery (fig. the active bleeding was stopped using gel - foam embolization of the two branches of the left hepatic artery (fig. 5) with a decision to embolize the whole left hepatic artery if bleeding did not stop while holding the heparin infusion. the patient 's liver function was preserved, and the bleeding stopped despite anticoagulation. figure 4:an angiographic scan showed reduced flow in the stented replaced right hepatic artery. figure 5:an angiographic scan showed left hap after a gel foam. a laparoscopic cholecystectomy is reportedly associated with an increased incidence of biliary and vascular injuries. specifically, biliary injuries are reported in 0.21% of procedures with a 10-fold increase when compared with open surgery, whereas vascular injuries occur in 0.250.5% of procedures. the symptoms may appear in the early postoperative period or as late as 120 days after operation. a pseudoaneurysm of the hepatic artery or its branches can lead to bleeding through the drain or in the form of hemobilia in 20% of cases. however, the classical clinical triad described by quinke of right upper quadrant pain, jaundice and hemobilia has been reported in 32% of patients with laparoscopic cholecystectomy - related hepatic artery pseudoaneurysms (haps). the clinical presentation of hap is bleeding, which might be intermittent bleeding if discovered early. if it is not identified, a massive hemorrhage may occur with a rupture, and the mortality rate could be as high as 50%. in our patient, direct vascular injury, erosion due to clip encroachment and diathermy shorting on clip - associated infections are likely to be precipitating factors. bile is cytotoxic and the amphipathic properties of bile acids make them powerful solubilizers of membrane lipids, causing cell death. a canine study by sandblom. showed that bile delays the healing of a liver wound, attributable to the fibrolytic or cytotoxic effects of bile. bile can therefore cause a weakening of suture lines or sites of surgical clips in vessels. the presence of an infection is another contributing factor for the development of the hepatic artery aneurysm. the most likely cause in our patient was a combination of an infection and a traumatic injury. the treatment of a hepatic artery aneurysm is an emergency because the patient may exsanguinate from a rupture at any time. patients with hematemesis or melena following the laparoscopic cholecystectomy should have an urgent endoscopy, and if no cause is identified, an abdominal ct and hepatic angiography should be performed. definitive treatment with radiologic embolization is the treatment of choice for hap, although some reports of conservative management have been recorded. in our case, the stenting of the replaced right hepatic artery and gel - foam embolization of the left hepatic artery were performed. when a patient presents with massive gi bleeding, a climbing total bilirubin level and recent hepatobiliary manipulation or intervention, a high index of suspicion is always needed. the added complexity in our patient was the presence of deep vein thrombosis and pulmonary embolism, the development of a left hap in the presence of a thrombosed replaced right hepatic artery that was stented and finally, bleeding from the left hap, which stopped with gel - foam embolization.	hepatic artery pseudoaneurysm is a rare complication of laparoscopic cholecystectomy. a high index of suspicion and early identification and therapy are important points needed to prevent rupture. we report a case of complex biliary and vascular injuries 4 weeks after a laparoscopic cholecystectomy. the patient had recurrent bleeding from a hepatic artery pseudoaneurysm that has been treated successfully with angiographic stenting and embolization.
as a key element in the eukaryotic gene expression, alternative splicing increases the coding capacity of the human genome that leads to the generation of numerous mrna variants which yield different polypeptides from a unique coding gene (1 - 3). it is estimated that more than 88% of human protein - coding genes are affected by this process (4). several studies demonstrate that changes in pre - mrna splicing play a significant role in human disease development (5 - 7). there are mounting evidences that many cancer - associated genes are regulated by alternative splicing. furthermore, the patterns of alternative splicing are changed in human cancers (8, 9). the cancer - associated genes involved in the key aspects of human malignancies including angiogenesis, invasion, differentiation, resistance to apoptosis (10) and metastasis are affected by alternative splicing (8, 11, 12). as the second most frequent cause of cancer death and a main public health concern, gastric cancer is a common disease worldwide affecting about one million people per year (13 - 15). it is more common in men than in women (the ratio is about 2:1). according to the national cancer registry data, gastric cancer is one of the first leading cause of iranian cancer - related deaths in men and the second one in women (16). an unhealthy lifestyle including a high concentration of salt in diet and smoking is the major environmental risk factors of this cancer in iran (17). the zfx gene on the human x chromosome is a member of zfy family that encodes a 90 kda zinc finger protein (c2h2-type) (18 - 20) this protein is a transcriptional regulator, identified as a putative stemness gene in embryonic and adult hematopoietic stem cells (hscs) (21). furthermore, zfx over expresses in various types of human malignancies including prostate adenocarcinoma (21), esophageal carcinoma (22), follicular lymphoma and diffuse large b - cell lymphoma, (23) and gliomas (24). therefore, it might be regarded as a cancer stem cell marker in the examined cancers as it confers a self - renewal - like property to cancer cells (25). recently, we showed that zfx (variant 1 and 3) overexpresses in the diffuse type of gastric cancer (27). due to the crucial role of alternative splicing and the lack of data concerning the expression of zfx isoform 3/variant 5 in gastric cancer, in the present study, we evaluated the clinicopathological relevance of the expression of zfx isoform 3/variant 5 gene in gastric tissue samples. this variant lacks two exons in the 5 ' utr and has an additional segment in the 3 ' cds, as compared to variant 1. furthermore, the deriving isoform is shorter and has a distinct c - terminus, as compared to isoform 1 (26). gastric tissue samples (tumoral and non - tumoral) were collected from iran tumoral bank (tehran, iran) as described previously (27). in brief, 30 paired tissue samples were examined for gene expression, of which 30 were adjacent non - tumoral and 30 were tumoral specimens of gastric from the same patients. all patients provided written informed consent to the iran tumoral bank prior to the participation. the study conforms to the code of ethics of the world medical association (declaration of helsinki). total cellular rnas from tumoral and adjacent non - tumoral tissue specimens were isolated with qiazol reagent and rneasy mini kit, following manufacturer 's instructions., dnase treatment was performed on - column using dnase set (qiagen, hilden, germany). the rna concentration was determined by optical density at 260 nm using nanodrop (biochrome, usa). two microgram of total rna from each sample was reverse transcribed to single - stranded cdna using mmlv reverse transcriptase (fermentas, vilnius, lithuania) with random hexamer primers (tag copenhagen). quantification of expression of zfx isoform 3/variant 5 was done by quantitative real - time rt - pcr (qrt - pcr) using maxima sybr green / rox qpcr master mix (fermentas, vilnius, lithuania) with specific primers for zfx isoform 3/variant 5 as follows : ggcagcagcttatggtaataattc and catggaactcgtgcgccctca and tbp (28). reaction mixes were subsequently run on the rotor - gene 6000 (qiagen, hilden, germany). the conditions of the pcr for zfx isoform 3/variant 5 consist of an initial denaturation step at 95c for 10 min, followed by 40 amplification cycles consisting of denaturation at 95c for 25 sec, annealing at 60.5c for 30 sec and an extension at 72c for 30 sec. to further verify the identity of the zfx isoform 3/variant 5 pcr product, agarose gel electrophoresis was performed. triplicate reactions were done for each independent preparation and the results were statistically analyzed by spss software, version 16 using student`s t - test and anova. gastric tissue samples (tumoral and non - tumoral) were collected from iran tumoral bank (tehran, iran) as described previously (27). in brief, 30 paired tissue samples were examined for gene expression, of which 30 were adjacent non - tumoral and 30 were tumoral specimens of gastric from the same patients. all patients provided written informed consent to the iran tumoral bank prior to the participation. the study conforms to the code of ethics of the world medical association (declaration of helsinki). total cellular rnas from tumoral and adjacent non - tumoral tissue specimens were isolated with qiazol reagent and rneasy mini kit, following manufacturer 's instructions., dnase treatment was performed on - column using dnase set (qiagen, hilden, germany). the rna concentration was determined by optical density at 260 nm using nanodrop (biochrome, usa). two microgram of total rna from each sample was reverse transcribed to single - stranded cdna using mmlv reverse transcriptase (fermentas, vilnius, lithuania) with random hexamer primers (tag copenhagen). quantification of expression of zfx isoform 3/variant 5 was done by quantitative real - time rt - pcr (qrt - pcr) using maxima sybr green / rox qpcr master mix (fermentas, vilnius, lithuania) with specific primers for zfx isoform 3/variant 5 as follows : ggcagcagcttatggtaataattc and catggaactcgtgcgccctca and tbp (28). reaction mixes were subsequently run on the rotor - gene 6000 (qiagen, hilden, germany). the conditions of the pcr for zfx isoform 3/variant 5 consist of an initial denaturation step at 95c for 10 min, followed by 40 amplification cycles consisting of denaturation at 95c for 25 sec, annealing at 60.5c for 30 sec and an extension at 72c for 30 sec. to further verify the identity of the zfx isoform 3/variant 5 pcr product, agarose gel electrophoresis was performed. triplicate reactions were done for each independent preparation and the results were statistically analyzed by spss software, version 16 using student`s t - test and anova. optimization of conventional and real - time rt - pcr for zfx isoform 3/variant 5 was performed on mcf7 cell line and a tumoral gastric tissue sample using specific primers. a single specific band with the expected size for the amplified zfx isoform 3/variant 5 (177 bp) was obtained using agarose gel electrophoresis (figure 1a). furthermore, real - time rt - pcr reaction for the examined gene showed a unique melting curve without primer dimers (figure 1b). qrt - pcr was performed to examine whether there is a significant difference in the expression of zfx isoform 3/variant 5 mrna in 30 paired cancerous and non - cancerous gastric tissue samples. real - time qrt - pcr results demonstrated that there was no significant difference in zfx isoform 3/variant 5 expression between tumoral and non - tumoral tissues (p - value : 1410). regarding fold changes in the tumoral and non - tumoral specimens, it was possible to categorize them into 3 classes : tumor tissues with significantly increased expression of the transcript (33.33%, p - value : 1810) (figure 2a), tumor samples with significantly decreased expression of the transcript (36.66%, p - value : 1410) (figure 2b), and the tissues without significant change in the transcript expression (30%) (figure 2c). of note, 60% of the first tissue category was high - grade diffuse - type gastric tumors. to examine the clinical importance of the zfx isoform 3/variant 5 expression, we investigated the correlation between clinicopathological status of gastric tumoral samples and the gene expression. analyses showed no significant association between the expression of zfx isoform 3/variant 5 and sex, age, n classification, lymphatic invasion, grade and type, except for a statistically significant correlation with tumor size (table 1). optimization of conventional and real - time rt - pcr for zfx isoform 3/variant 5 was performed on mcf7 cell line and a tumoral gastric tissue sample using specific primers. a single specific band with the expected size for the amplified zfx isoform 3/variant 5 (177 bp) was obtained using agarose gel electrophoresis (figure 1a). furthermore, real - time rt - pcr reaction for the examined gene showed a unique melting curve without primer dimers (figure 1b). qrt - pcr was performed to examine whether there is a significant difference in the expression of zfx isoform 3/variant 5 mrna in 30 paired cancerous and non - cancerous gastric tissue samples. real - time qrt - pcr results demonstrated that there was no significant difference in zfx isoform 3/variant 5 expression between tumoral and non - tumoral tissues (p - value : 1410). regarding fold changes in the tumoral and non - tumoral specimens, it was possible to categorize them into 3 classes : tumor tissues with significantly increased expression of the transcript (33.33%, p - value : 1810) (figure 2a), tumor samples with significantly decreased expression of the transcript (36.66%, p - value : 1410) (figure 2b), and the tissues without significant change in the transcript expression (30%) (figure 2c). of note, 60% of the first tissue category was high - grade diffuse - type gastric tumors. to examine the clinical importance of the zfx isoform 3/variant 5 expression, we investigated the correlation between clinicopathological status of gastric tumoral samples and the gene expression. analyses showed no significant association between the expression of zfx isoform 3/variant 5 and sex, age, n classification, lymphatic invasion, grade and type, except for a statistically significant correlation with tumor size (table 1). zfx has five different variants that encode 3 different isoforms (26). in the present study, for the first time we assessed and quantified the expression of zfx isoform 3/variant 5 transcript in cancerous and non- cancerous gastric tissues using qrt - pcr. our results demonstrated that the expression of this transcript was heterogeneous in gastric specimens, while its expression was significantly increased in some specimens (33.33%), and it was underexpressed in the others (36.66%). of note, 60% of overexpressed class was high - grade diffuse - type gastric tumors. however, there was a positive correlation between the zfx isoform 3/variant 5 gene expression and tumor size, but not with other clinicopathological features of gastric tumors. recently, we demonstrated that zfx (variant 1 and 3, almost exclusively isoform 1) was differentially expressed in tumoral and non - tumoral tissues, in different tumor types (intestinal vs diffuse) and grades (27). comparing the expression of zfx isoform 3/variant 5 transcript in gastric tissue samples with zfx isoform 1, it appears that these two isoforms expressed differentially in different tumor types, while isoform 1 expression increased significantly in diffuse - type, and zfx isoform 3/variant 5 expression increased in intestinal - type gastric cancer, although not statistically significant. pre - mrna alternative splicing is the flexible point in the control of gene expression in human. this process creates protein isoforms with different, even contradicting, functions from a unique gene. cancer cells often exploit this process to produce proteins that promote survival and growth (29). notably, alternative splicing can shift in the expression of a proapoptotic isoform to an antiapoptotic one, and vice versa. for example, the pre - mrna of bcl - x can alternatively splice and produce two isoforms : bclx (s), which promotes apoptosis and bcl - x (l), which has anti - apoptotic effects (30). for instance, gunthert showed that two variants of cd44 express exclusively in a metastasizing pancreatic carcinoma cell line, but not in the parental tumor (31). moreover, overexpression of serine / arginine - rich protein sf2/asf can alter the ratios between isoforms of key regulators of cell growth and results in the transformation and tumor formation (32). collectively, our results may provide a preliminary clue to the function of various zfx isoforms / variants during tumorigenesis that elucidation of the precise molecular mechanisms governed by the zfx isoforms / variants needs further investigation. this report shows that the expression of zfx isoform 3/variant 5 transcript was heterogeneous in gastric specimens. furthermore, there was no significant association between zfx isoform 3/variant 5 expression and most of clinicopathological features of gastric tumors except a positive correlation with tumor size. taken together, our results call for further studies to precisely define the role of various zfx isoforms / variants during tumorigenesis.	objective(s) : previous studies demonstrate that changes in pre - mrna splicing play a significant role in human disease development. furthermore, many cancer - associated genes are regulated by alternative splicing. there are mounting evidences that splice variants which express predominantly in tumors, have clear diagnostic value and may provide potential drug targets. located on the x chromosome, zfx gene functions as a transcription regulator for self - renewal of stem cells. this gene has 5 splice variants that encode 3 isoforms. in the present study, we evaluated the clinicopathological relevance of the expression of zfx isoform 3/variant 5 gene in gastric carcinoma.materials and methods : a total of 60 tumoral and non - tumoral gastric specimens were evaluated for zfx isoform 3/variant 5 gene expression using quantitative real - time pcr.results : our results showed that the expression of zfx isoform 3/variant 5 transcript was heterogeneous in gastric specimens. we further showed that there was a positive correlation between the variant expression and tumor size, but not with other clinicopathological features of gastric tumors.conclusion : this report shows that the expression of zfx isoform 3/variant 5 transcript was heterogeneous in gastric specimens. furthermore, there was no significant association between zfx isoform 3/variant 5 expression and most of clinicopathological features of gastric tumors except for a positive correlation with tumor size. the elucidation of the precise molecular mechanisms governed by the zfx isoforms / variants needs further investigation.
tuberculosis is a leading cause of death, especially in low and middle income countries (1). the latest estimates included in the global tb report of 2014 that there were 9.0 million new tb cases in 2013 and 1.5 million tb deaths, among them china accounted for 11% new tuberculosis (2). totally 37 million lives were saved between 2000 and 2013 through effective diagnosis and treatment (2). however recent studies showed extensive delay in tb diagnosis and treatment were severe (3). hence, the crucial point to eradicate the disease is improving the diagnosis of tuberculosis. in high prevalence of tb areas, the sputum smear microscopy test, with a sensitivity of 50%70%, is available and affordable (4). although smear - negative cases are less infectious than smear - positive cases, at least 17% of tb cases are affected by smear - negative cases (5). the mycobacterium culture is the golden standard for tb diagnosis, but the detection results cost a long time and have a low positive rate, the sensitivity of 43%83% (6). pcr test has found an increasingly wide utilization in tb diagnosis, but in developing areas its utilization remains limited because of the high cost of detection and the deficiency of special equipment and professional staffs (7, 8). therefore, the cheap, accurate and rapid diagnostic tests for tb are much - needed (9). in recent years, serum tumor markers were an effective and non - invasive diagnostic tool (10), and became a common clinical method for screening tumor (11). interestingly, a study in 1995 reported that ca125 was increased in ptb (12), since then several studies reported that the serum levels of ca125, ca199, cea in ptb were higher than those in normal population (13 16). the results provided a clue that tumor markers had potential clinical value for the diagnosis of ptb possibly. however, the study on the predictive power of tumor markers and which tumor marker has higher sensitivity and specificity in diagnosis of ptb are uncleared. in this study, we aimed to evaluate the sensitivity, specificity and cut - off of serum tumor markers to ptb diagnosis by roc, and expected to evaluate the correlations between serum tumor markers and clinically relevant parameters of ptb. this study was designed as a case - control study with 565 subjects who visited the yijishan hospital from jun to dec in 2014. case group was selected from subjects with ptb diagnosed by physicians according to reference (17). cases were excluded if there was a change in diagnosis during the treatment period, and the smokers were not enrolled in this study. control group was selected from individuals who underwent physical examination in center of health examination of yijishan hospital during the same period. if the subjects with systemic infections, history of pulmonary tuberculosis, closely contact with ptb patients, tumor, neoplastic diseases and gynecological disorders were excluded from control group. we also excluded the subjects who were in inflammatory conditions (confirmed by standard diagnostic criteria) with clinical features that could overlap the tb. for example, subjects with differential diagnosis of ptb, and other severe respiratory infections representing patients who had non - tuberculous destructive pulmonary pathology. the distribution of ptb cases and controls was not intended to reflect a particular population or epidemiological setting, but to encompass a broad range of symptomatically overlapping clinical presentations. the protocol of this study was approved by medical ethics committee of wannan medical college, all participants had been informed about the study and gave their consent. all participants were characterized by sputum smear microscopy and culture including subsequent antigen or molecular confirmation of m. tuberculosis. serum samples were collected within 2 days of the first presentation and before initiation of treatment for ptb patients. three milliliter venous blood was collected from the patients fasted for 12 h and then centrifuged and stored at 70 c until assayed. serum cea, ca125 and ca199 were detected by roche electrochemiluminescence instrument at department of nuclear medicine of yijishan hospital. all detection reagents were the corresponding bundled reagents, and its operation was in strict accordance with the manual. the normal reference values were as follows : cea5ng / ml, cal2535 u / ml, and cal99 37 u / ml. before analysis, means (sd) or median (25th percentile, 75th percentile) was calculated for continuous variables and proportions for categorical variables. chi - square test was conducted to compare the differences of demographic characteristics between case and control groups. and the mean concentrations of ca125, ca199 and cea between initial treatment and retreatment, case group and control group were tested by kruskal - wallis method of nonparametric test. in our study, the concentrations data of ca125, ca199 and cea like other references are not normal distribution, and then we selected the non - parametric tests. furthermore, the mann - whitney u test was applied to determine the abnormal rates of three tumor markers between case group and control group. then the roc curve analysis was conducted to assess the difference between areas under the curve (auc) which belonged to respective tumor marker and to evaluate the diagnostic sensitivity and specificity at the optimal cut - off. data management and all analyses were performed using r software program, version 3.0.0 (http://www.r-project.org). this study was designed as a case - control study with 565 subjects who visited the yijishan hospital from jun to dec in 2014. case group was selected from subjects with ptb diagnosed by physicians according to reference (17). cases were excluded if there was a change in diagnosis during the treatment period, and the smokers were not enrolled in this study. control group was selected from individuals who underwent physical examination in center of health examination of yijishan hospital during the same period. if the subjects with systemic infections, history of pulmonary tuberculosis, closely contact with ptb patients, tumor, neoplastic diseases and gynecological disorders were excluded from control group. we also excluded the subjects who were in inflammatory conditions (confirmed by standard diagnostic criteria) with clinical features that could overlap the tb. for example, subjects with differential diagnosis of ptb, and other severe respiratory infections representing patients who had non - tuberculous destructive pulmonary pathology. the distribution of ptb cases and controls was not intended to reflect a particular population or epidemiological setting, but to encompass a broad range of symptomatically overlapping clinical presentations. the protocol of this study was approved by medical ethics committee of wannan medical college, all participants had been informed about the study and gave their consent. all participants were characterized by sputum smear microscopy and culture including subsequent antigen or molecular confirmation of m. tuberculosis. serum samples were collected within 2 days of the first presentation and before initiation of treatment for ptb patients. three milliliter venous blood was collected from the patients fasted for 12 h and then centrifuged and stored at 70 c until assayed. serum cea, ca125 and ca199 were detected by roche electrochemiluminescence instrument at department of nuclear medicine of yijishan hospital. all detection reagents were the corresponding bundled reagents, and its operation was in strict accordance with the manual. the normal reference values were as follows : cea5ng / ml, cal2535 u / ml, and cal99 37 u / ml. before analysis, means (sd) or median (25th percentile, 75th percentile) was calculated for continuous variables and proportions for categorical variables. chi - square test was conducted to compare the differences of demographic characteristics between case and control groups. and the mean concentrations of ca125, ca199 and cea between initial treatment and retreatment, case group and control group were tested by kruskal - wallis method of nonparametric test. in our study, the concentrations data of ca125, ca199 and cea like other references are not normal distribution, and then we selected the non - parametric tests. furthermore, the mann - whitney u test was applied to determine the abnormal rates of three tumor markers between case group and control group. then the roc curve analysis was conducted to assess the difference between areas under the curve (auc) which belonged to respective tumor marker and to evaluate the diagnostic sensitivity and specificity at the optimal cut - off. data management and all analyses were performed using r software program, version 3.0.0 (http://www.r-project.org). the case group was completed on 113 cases, including 58 ptb initial treatment patients (37 men, 21 women, age ranged from 16 to 81 yr, average age : 38.1618.86) and 55 retreatment patients (35 men, 20 women, age ranged from 19 to 91 yr, average age : 52.1821.22). matched controls were 452 healthy people (288 men, 164 women, age range 16 to 91 yr old, average age : 45.1720.97). there were no significant differences of age, sex and smoking between case and control group. in terms of drink and marital status, there were significantly different between two groups (table 1). demographic characteristics of participants the concentration of serum ca125, ca199 and cea in case group were found significantly higher than those in control group (p<0.001) (table 2). we also accessed the abnormal rates of three tumor markers between two group, the abnormal rates of ca125 (case group : 48.67% vs control group : 0.22%) and cea (case group : 7.08% vs control group : 0.88%) in case group were significantly higher than in control group (p<0.001). the abnormal rate of ca199 was no significant different between two groups (table 3). the comparison of concentration levels of three tumor markers between case and control group the comparison of abnormal rates of three tumor markers between case and control group (n, %) to understand whether the retreatment of ptb effect the three tumor markers, we test the levels of serum ca125, ca199 and cea, there were no significant differences between initial treatment group and retreatment group. the logistic regression analysis was performed to evaluate the association of three tumor markers with ptb, ptb (case group=1, control group=0) was selected as dependent variable, and ca125, ca199 and cea were selected as independent variables. the results showed that ca125 was significantly related to ptb (or = 1.133, p<0.001) (table 4). the evaluation of association of three tumor markers with ptb by binary logistic regression the roc analysis revealed that for ca125, the auc whose 95% confidence interval ranges from 0.951 to 0.981 was 0.966, and the cut - off value was 10.30, with the sensitivity, specificity 0.956 and 0.850, respectively. the case group was completed on 113 cases, including 58 ptb initial treatment patients (37 men, 21 women, age ranged from 16 to 81 yr, average age : 38.1618.86) and 55 retreatment patients (35 men, 20 women, age ranged from 19 to 91 yr, average age : 52.1821.22). matched controls were 452 healthy people (288 men, 164 women, age range 16 to 91 yr old, average age : 45.1720.97). there were no significant differences of age, sex and smoking between case and control group. in terms of drink and marital status, there were significantly different between two groups (table 1). the concentration of serum ca125, ca199 and cea in case group were found significantly higher than those in control group (p<0.001) (table 2). we also accessed the abnormal rates of three tumor markers between two group, the abnormal rates of ca125 (case group : 48.67% vs control group : 0.22%) and cea (case group : 7.08% vs control group : 0.88%) in case group were significantly higher than in control group (p<0.001). the abnormal rate of ca199 was no significant different between two groups (table 3). the comparison of concentration levels of three tumor markers between case and control group the comparison of abnormal rates of three tumor markers between case and control group (n, %) to understand whether the retreatment of ptb effect the three tumor markers, we test the levels of serum ca125, ca199 and cea, there were no significant differences between initial treatment group and retreatment group. the logistic regression analysis was performed to evaluate the association of three tumor markers with ptb, ptb (case group=1, control group=0) was selected as dependent variable, and ca125, ca199 and cea were selected as independent variables. the results showed that ca125 was significantly related to ptb (or = 1.133, p<0.001) (table 4). the roc analysis revealed that for ca125, the auc whose 95% confidence interval ranges from 0.951 to 0.981 was 0.966, and the cut - off value was 10.30, with the sensitivity, specificity 0.956 and 0.850, respectively. in this study we investigated the diagnostic value of three common clinical serum tumor markers, the ca125, ca199 and cea in case group were found significantly higher than those in control group, but only ca125 was the positive association factor with ptb, the value of predictive power (auc) was 0.966, the cut - off, sensitivity and specificity of ca125 were 10.30 u / ml, 0.956 and 0.850, respectively. ca125 was identified by molecular cloning (18) and presents in the tracheal, bronchial, bronchiolar, terminal bronchiolar epithelium (19), as a cell surface glycoprotein involved in promoting ovarian cancer cell growth, and it was considered as a specific biological marker for ovarian cancer. however, our study suggested that ca125 was significantly higher ptb patients both in treatment and retreatment, and had higher sensitivity and specificity compare to another study (15). the possible reason was that severe ptb was associated with more bronchial epithelial cells destruction, which induced the elevated ca125 level in ptb patients (16). a study in germany explained that it was the inflammatory mesothelial cell proliferation, which induced the secretion of ca125 in patients with tb (20). ca199 is another common clinical tumor marker in diagnosis pancreatic cancer (21) and gastric cancer (22). we selected ca199 and tested the diagnosis of predictive value to ptb, since there several case reports showed that ca199 was increased in a ptb patient (23) and in an endobronchial tb patient (24). however, this study found that ca199 had no significant to ptb in multivariables logistic aggression analysis. canturk tasci,. also reported that there were no statistically significant in ca199 values of ptb group before treatment when compared with that of the healthy control group, and the difference found in ca199 levels before and after treatment in patients with ptb was not statistically significant (p<0.08) (25). the reason has been unknown, whatever the results of case report may influenced by some casual factors. a report in 1993 demonstrated that elevated serum cea was found in 16.9% of patients with active pulmonary tuberculosis (26). especially in female, a report showed that among 6 to 7 female patients with miliary tuberculosis, cea had elevated (27). these finds were similar to our results, but in our study, cea in male (1.35) was significantly higher than that in female (0.89), and the auc of cea was only 0.710. differentiating pulmonary tumor from ptb is difficult, in our study there 48.67% ptb case were abnormal according to ca125 normal reference, interestingly, the cut - off in roc curve of ca125 was 10.30, and it was significantly lower than its normal references of tumor 35u / ml. in other words, it may be a potential bio - marker which can differentiate the pulmonary tumor from ptb. firstly, the patients in case group of this study were recruited only with ptb, thus the results may not be applicable in other type tb. secondly, both case and control groups, we excluded patients according to imaging findings and clinical experiences instead of tissue biopsy, it was inevitable that the ptb and tumor patients incubation period were enrolled this study. thirdly, all patients in our study were characterized by sputum smear microscopy and culture including subsequent antigen or molecular confirmation of mycobacterium tb to determine the reference standard, so these results may be not applicable in smear - negative ptb. the mechanism of the association of the serum tumor markers with ptb remains to be further explored. serum ca125 has good diagnostic performances for ptb, this diagnostic accuracy would surpass that of other available immediate diagnostic options, and could yield a result much more rapidly than cultures. 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.	background : the diagnosis of pulmonary tuberculosis (ptb) is complicated and time - consuming currently. there was association of ptb with serum tumor markers. in this study we aimed to evaluate the predictive role of serum ca125, ca199 and cea as diagnostic tools for ptb.methods:this study was designed as a case - control study with 565 subjects who visited the yijishan hospital from jun to dec in 2014.this case - control study matched as for age and sex with 113 cases and 452 controls. serum ca125, ca199 and cea levels were detected by electrochemiluminescence instrument. the area under the curve (auc) of the receiver operating characteristic (roc) curve was performed to evaluate the diagnostic value on ptb.results:serum levels of ca125, ca199 and cea in ptb patients were significantly higher than those in control group (p<0.001). there was no significantly different of three tumor markers between initial treatment group and retreatment group. the logistic regression analysis showed that ca125 was an impact factor to ptb. the roc analysis revealed that auc of ca125 was 0.966 (95%ci : 0.9510.981), the sensitivity, specificity in serum and cut - off were 95.6%, 85.0% and 10.30 u / ml, respectively.conclusion:the serum ca125 has potential good diagnostic performance for ptb.
a nonsmoker 51 year - old man used sildenafil citrate (viagra) for erectile dysfunction. the patient did not clarify the quantity of the drug that he used the previous day, but he mentioned during history recording that he had been using this specific agent for the last 6 months at least once a week. the following morning, he noted painless blurred vision in his right eye and he was referred to the university eye clinic of ioannina. ophthalmologic examination revealed a corrected visual acuity of 1/10 for the right eye and 10/10 for the left eye. the color vision score for the affected eye was 10 out of 15 ishihara plates while the unaffected eye had a score of 15 out of 15. dilated fundus examination revealed swelling of the right optic disk while the vessels, macula, and the peripheral retina were normal. fluoroangiography revealed hyperfluorescence of the right optic disk and leakage from it, indicating edema. the patient was not hyperopic and the cup - to - disk ratio in the fellow eye was 0.3. syphilis screening tests, antinuclear antibodies, antinuclear cytoplasmic antibodies, and anticardiolipin antibody tests were all negative. in addition, laboratory screening revealed that erythrocyte sedimentation rate, c - reactive protein, and blood count were in the normal range. a magnetic resonance image scan of the brain and orbits with gadolinium demonstrated normal optic nerves and no white matter lesions. the above mentioned results led to the conclusion that the patient had experienced a naion attack on his right eye. the patient was subjected to three subtenon injections of beta - methoxazone with a 20-day interval in between. visual acuity at the last follow - up evaluation, 1 year after the initial attack, improved to 8/10 for the right eye with ensued optic disk atrophy yet without any significant improvement concerning the impaired visual field. naion is the most common acute optic neuropathy in older age groups with an estimated annual incidence of 2.3 per 100,000.1,2 it is presumed to result from circulatory insufficiency within the optic nerve head, but the specific mechanism of the vasculopathy remains unproven. therefore, the most important considerations in understanding the development of naion are the factors that influence blood flow in the optic nerve head. hypovolemia, mainly due to severe surgical procedures,3,4 atherosclerotic risk factors,5 hypercoagulable states,68 and crowded optic disk,9 in possible combination with regional vascular endothelial disorders has been implicated in the pathophysiology of naion. over the last few years there has been an increasing number of case reports concerning patients who have developed naion soon after the use of sildenafil and other phosphodiesterase type 5 inhibitors.1013 it has been hypothesized that these agents might exaggerate the physiologic nocturnal hypotension resulting in ischemia to the optic nerve head or that they might interfere with the autoregulation of blood flow thereby decreasing perfusion to the optic nerve head.14 in most of the reported cases of naion after the use of sildenafil, patients detected visual loss upon awakening in the morning. it is usually described 636 hours after use of the agent, as it was in this case. the majority of affected users of phosphodiesterase type 5 inhibitors suffer already from other possible risk factors for naion. in a retrospective matched case - control study, 38 patients with naion and age - matched controls without previous history of naion were questioned for the use of erectile dysfunction agents. the study showed that men with a history of myocardial infarction or hypertension are at increased risk for naion when using sildenafil or taldenafil.15 in this case, the patient had mild hypercholesterolemia without any other systemic or vascular predisposing risk factors based on clinical examination and the rest of the screening as described above. however, the family history of his father having suffered from bilateral attacks of naion indicates the possible presence of anatomical or other unidentified risk factors for the development of naion. the role of hereditary factors in familial naion remains unknown and the only clinical difference between classical and familial naion is that the familial type seems to have an earlier onset and a higher frequency of bilateral disease.16 this case might support all previous indications of the association between naion and the use of erectile dysfunction drugs. since it seems that sildenaf il can provoke naion in some individuals who have a risk profile, the physician might need to investigate the presence of a family history of naion among other risk factors before prescribing erectile dysfunction drugs.	a 51-year - old male was referred to the university eye clinic of ioannina with nonarteritic anterior ischemic optic neuropathy (naion) 12 hours after receiving sildenafil citrate (viagra). examination for possible risk factors revealed mild hypercholesterolemia. family history showed that his father had suffered from bilateral naion. although a cause - and - effect relationship is difficult to prove, there are reports indicating an association between the use of erectile dysfunction agents and the development of naion. physicians might need to investigate the presence of family history of naion among systemic or vascular predisposing risk factors before prescribing erectile dysfunction drugs.
vulvar carcinomas are rare malignancies, and squamous cell carcinoma (scc) of the vulva account for about 80% of such cases.1 vulvar intraepithelial neoplasia and vulvar bowen s disease are considered as possible precursors of vulvar scc.2 there are two major ways vulvar scc can develop : (a) 8.6%40% of cases emerge due to infection with carcinogenic subtypes of human papilloma virus (hpv) and (b) 60%70% of tumors are not related to hpv.35 among hpv types, hpv-16 seems to be the most commonly isolated in vulvar carcinomas with hpv-11 and hpv-6 being less common.4 vulvar scc with a warty surface has been strongly linked with hpv-33, hpv-45, hpv-52, hpv-18, and hpv-16.6 several conditions of either autoimmune or infectious origin and miscellaneous disorders have been associated with development of vulvar scc, such as hailey hailey disease,7 pemphigus vulgaris and systemic lupus erythematosus,8 lichen sclerosus and langerhans cell histiocytosis,9 crohn s disease,10 fanconi s anemia,11 and infection with human immunodeficiency virus (hiv).12 in women aged less than 40 years, vulvar scc is extremely rare.13 the availability of hpv vaccination may lead to a reduction in hpv - associated vulvar scc.4 scc is an important clinical and histological differential diagnosis in anogenital condylomata accuminata.14 however, scc may be misdiagnosed as condyloma. we report two middle - aged females presenting with condyloma - like vulvar midline ssc. a 41-year - old woman was seen in consultation for the combination of an asymptomatic hyperpigmented plaque type vulvar lesion on the right side and a painful exophytic nodule of the contralateral site arising from a larger flat leucoplakic lesion (figure 1). the lesions were noted for > 1 year (plaque) and 3 months (nodule). the nodule on the left side of the vulva was about 4 cm in diameter, was pedunculated, had a firm consistency, was covered in whitish film - like material and exhibited a verrucous whitish cover. histopathological examination of the nodule revealed an epithelial proliferation with the formation of horn pearls, keratinocytes with nuclear polymorphism, and increased mitotic activity (figure 2). the other lesion was diagnosed as pigmented bowen s disease with pagetoid growth of enlarged intraepithelial keratinocytes developing into scc. the tumor was staged as stage ii according to international federation of gynecology and obstetrics staging guidelines and t2n0m0 according to union for international cancer control guidelines (table 1).15 the patient was referred to the gynecologist for vulvectomy. a 44-year - old female presented with asymptomatic condyloma - like protrusions affecting labia majora and perineum and she noted > 2 years of slow enlargement (figure 3). she was a smoker for > 20 package - years but her medical history was unremarkable. magnetic resonance imaging revealed a deep subcutaneous neoplasia, extruding vagina and anus, and penetrating urogenital diaphragm including the musculus peroneus profundus measuring 33 43 42 mm. the tumor was staged as stage iiia according to international federation of gynecology and obstetrics staging guidelines and t3n1m0 according to union for international cancer control guidelines (table 1).15 surgery was performed for tumor debulking (figure 4). a 41-year - old woman was seen in consultation for the combination of an asymptomatic hyperpigmented plaque type vulvar lesion on the right side and a painful exophytic nodule of the contralateral site arising from a larger flat leucoplakic lesion (figure 1). the lesions were noted for > 1 year (plaque) and 3 months (nodule). the nodule on the left side of the vulva was about 4 cm in diameter, was pedunculated, had a firm consistency, was covered in whitish film - like material and exhibited a verrucous whitish cover. histopathological examination of the nodule revealed an epithelial proliferation with the formation of horn pearls, keratinocytes with nuclear polymorphism, and increased mitotic activity (figure 2). the other lesion was diagnosed as pigmented bowen s disease with pagetoid growth of enlarged intraepithelial keratinocytes developing into scc. the tumor was staged as stage ii according to international federation of gynecology and obstetrics staging guidelines and t2n0m0 according to union for international cancer control guidelines (table 1).15 the patient was referred to the gynecologist for vulvectomy. a 44-year - old female presented with asymptomatic condyloma - like protrusions affecting labia majora and perineum and she noted > 2 years of slow enlargement (figure 3). she was a smoker for > 20 package - years but her medical history was unremarkable. magnetic resonance imaging revealed a deep subcutaneous neoplasia, extruding vagina and anus, and penetrating urogenital diaphragm including the musculus peroneus profundus measuring 33 43 42 mm. the tumor was staged as stage iiia according to international federation of gynecology and obstetrics staging guidelines and t3n1m0 according to union for international cancer control guidelines (table 1).15 surgery was performed for tumor debulking (figure 4). vulvar cancer can be differentiated as two types : type 1 is seen in patients younger than 65 years old with frequent detection of hpv dna and a strong association to preexisting condyloma, vulvar intraepithelial neoplasia, sexually transmitted diseases, and cigarette smoking. scc is seen more often in type 2 vulvar cancer.16 our patients, however, belonged to the age - group of type 1 and both had scc broder grades ii and iii. when symptoms are present, the most common include pruritus vulvae, vulvar bleeding or pain, swelling, or vaginal discharge. 16,17 often, the patient does not complain due to cultural reasons, the so - called culture of silence, due to embarrassment, or for economic reasons. vulvar scc may be confused with condyloma14 and therefore may be inadequately treated by various modalities. the 5-year survival rates for vulvar scc have been estimated as 41% for india,18 68% for south korea,19 and 86% in the united states.1 the most important negative prognostic factor for vulvar scc is the inguinofemoral lymph node status at the time of initial diagnosis.20 surgery remains the cornerstone of therapy. individualized vulvectomy should be accompanied by groin lymph node dissection for tumors with a diameter of at least 2 cm and those invading 1 mm or deeper. medially located tumors may spread bilaterally.21,22 surgery, tumor stage, and stromal invasion of less than 9 mm were the most dominant predictors for relapse - free survival in a recent study in italy.23 the distance of the surgical margins is critical for the prevention of local relapses. in a study analyzing 93 patients with vulvar carcinoma, a margin of more than 8 mm did not improve recurrence rate.24 in advanced cases, neoadjuvant chemotherapy is a beneficial option if surgery can be performed thereafter.25 the combination of chemotherapy and radiation is an option for patients where surgery is impossible.26,27 in our cases, vulvectomy is planned for case 1 and combined chemoradiotherapy after debulking surgery is planned for case 2. in conclusion, it is important for dermatologists to be aware of vulvar scc and consider this malignancy when dealing with large vulvar condylomas. early recognition with reference to treatment by gynecologists, oncologists, and radiologists helps to improve outcome.	vulvar cancer is uncommon and may be confused with genital condylomata. we report two cases of middle - aged women presenting with exophytic vulvar tumors of the midline for which diagnosis of a vulvar squamous cell carcinoma was confirmed by histopathology. risk factors, staging, and treatment options are discussed.
the most frequent causative microorganisms are staphylococcus aureus, streptococcus species, and pseudomonas aeruginosa that are commonly found in patients under intravenous medication. in this paper, a rare sacroiliitis case that developed due to salmonella typhi is discussed. a woman at the age of twenty applied to our clinic with complaints of fever, headache and diarrhea with which she had been suffering for five days. on physical examination, she had a slight fever, with a body temperature of 38.6c. although sacroiliitis arising from salmonella typhi infection is a rare entity, it should not be ignored in patients who have a clinical history for sacroiliitis. septic arthritis is an inflammatory disease of the joints that develops after infection with various microorganisms. the most frequent microorganisms causing pyogenic infections in the sacroiliac joint are staphylococcus aureus, streptococcus species and pseudomonas aeruginosa, the latter being more frequent in patients under intravenous medication. salmonella typhi sacroiliitis is observed quite rarely and only a few cases have been reported in the clinical literature up to now[26 ]. to our knowledge, this case report is the second experience of salmonella typhi sacroiliitis in turkey, and the seventh reported in the clinical literature. this case comes from the erek town of the province of the van in the republic of turkey where a small - scale salmonellosis epidemic is seen. a woman at the age of twenty applied to our clinic with complaints of fever, headache and diarrhea with which she had been suffering for five days. on physical examination her arterial blood pressure was 110/70 mm hg and her heart rate was 76 beats / minute. laboratory analyses of the patient 's blood revealed a white blood cell (wbc) count of 6200/mm [70% polymorphonuclear leukocytes (pmnl), 30% mononuclear leukocytes (mnl) ], hemoglobin 13g / dl, hematocrit 38%, platelet count 103.000/mm, erythrocyte sedimentation rate (esr) 34 mm / hour, c - reactive protein (crp) 44.6 mg / l, aspartate aminotransferase (ast) 35 u / l, alanine aminotransferase (alt) 21 u / l, alkaline phosphatase (alp) 148 u / l, and direct / indirect bilirubin 1.6/0.2 mg / dl. the woman was hospitalized in the department of infectious diseases and clinical microbiology with a presumptive diagnosis of salmonellosis. after the culture of a blood sample was initiated, treatment with oral ciprofloxacin at 500 mg, 1 tablet twice daily, was started. in the third day of her hospitalization, the patient 's fever reduced. from her blood culture, salmonella typhi was isolated with sensitivity to ciprofloxacin. on abdominal sonography, hepatomegaly (mid - clavicular and in caudocranial 175 mm) the patient was discharged in the seventh day of her hospitalization to complement her therapy to 14 days. five days later, she returned with complaints of fever, asthenia, abasia and pain on the left hip. her blood pressure was 120/80 mm hg and heart rate was 82 beats / minute. a locomotor system examination of the patient revealed pain during left hip movement, lack of motor power, left sacroiliac compression and distraction, and positive results in the faber tests. laboratory analyses of the patient revealed : wbc 9400/mm (76% pmnl, 24% mnl), hemoglobin 13g / dl, hematocrit 38%, platelet count 206,000/mm, esr 60 mm / hour, crp 79 mg / l, ast 27 u / l, alt 23u / l, alp 192 u / l, and direct / indirect bilirubin 1.4/0.4 mg / dl. tests for hepatitis b (hbsag), hepatitis c (anti - hcv), and human immunodeficiency virus (anti - hiv) were negative, as was the test for brucellosis by the brucella tube agglutination. the woman was re - hospitalized with a presumptive diagnosis of sacroiliitis due to salmonella typhi. no bacterial reproduction was observed in the hemoculture and bone marrow aspiration cultures of the patient. tests for hla - b27 antigen, antinuclear antibody (ana) and anti - dsdna antibodies were negative. while no pathology was found in the direct sacroiliac radiography of the patient, her sacroiliac diffusion - weighted magnetic resonance imaging (mri) examination revealed signal changes consistent with edema in the bones adjacent to the left sacroiliac joint and inflammation in the adjacent paraspinal muscles, corresponding to left sacroiliitis (fig. 1). on the three - phased and whole body bone scintigraphy, on the late static images, activity retention which increased in focal terms in the left sacroiliac joint region, consistent with sacroiliitis, 2). diffusion - weighted magnetic resonance imaging in axial plane demonstrating edema (arrows) in the left sacroiliac joint (sacroiliitis). three - phasic whole body bone scintigraphy (tc99 m mdp) : in late static images, focal increased activity on the left sacroiliac joint (arrow) compatible with sacroiliitis is seen. with these symptoms and findings, the patient had a diagnosis of salmonella typhi sacroiliitis, and intravenous ceftriaxone, 1 g twice daily medication was started. a significant reduction in the fever of the patient the patient 's pain was reduced, she became better in clinical terms, and started to walk after a week. in the 21 day of the therapy, it was observed that her body temperature was in normal range, her complaints were reduced, and she was discharged with recovery. a control diffusion - weighted mri examination 10 weeks later found no pathology except minimal edema. salmonella infections cause acute gastroenteritis, bacteremia, enteric fever, and salmonella - bearing and localized infections. localized bone and joint infections are seen in less than 1% of all salmonella infections, and such localized infections are generally observed with hiv infection, systemic lupus erythematosus, sickle cell anemia, and immunosuppressive therapy fields. only a few sacroiliitis cases due to salmonella typhi infection have been reported in clinical literature. in the cases of salmonella typhi sacroiliitis, the reported clinical findings were high fever, fever - heart rate discordance, waist and hip pain, pain attacking the femur, and difficulty in walking[26 ]. in the case reported here, the patient applied to our hospital with complaints of fever, asthenia, abasia and left hip pain. there was no history of trauma, but there was fever - heart rate discordance, like the other cases reported in the literature. when direct radiography reveals no specific pathology, scintigraphy is a method which is preferred in the early diagnosis of pyogenic sacroiliitis, and computed tomography (ct) and magnetic rersonance imaging (mri) analyses are auxiliary to the early diagnosis. consistent with the clinical literature reports, no pathology was found on direct sacroiliac joint radiography in this case, but findings consistent with left sacroiliitis facilitating the diagnosis were observed on bone scintigraphy and sacroiliac diffusion - weighted mri. it has been reported that compared with other salmonella infections, salmonella typhimurium more frequently causes reactive arthritis. in this case, in the blood culture which was taken for the first diagnosis, salmonella typhi multiplied. in the second blood and bone marrow samples, no bacterial reproduction was observed, which was thought to be due to the intake of ciprofloxacin and ceftriaxone which were initiated before the second samples were taken. reactive arthritis is an acute, sterile joint inflammation which usually appears in 1 to 4 weeks following an infection. tests for hla b27 antigen are generally positive in 60% to 90% of the post - enteric reactive arthritis cases. extraarticular findings (mucocutaneous symptoms, ocular inflammation, cardiac retention, renal retention, cervicitis, and urethritis) can be observed in cases with reactive arthritis[912 ]. acute asymmetric inflammatory oligo - arthritis (on average three joint retentions) is generally observed in reactive arthritis. in the early stages, lower extremity joints such as knee and ankle are involved, and upper extremity and spine (back bone) joints are involved later. in our case, monoarticular (left sacroiliac joint) retention was existent, no extraarticular findings were observed, and hla b27 antigen was negative. due to the high fever, abasia, observance of the complaints of the pain attacking the waist and the femur, and pathological findings in radiology and scintigraphy, we think that this case was not a reactive arthritis but a sacroiliitis complication which developed in connection with inadequate therapy or relapse. in the clinical literature of cases with salmonella typhi sacroiliitis, the etiologic microorganism grew in blood culture, and all of the cases responded to antibiotic therapy. in recent years, due to antibiotic resistance in salmonella infections, quinolones came to be the most recommended antibiotics instead of the classic medications : ciprofloxacin and ofloxacin for adults, and cephalosporin for children and pregnant women, used for 14 days. for complicated cases such as the development of septic arthritis, intravenous treatment is started for 5 - 7 days, and if treatment response is positive, oral therapy can be initiated[1315 ]. although antibiotic use for reactive arthritis is still not justified, use of long - term combinations of rifampicin and doxycyclin may be beneficial.the use of anti - inflammatory agents and steroid or immunosuppressive therapies are recommended. for the patient in this case, third - generation cephalosporin treatment for three weeks was enough for recovery without anti - inflammatory agents. in conclusion, although salmonella typhi sacroiliitis is a rare entity, it should not be ignored in patients who have a clinical history for sacroiliitis. though no pathology was found in the direct radiography of the sacroiliac joint, bone scintigraphy (tc99 m mdp) and diffusion - weighted mri look like beneficial methods for the early diagnosis of this type of sacroiliitis.	context : pyogenic infections of the sacroiliac joint are observed quite rarely. the most frequent causative microorganisms are staphylococcus aureus, streptococcus species, and pseudomonas aeruginosa that are commonly found in patients under intravenous medication. in this paper, a rare sacroiliitis case that developed due to salmonella typhi is discussed.case report : a woman at the age of twenty applied to our clinic with complaints of fever, headache and diarrhea with which she had been suffering for five days. on physical examination, she had a slight fever, with a body temperature of 38.6c. she was hospitalized, and salmonella typhi was isolated from her blood culture. later on, the patient described pain during left hip movement. diffusion - weighted magnetic resonance imaging and scintigraphic examinations revealed left sacroiliitis.conclusion:although sacroiliitis arising from salmonella typhi infection is a rare entity, it should not be ignored in patients who have a clinical history for sacroiliitis.
atherosclerosis is a chronic inflammatory reaction of the vascular wall in response to dyslipidemia and endothelial distress involving the inflammatory recruitment of leukocytes and the activation of resident vascular cells. atherosclerosis is a prerequisite for the development of pathological changes in the coronary blood vessels. atherothrombotic cardiovascular disease is a leading cause of death and disability not only in rich countries but globally and, as such, has a large economic and public health impact (2). a lot of studies is conducted that have analyzed the risk factors which lead to the formation of atherosclerosis changes in the blood vessels. many years of testing revealed a large number of risk factors, which are set based on monitoring of each risk factors responsible for the occurrence of coronary heart disease, combined or individual, which led to the formation of various guidelines of the european cardiology association and the american association of cardiologists (3, 4, 5). the framingham study as a largest longitudinal epidemiological study of coronary disease in the world, based on the concept of the risk factors estimate, is the basis for the treatment of patients, and thus lowering the incidence of coronary artery disease (pitt b. 1999). the complex etiology of coronary heart disease is connected to smoking, high blood pressure, high levels of fat and sugar in the blood, increased body weight, stress and lack of physical activity (5,6). the age, gender and family history, greatly complicate the risks for developing the disease (hf epstein, 1995). despite many limitations, distribution and frequency of the heart and blood vessels diseases are analyzed on the basis of mortality statistics data and the results obtained after the who monica project (multinational monitoring of trends and determinants in cardiovascular disease) covering a population from 35 to 64 years in 35 countries of the world. in industrialized countries, heart and blood vessels diseases make up one - third to one - half of all deaths (33% in france and 39% in japan up to 48% in england and wales and 52% in finland) (tolonen h 2005). an important shift in the primary prevention of coronary heart disease is an attempt to observe all risk factors that contribute to the creation and development of atherosclerotic changes in blood vessels as well as the mutual interaction of various risk factors. the aim of this study is that by the results obtained using a modified algorithm with tables adopted by the european society of cardiology demonstrate the possibilities for assessment of the risk of coronary heart disease degree, for application to the targeted individual or risk factors groups. are two groups of 200 respondents (experimental and control group) at approximately same age, socio economic conditions and provided health care. the modified algorithm for risk of cardiovascular disease estimation the modified algorithm for risk of cardiovascular disease estimation involves grading of differences on the basis of the intensity within the four groups : without presence of risk, risk is present, expressed risk, high risk expressed, without presence of risk, in all patients is estimated absolute risk, by summing the points score for the presence of risk factors, so that all the participants are divided into four groups : low risk for coronary heart disease, 0 pointspresent risk for coronary disease, 1- 3 pointspronounced risk for coronary disease, 46 pointshigh risk for coronary disease, 79 points low risk for coronary heart disease, 0 points present risk for coronary disease, 1- 3 points pronounced risk for coronary disease, 46 points high risk for coronary disease, 79 points after this the total risk score for developing coronary disease in all patients was determined according to two tables (the european table for coronary risk from 1998 and european heart score system (systematic coronary risk evaluation)). the results show that there is low risk in 20% of respondents, is present in 50% of respondents, and expressed in 20% of respondents. estimate of coronary risk by testing a sample of healthy respondents show that low level of risk has 58% of healthy respondents, present risk in 37%, expressed risk in 4% and expressed high risk in 1% of healthy subjects. comparison of the risk assessment results of according to the european table for both groups, the results show that respondents with coronary heart disease have a higher risk score in the group present, expressed and expressed high risk compared to healthy respondents who have a higher presence of low risk score. the new european table for risk (score) includes : systolic blood pressure, cholesterol values, cigarette smoking, age and gender. risk assessment by score table shown that among patients suffering from coronary heart disease in low - risk group there was 35% respondents, present risk has 20%, expressed risk 25%, and highly expressed 10% of the respondents. according to the new european table for risk (score) the results of the risk assessment showed that among healthy respondents the low level of risk was present in 57% of the respondents, present risk has 34%, expressed risk 6% and expressed high risk 3% of healthy respondents. according to score tables for risk assessment by comparing the results of two research groups have shown that a higher percentage of risk groups have patients with expressed (25% : 6%) and a high risk (10% : 3%), while in group of healthy respondents a higher percentage have the low risk (57% : 20%), and present risk (34% : 20%). when we compared the results of risk assessment by groups of european and score table we got the results showing that by about same percentage are represented groups according to risk and of course higher risk have patients in the group of expressed and expressed high risk, while in the group of low risk is present a higher percentage of healthy respondents. in this study is also used modified algorithm that includes following of nine risk factors : age, hereditary factors, high blood pressure, elevated blood cholesterol, elevated blood sugar, exercise habits, smoking, bmi, and the ratio of the volume of abdominal obesity and hip circumference. assessment of coronary risk by our modified algorithm in patients with coronary heart disease showed that low risk has only 3% of respondents, a high percentage 9%, while 43% belongs to the group of present risk and 44% to expressed risk. assessment of coronary risk by modified algorithm in healthy subjects show that low risk has 5% of respondents, present risk 66%, 28% expressed and highly expressed 1% of respondents. according to our modified risk assessment algorithm comparison of results in two research groups have shown that a higher percentage of risk groups have patients with pronounced (or=2.0) and a highly expressed risk (or=9.1), while in healthy respondents group was higher percentage with low and present risk. however, when we compared levels of risk for coronary disease development in both groups, according to these three tables, we obtained the results that in the group with present and expressed risk there is a higher percentage representation as a result of inclusion of more risk factors in calculation in relation to the european table and score table, and on the other hand the presence of risk in the healthy population is much higher in our population than the population of the eu countries for who these tables are adapted. by comparing the presence of risk factors according to the representation of groups of diseases, or myocardial infarction, results showed that the european score table have significantly lower levels of risk factors, or those with sustained myocardial infarction are ranked in groups of low and present risk in relation to our algorithm which patients with myocardial infarction ranked as high and pronounced degree of risk (figure 1). comparison of the results of the risk assessment according to all three tables among patients with myocardial infarction when we compared the presence of risk groups according to the representation of diseases groups, or, coronary insufficiency, the results showed that the european and score table significantly reduce the levels of risk or the persons with heart failure have been ranked in the group of low and present risks in relation to our algorithm which patients with coronary insufficiency ranked as pronounced degree of risk (figure 2). comparison of the results of the risk assessment according to all three tables in relation to the patients with coronary insufficiency overall assessment of coronary risk the proposal of modified algorithm after the conducted study, by review of the data received, was made the risk assessment for all asymptomatic respondents older than 35 years if there is a positive family history of early coronary disease, elevated blood lipids, that smoke, have high blood pressure, with increased bmi, present symptoms of coronary disease and diabetes (table 1 and 2). in our study is used a modified algorithm that includes follow up of nine risk factors : age, hereditary factors, high blood pressure, elevated blood cholesterol, elevated blood sugar, exercise habits, smoking habits, bmi, the ratio of the volume of abdominal obesity and hip circumference. when we investigate all nine risk factors results showed that the prevalence of standard risk factors : high blood pressure (sd=4.5), the level of blood sugar (or=3.1), smoking (or=2.0), cholesterol (or=1.9), obesity (or=1.8), lack of physical activity (or=1.7), waist - hip ratio in women (or=1.3), waist - hip ratio men (or=1.6) was higher in patients with coronary disease, compared to the control group. the main risk factors are present high blood pressure, elevated blood sugar, smoking, elevated cholesterol levels in the blood, which was confirmed in interhealth study emphasizes just these five risk factors (yusuf s.2005). also, the studies of other authors confirm our research because they stated that among patients with coronary heart disease, at least one of the four conventional risk factors smoking, elevated blood sugar, elevated blood pressure, elevated blood lipids, were present in 84.6% of women and 80.6% of men (umesh n 2003). once we have determined the presence of risk factors, their degree of influence on the occurrence of coronary disease and regardless of the fact that some risk factors have not yet found their place in certain tables, but our experience shows that there is the need to include all known and easily measurable risk factors in the assessment of the overall risk. the studies of various authors have shown that in order to identify risk factors and assess absolute short - term (ten years) and long - term risk of coronary heart disease is the first step in the primary prevention and we know that certain risk factors can be eliminated, and the other alleviated, thus slowing down the progression of the disease, delay occurrence of clinical events and improve prognosis (isles cg, 2000). when we compared the results of the risk assessment to the european table of both groups, the results show that people with coronary artery disease have higher risk score in the group of present risk (50% : 39%), expressed risk (20% : 4%) and a highly expressed risk (10% : 1%) relative to healthy respondents who have more present low risk score (20% : 1%). assessment of coronary risk by our modified algorithm in patients with coronary heart disease showed that low risk is present in only 3% of respondents, highly expressed risk 9% while with present risk there is 43% and 44% have expressed risk. assessment of coronary risk by modified algorithm in healthy subjects show that low risk has 5% of respondents, present risk 66%, 28% expressed and highly expressed 1% of respondents. according to our modified risk assessment algorithm for comparing results of two research groups have shown that a higher percentage of risk groups have patients with expressed (45% : 28%) highly expressed risk (9% : 1%), while in healthy subject s greater percentage has a group of low (5% 2%) and the present risk (66% : 43%). when we compared the presence of risk groups by the representation of groups of diseases, or myocardial infarction, results showed that the european and score table significantly reduce the levels of risk, and those with sustained myocardial infarction are ranked in groups of low, present risk in relation to our algorithm that patients with myocardial infarction ranked as under high and expressed degree of risk. our research has shown that in a group of healthy participants we have 2x higher present risk and risk 4,5x higher estimated risk in relation to the european table and 7x higher estimated risk in relation to the score table. this is confirmation of the prevalence of risk factors in the general population in our country and coincides with the information from neighboring countries. however, in people who have suffered coronary heart disease, the results obtained by measuring risk factors show that at high risk for developing the disease results were equal to other tables. but the group with expressed risk in patients with coronary heart disease shows 2x higher value in relation to the european table and score table. because we see that in the group of healthy respondents, the group with expressed risk have 4,5x and 7x higher representation in comparison with comparative tables used, it confirms our thesis that the use of modified algorithm is extremely important for our region, as it allows us to significantly earlier identify a group of risk patients in need of reduction, so that the obvious risks of developing coronary disease are reduced and eliminated. by including more risk factors for coronary disease is certainly significant and leads to a more accurate assessment of coronary risk, in relation to the european table, the table score, in our study we included a family history of the disease, sedentary lifestyle, obesity and the waist - hip ratio. similar results were obtained in procam study based on a large cohort study of the population of europe, which recommended the inclusion of hdl cholesterol as one of the most important cardiovascular risks (assmann gerd 2005). thanks to the continuous implementation of prevention programs, researching and active search for people with an increased risk for coronary disease reduction and the elimination of risk factors, changing lifestyles and the use of therapeutic pharmacological measures have significantly reduced the trend of morbidity and mortality from coronary heart disease in developed countries (raljevi 2007). determination of the overall risk of coronary disease, enables the health professionals for planning the intensity of preventive action, or when dietary advice should be more specific, when physical activity should be more individualized, when drugs should be prescribed, dose adjustment or introduce a combination for the risk factors control. the conclusion is that by providing continuous implementation of prevention programs, at all levels of health care, by research and the active search for people at risk for coronary artery disease, continuous implementation of the reduction and elimination of risk factors, changing lifestyles, and the use of therapeutic pharmacological measures, will significantly reduce the trend of morbidity and mortality from coronary heart disease in our area. the activities of primary prevention of risk factors, or already resulting disease, may be helpful in assessing the reduction in economic costs in healthcare, both due to lower morbidity, and reducing the total cost of treatment of patients with coronary disease.	introduction : determination of the overall risk of coronary disease, enables the health professionals for planning the intensity of preventive action.aim:the aim of this study is that by the results obtained using a modified algorithm with tables adopted by the european society of cardiology demonstrate the possibilities for assessment of the risk of coronary heart disease degree, for application to the targeted individual or risk factors groups.material and methods : the study was conducted as a retrospective, prospective and controlled (included two groups of 200 respondents).results : by comparing the presence of risk factors according to the representation of groups of diseases, or myocardial infarction, results showed that the european and score table have significantly lower levels of risk factors, or those with sustained myocardial infarction are ranked in groups of low and present risk in relation to our algorithm which patients with myocardial infarction ranked as high and pronounced degree of risk. results showed that the european and score table significantly reduce the levels of risk or the persons with heart failure have been ranked in the group of low and present risks in relation to our algorithm which patients with coronary insufficiency ranked as pronounced degree of risk.conclusion:determination of the overall risk of coronary disease, enables the health professionals for planning the intensity of preventive action. the activities of primary prevention of risk factors, or already resulting disease, may be helpful in assessing the reduction in economic costs in healthcare, both due to lower morbidity, and reducing the total cost of treatment of patients with coronary disease.
comparative genomics has benefited during the last decade from several specialized databases like hovergen (1) and hobacgen (2) to access growing amounts of sequence data. these databases have facilitated the biologists ' work by giving access to information on between - species similarity / homology. no such tool, however, exists for within - species polymorphism data, which are currently dispersed within huge generalist databases, making it very difficult to access and re - analyse them first, it would help population geneticists to quickly review existing datasets and bibliography in species / taxa of interest. the task of building a complete polymorphism sequence dataset in a taxon of interest (e.g. drosophila), or for a locus of interest second, it would make it possible to set up data sets for meta - analyses. multi - locus analysis is a powerful way to detect adaptation at the population level, and discriminate between selection and demographic effects selection acts locally in the genome, while genetic drift and migration affect all loci simultaneously (36). large datasets are also required to understand the role of the various forces driving molecular evolution such as mutation, selection, recombination and gene conversion (710). finally, a polymorphism database would allow one to compare amounts and patterns of sequence polymorphism in distinct species, and investigate correlations with species ' ecology and life history traits (11,12). currently, one can retrieve sequence polymorphism datasets in the popset section from the entrez web interface developed at the ncbi. this database stores alignments submitted by authors, but there is no attempt to cluster sequences in homologous families, and phylogenetic studies are mixed with studies of intra - species polymorphism. the ebi hosts a similar alignment database, embl - align, accessed by the srs web interface. alfred (allele frequency database) stores allelic frequencies, source of the samples and phenotype information, but not sequence alignment (13). mendb (14) contains information about sequence variation, primers and pcr conditions for laboratory work. some basic descriptive statistics are also given, such as intra- and inter - population diversity. finally, pgdb (population genetics database) is a prototype mitochondrial database (15), but again, relying on authors submissions. none of these databases allow queries that make use of all the information available in embl / genbank. polymorphix is an acnuc database (16) organizing genbank / embl sequences into within - species sequence polymorphix can be accessed via a client system with a dedicated web interface (http://pbil.univ-lyon1.fr/polymorphix/query.php), or using query_www, an expert web interface allowing complex queries of data on several sequence families databases (17). we used all the eukaryotic genomic dna sequences in embl release 73 after removing the est, htg, gss, syn, sts and htc sections. tandem repeat and satellite repeat. to build families in a given species, we first extracted all the sequences of length between 100 and 20 000 bp from the species, and performed a similarity search of all sequences against themselves, using the megablast program. two sequences were clustered into the same family if they shared a > 100 bp - long fragment with similarity > 80%. two criteria were used to remove paralogous sequences, and to split paralogous loci into distinct families. first, two sequences were not clustered if there was a mismatch of > 50 nt with 78% similarity to the consensus sequence. we refrained from including too distant outgroup, which are useless for the goal of orienting polymorphic sites within the ingroup. outgroup sequences were added without altering the ingroup alignment, by using the profile and quicktree options in clustalv1.8 (19). a nj (20) phylogenetic tree (k2p distances) was built for each family. this treatment was repeated for each species in embl subspecies are considered as the same taxon in polymorphix. then the embl flat files describing the selected sequences were modified. several keywords were added to each sequence in polymorphix, namely the name of the family(ies) it belongs to (e.g. hsa000001 for family number one in homo sapiens), and keywords ingroup and outgroup when appropriate. a sequence can be an ingroup in a family and outgroup in one or several other families. an important issue is the problem of updating polymorphix as new genbank / embl versions are released. currently, polymorphix updates are achieved by re - building families de novo for all the species for which new sequences have been added since the previous embl version. polymorphix database contains 244 million bases, 217 271 sequences and 18 445 bibliographic references. polymorphix allows simple or elaborate queries on embl / genbank fields (species names, keywords, etc.), and a blast - assisted selection of families. the coding regions of sequences and their ingroup / outgroup status are annotated in the header of mase (21) alignment files. trees are less obviously useful than in between - species homology databases, but they help in visualizing potential population structure, distance to outgroup and in checking paralogy. for each family, a short description appears giving the name of the ingroup species and a locus identifier. users of the elaborate query_www interface have to keep in mind that queries are made on sequences, and not on families. this means that a family will be selected as soon as at least one sequence of the family satisfies the query. the web interface allows one to select a subset of sequences within a family if required (e.g. ingroup only). table 1 shows family distribution among the major lineages of animals, plants and fungi, for the nuclear and organellar genomes. two tables in supplementary material give the detailed family distribution within mammals and angiosperms, respectively. in many animal taxa and mammalian orders, the number of mitochondrial families is higher than the nuclear one, despite the genome size being 2 10 times smaller, reflecting the prevalence of mitochondrial dna as a tool for phylogeographic studies. this is not true for primates and rodentia, because of a greater effort in human and mouse nuclear polymorphism studies, and because of a higher proportion of mitochondrial dna polymorphism has hardly ever been explored, probably because of technical problems the plant mitochondrial genome is relatively large, and highly repetitive. we also plotted the year - wise distribution of sequences publication in polymorphix (figure 1) separately for nuclear, mitochondrial and chloroplastic dna. the number of nuclear and mitochondrial sequence polymorphism publications has been increasing exponentially since 1990. however, in 2003, this number is lower than in 2002, probably because of the delay between sequence submission (usually prior to publication) and update of references by authors in genbank / embl. the polymorphix database provides a simple access to sequence polymorphism data for eukaryotic species present in genbank / embl. one important problem when searching for polymorphism data in genbank / embl is to distinguish polymorphic variants from paralogous sequences. in polymorphix, we used two criteria to try to exclude paralogues : (i) sequence similarity and (ii) bibliographic references. the similarity threshold that we used (> 80% identity) is not very stringent, because in some cases, allelic variants can be very divergent (22). but to be included in a family, we require that the bibliographic references of every sequence in the family, is shared with at least three other members of the family. this criteria allows us to exclude most of paralogues and to retain most of allelic variants (except for sequences that are not yet associated with a published article). note, however, that some paralogous sequences might still be present in polymorphix families. true polymorphic loci in polymorphix is > 95% for drosophila melanogaster nuclear data and mammalian mitochondrial data, but hence, it is necessary for the user to manually curate the data (sequence annotations, bibliographic references) to exclude paralogues. the web interface provides a number of tools for rapidly checking and manipulating families, including alignments, trees and links to publications. the main advantage of polymorphix is that it contains virtually all the polymorphism sequence datasets that were submitted to genbank / embl. polymorphix will therefore help population geneticists and molecular evolutionary biologists in building exhaustive datasets for meta - analyses. as an illustration, a one - day long manual survey and cleaning of polymorphix was enough to build a dataset of 220 non - coding polymorphic loci in d.melanogaster, a dataset three or four times larger than in published meta - analyses (9,23). polymorphix can also be useful as a bibliographic tool, e.g. for performing a quick review of the literature of sequence polymorphism in a taxon of interest. polymorphix does not include single nucleotide polymorphism (snp) data, for which species - specific dedicated databases are available (e.g. dbsnp at ncbi). although typically more voluminous than sequence polymorphism datasets, snp data are of limited interest for population genetics analyses. this is because (i) they do not provide access to haplotype / allele genealogies, on which many methods are based, (ii) they are available in a small number of taxa and (iii) they do not allow characterization of the molecular evolution of specific genes of interest. however, snp data are useful for characterizing molecular microevolutionary patterns at the genome level (24,25). one limitation of polymorphix is that it does not provide information about the sampling strategy (population subdivision, individual location), or about allele frequencies. these data are crucial for many studies of population genetics, and will have to be obtained from the literature by users prior to data analysis. some questions, however, can be addressed just with haplotype or allelic type data, such as the use of the mcdonald it should be noted, however, that the embl format supports the submission of this information through the feature key /country=. we did not make use of this information because of the low number of entries using these features. we would, however, encourage population geneticists to submit these data, as well as alignments, together with sequences, since this low - cost effort can greatly help in the future re - analysis or meta - analysis of polymorphism sequence data. we are grateful to deborah charlesworth and sylvain glemin for critical reading of the manuscript and for their helpful suggestions.	within - species sequence variation data are of special interest since they contain information about recent population / species history, and the molecular evolutionary forces currently in action in natural populations. these data, however, are presently dispersed within generalist databases, and are difficult to access. to solve this problem, we have developed polymorphix, a database dedicated to sequence polymorphism. it contains within - species homologous sequence families built using embl / genbank under suitable similarity and bibliographic criteria. polymorphix is an acnuc structured database allowing both simple and complex queries for population genomic studies. alignments within families as well as phylogenetic trees can be download. when available, outgroups are included in the alignment. polymorphix contains sequences from the nuclear, mitochondrial and chloroplastic genomes of every eukaryote species represented in embl. it can be accessed by a web interface (http://pbil.univ-lyon1.fr/polymorphix/query.php).
caliceal diverticuli are nonsecretory urothelium lined cystic cavities within the renal parenchyma that harbor static urine. upto 0.45% of routine intravenous urogram (ivu) may show the presence of caliceal diverticulum. a narrow neck usually communicates between the diverticulum and the rest of pyelocaliceal system (pcs) and allows passive filling with urine. the indications for treatment include flank pain, pyuria, and urinary tract infection (uti). in the era of minimally invasive surgery, various procedures like shockwave lithotripsy (swl), percutaneous nephrolithotomy (pcnl), ureterorenoscopic surgery (urs), and laparoscopy have been described for the treatment of this entity, but the stone - free and symptom - free rates are found to be best with pcnl. during pcnl, in order to prevent urinary stasis and recurrent stone formation, either the diverticular neck is dilated or the wall is fulgurated. there is scant literature regarding the outcome, complications, and recurrence rates using these two methods. we present our experience with caliceal diverticular stones managed with pcnl and describe the outcomes of these two techniques to deal with diverticula after stone retrieval. we retrospectively analyzed data of 44 consecutive patients, who underwent pcnl for caliceal diverticular stones (cds) from january 2001 to may 2011 at our tertiary care center. all patients were evaluated preoperatively with ivu to assess the location of diverticula and the size of stone. percutaneous access was achieved by direct stone - guided puncture with the bull 's eye technique. when appropriate coiling of guide wire was not possible, we used the skipping technique in which we replaced the thin hydrophilic wire with the stiff guide wire. this stiff guide wire facilitates one - stage tract dilation without the risk of bending. after complete removal of the stones, the diverticular cavity was inspected to locate the neck of diverticula. if the neck could not be localized, methylene blue dye was instilled through the ureteric catheter placed preoperatively to aid visualization. the diverticula were treated using one of two techniques. if the guide wire could be negotiated through the neck of diverticulum, we dilated the neck and stented it with either a ureteric catheter or double j stent (djs). if the neck couldnot be canulated or dilated, the cavity was fulgurated using a 24 french resectoscope equipped with roller - ball electrode. a nephrostomy tube was placed for 1 to 2 days. an x - ray kidney ureter bladder (kub) postoperative follow - up included an ivu at around 3 months to assess diverticular resolution or decrease in its size and annual plain films thereafter to look for any stone recurrence. of the 44 patients studied, 24 (55%) were males and 20 (45%) were females. the mean age was 38 years (range : 23 - 63 years, table 1 ]. flank pain was the presenting symptom in all patients, whereas six patients had recurrent urinary infection, three had pyuria, and one had hematuria. most of the diverticula were located at the upper calyx, followed by the middle and lower calyces. the size of stones ranged from 1.1 to 3 cm (mean 1.9 cm). patient characteristics percutaneous access was obtained through direct stone - guided puncture in all patients. stones were fragmented with a pneumatic lithoclast in 39 and with a holmium laser in 3 patients. in two patients, stones were removed in toto. we were able to cannulate and dilate the neck of diverticula in 35 (79.5%) patients, among which the ureteric catheter was placed in 29 and djs was kept in 6 patients. in the remaining nine (20.5%) patients, the neck couldnot be localized and the walls of diverticula were fulgurated and 20 fr nephrostomy tube was placed. the mean operative time was 87 min (range : 60 - 130 min). the average hospital stay was 3 days (range : 2 - 5 days). the nephrostomy output from fulgurated cavity ranged from 50 to 200 ml (mean : 78 ml). pneumothorax occurred in one patient with a supracostal puncture, which necessitated the placement of an intercostal drain for 4 days. one patient had significant bleeding, which was diagnosed intraoperatively and responded to nephrostomy clamping and blood transfusion. four patients with incomplete clearance were successfully cleared of stones with either single (in three patients) or two (in one patient) applications of swl. the size of residual stones ranged from 8 to 10 mm. in all of these cases, postoperative ivu showed obliteration of diverticula in 7 patients and the improved drainage in 37 patients. the goals of treatment of cds are complete removal of stone and obliteration of the diverticulum. among various available options like swl, pcnl, urs, and laparoscopy, percutaneous management offers the highest symptomatic relief and stone - free rates. only group, 1 of 26 patients (4%) became completely stone free, although 9 (36%) became asymptomatic. moreover, 10 patients (38.5%) of swl group ultimately required pcnl. on the contrary, in the pcnl group, 13 of 14 patients (92.85%) were stone free and all became symptom free. the other attractive option for treatment of cds is urs, but various studies have found only 20%-55% of stone - free rates. auge., compared the outcome of pcnl and urs and found a higher stone - free (78% vs. 19%) and symptom - free (86% vs. 35%) rates in the pcnl group. the preferred approach to pcnl is to puncture directly in the diverticula and to advance the guidewire through the diverticular neck. lancini., achieved stone - free rate of 95% and symptom - free rate of 100% with this technique. among 40 patients treated, 35 had complete resolution of their diverticulum and remaining 5 patients had around 50% decrease in diverticular size without any recurrent stone formation. donnellan., found similar stone - free rate and diverticular resolution in their series, but around 14% patients had recurrent stones, which were managed by swl. several authors have described the fulguration of diverticular cavity at the time of pcnl without making any effort to traverse the neck and establish the continuity with the remainder collecting system. usually, a small nephrostomy tube is kept in the diverticulum after the procedure. in the series of kim., all 16 patients who had postoperative ivu showed a decrease in the size of diverticula and 14 (87.5%) had complete resolution. other authors also described similar results with no recurrence of symptoms, stones, or infection. krambeck and lingeman compared outcomes in patients undergoing fulguration alone to patients who underwent diverticular neck dilatation and found a shorter hospital stay, fewer complications, and higher stone - free rates in the first group. some authors have combined diverticular fulguration with the conventional technique of diverticular neck cannulation or dilatation in a group of patients with encouraging results. in cases where the caliceal neck can not negotiated, some authors recommend the trans - diverticular approach with creation of a neo - infundibulum. ndez., compared these two techniques and found almost similar stone - free rates without any added complication. proponents of this technique argue that this technique eliminates the need of prolonged probing with a wire for the neck of diverticulum. we have, however, never performed this technique. among management options for cds, the most versatile approach with maximum stone - free and symptom - free rates is pcnl. we found the stone clearance of 90.90% in our series, which is comparable with that reported in the literature. the importance of our data lies in the fact that we analyzed a relatively large number of patients. we suggest using a method to deal with diverticula after stone extraction depending on the neck of diverticula. if we are able to canulate the neck, drainage of the diverticula to the remaining pcs can be performed by the ureteric catheter or djs, otherwise simple fulguration of diverticular wall with external drainage by a small nephrostomy tube is a good option. after stone retrieval, the diverticula may be drained into the pcs if the neck can be negotiated, otherwise it can be fulgurated.	introduction : caliceal diverticulae are a frequent surgical problem. we present our experience with caliceal diverticular stones (cds) managed with percutaneous nephrolithotomy (pcnl) and describe the two different techniques to deal with diverticula after stone retrieval.materials and methods : we retrospectively analyzed 10-year data of 44 consecutive patients who underwent pcnl for cds. during pcnl, if the guide wire could be negoted through the neck of the diverticula, we dilated and stented it. if we couldnot find the neck, we fulgurated the diverticular walls. follow - up included intravenous urogram at 3 months and annual plain films thereafter. we analyzed the outcome, complications, and recurrence rate.results:total stone clearance was obtained in 40 (90.90%) patients. we dilated and stented the diverticula in 35 (79.5%) patients and fulgurated the walls in nine (20.5%) patients. complications occurred in three patients. the postoperative intravenous urogram showed obliteration of diverticula in seven patients and the improved drainage in 37 patients. at the average follow - up of 2 years, 41 (93.18%) patients were asymptomatic and two (4.5%) patients showed the recurrence of stone.conclusions:pcnl can clear calculi from caliceal diverticula in most cases with minimal morbidity. after stone retrieval, the diverticula may be drained into the pyelocaliceal system, if the neck is negotiable and fulgurated if the neck can not be dilated.
high - valent nonheme iron - oxo species are implicated as key oxidants in the catalytic cycles of nonheme o2-activating enzymes, catalytic oxidation of inert c mononuclear iron(iv)-oxo intermediates have been detected and spectroscopically characterized as active oxidants in -ketoglutarate - dependent taurine dioxygenase (taud), tyrosine hydroxylase, pterin - dependent phenylalanine hydroxylase, and nonheme iron - dependent halogenases. in parallel with their discovery in biological systems, a number of synthetic iron(iv)-oxo species have been prepared. the spectroscopic and structural properties of synthetic iron(iv)-oxo species have been analyzed in detail, and their oxidizing abilities have been a matter of intense studies. although some of these compounds are sufficiently powerful oxidants to oxidize even the strong c h bonds of cyclohexane, the factors that determine the reactivity of the oxoiron(iv) unit are of central interest, and extensive efforts have been directed toward the use of coordination complexes as synthetic models. the use of oxygen atom donors such as iodosobenzene (phio) is the most common strategy for the chemical synthesis of model iron(iv)-oxo species. regard, nam and co - workers recently reported the generation of [fe(o)(n4py) ] (n4py = n, n - bis(2-pyridylmethyl)-n - bis(2-pyridyl)methylamine) from [fe(n4py)(ch3cn)](otf)2 (otf = trifluoromethanesulfonate anion) using either a strong oxidant (cerium(iv) ammonium nitrate, can) or a photochemically generated oxidant. in particular, it was reported that [ru(bpy)3 ] could be used as a photosensitizer in combination with [cocl(nh3)5 ] as the terminal electron acceptor to generate the iron(iv)-oxo compound. herein, we show that the new complex [fe(o)(mepy2tacn) ] (2, mepy2tacn = n - methyl - n,n-bis(2-pyridylmethyl)-1,4,7-triazacyclononane, scheme 1) can be generated from [fe(mepy2tacn)(s) ] (1, s = solvent) by reaction with oxygen atom transfer oxidants (phio or n - bu4nio4) in ch3cn or by using water as the source of oxygen in combination with 1e oxidants (can or [ru(bpy)3 ]) (scheme 1). the transformation of 1 to 2 can also be photocatalyzed by [ru(bpy)3 ] in the presence of na2s2o8. importantly, the oxygen - atom transferability of the low - spin (s = 1) iron(iv)-oxo complex 2 is enhanced upon irradiation at 447 nm in the presence of [ru(bpy)3 ]. the origin of this rate enhancement is investigated through nanosecond - time - resolved absorption spectroscopy. reaction of the pentadentate ligand mepy2tacn (figure 1) with an equimolar amount of [fe(otf)2(ch3cn)2 ] in thf under anaerobic conditions afforded [fe(mepy2tacn)(ch3cn)](otf)2 (1) as a deep red solid. slow diffusion of diethyl ether over a saturated ch2cl2/ch3cn solution yielded dark red crystals of 1 in 60% yield. x - ray analysis revealed a ferrous center octahedrally coordinated to the five nitrogen atoms of the ligand and to one exogenous acetonitrile molecule (figure 1, see supporting information (si) for crystallographic details). the two pyridine moieties are arranged perpendicular to one another n distance is 1.98, indicative of a low spin iron(ii) center (s = 0). accordingly, a diamagnetic h nmr spectrum is obtained for 1 in acetonitrile - d3, indicating that the low - spin structure is retained in solution (si figure s1), and its mssbauer spectrum exhibited an isomer shift (= 0.44 mms) and a quadrupole splitting (\|eq\| = 0.41 mms) characteristic of a low - spin iron(ii) center (si figure s2, table s5). left : schematic representation of ligand mepy2tacn. iron(iv)-oxo compound 2 was obtained by direct oxidation of 1 with excess phio or 1.2 equiv of n - bu4nio4 in ch3cn, as previously reported for structurally related iron(iv)-oxo compounds (route a, scheme 1a). the uv / vis absorption spectrum of 2 in ch3cn (figure 2 top) is characterized by an absorption band with a maximum at 736 nm (= 310 m cm), a common feature in s = 1 iron(iv)-oxo species. complex 2 was further characterized by mssbauer spectroscopy using fe - enriched samples. as shown in figure 3, the spectrum recorded at 80 k under zero - applied magnetic field is the superposition of two doublets. the minor one constitutes 18% of the sample (isomer shift = 0.48 mms, quadrupole splitting eq = 1.57 mms), and it is attributed to an oxo - bridged diferric decomposition product that frequently constitutes a thermodynamic sink for the present chemistry. the major one, corresponding to 2, represents 82% of the total iron content and exhibits parameters (= 0.01 mms and eq = 0.93 mms) that are consistent with an iron(iv) center in a low spin (s = 1) configuration. electrospray ionization mass spectrometry (esi - ms) of 2 in acetonitrile showed a single major peak at m / z 546.12, with an isotopic pattern that corresponds to { [fe(o)(mepy2tacn)](otf) } (figure 2, bottom). furthermore, this peak shifted by two m / z units when h2o was added to 2, thus further confirming the presence of an oxo ligand that readily exchanges with water. the h nmr spectrum of 2 in cd3cn shows paramagnetically shifted signals and resembles the structurally related iron(iv)-oxo species [fe(o)(n4py) ] and [fe(o)(bntpen) ] (bntpen = n - benzyl - n, n,n-tris(2-pyridylmethyl)-1,2-diaminoethane), the signals of the pyridine moiety being the most distinctive feature (si figure s7). a h nmr cosy experiment shows two distinguishable sets of signals exhibiting particular shift patterns (si figure s8) : one set corresponds to a pyridine ring placed perpendicular to the fe = o bond (11, 2, 1 ppm), and the other set, to the pyridine ring parallel to the fe = o axis (47, 13, 13 ppm), assigned by comparison with previous studies. the extinction coefficient was determined according to the purity of the sample measured by mssbauer spectroscopy (82%). bottom : esi - ms spectrum of 2 exhibiting a base peak at m / z 546.1. inset right : experimental and simulated peak at m / z 546.1 corresponding to { [fe(o)(mepy2tacn)](otf)}. inset left : experimental and simulated peak at m / z 548.1 corresponding to { [fe(o)(mepy2tacn)](otf) } obtained after reaction of 2 with 1000 equiv h2o. for the latter, the slight mismatch between the experimental and the calculated mass spectrum is due to the formation of iron(iii)-hydroxo species as a byproduct under the experimental conditions (m / z 549.1 { [fe(oh)(mepy2tacn)](otf) }). mssbauer spectrum of 2 recorded at 80 k. the experimental data are the hatched bars, and the dark and light gray lines represent the contributions of 2 and a decomposition diferric product, respectively. x - ray absorption spectroscopy (xas) provided insight into the structure of 2, which was compared with those of related complexes supported by pentadentate ligands, including 3 (scheme 1b), [fe(o)(n4py) ] and [fe(o)(bntpen) ]. the rising fe k - edge energy for 2 was found to be at 7124.2 ev and is comparable to the corresponding values measured for 3, [fe(o)(n4py) ], and [fe(o)(bntpen) ] at 7124.7, 7124.0, and 7123.7 ev, respectively. as expected, a 1s 3d transition can be observed at 7114.1 ev in the near - edge region. the normalized area of the pre - edge peak for 2 was found to be 26 units, compared with pre - edge areas of 2931 units for 3, [fe(o)(n4py) ], and [fe(o)(bntpen) ], suggesting that the ligand environment of 2 is slightly less distorted than the others. analysis of the extended x - ray absorption fine structure (exafs) data (figure 4, si table s4) shows that 2 has a single o scatterer at 1.63 corresponding to the oxo group and a shell of nitrogen scatterers at 2.00 arising from the pentadentate supporting ligand, similar to the data obtained for 3. interestingly, fitting of the outer sphere region of 2 required the inclusion of two shells of carbon scatterers at 2.81 and 2.95, in contrast to 3, for which a single shell of carbon scatterers at 2.90 was sufficient to fit the data. this difference likely arises from the fact that the two pyridine rings of the mepy2tacn ligand in 2 are coordinated to the iron center in somewhat different modes, as highlighted by the h nmr cosy experiment (si figure s8). thus, the xas data is consistent with the structure of 2 shown in scheme 1. taken together, the spectroscopic data support the formulation of 2 as an s = 1 iron(iv)-oxo compound with the general formula [fe(o)(mepy2tacn) ]. best fit (red solid line) to the experimental (black dashed line) unfiltered exafs data (inset) and corresponding fourier transform of 2. inspired by the recently described photochemical method for the generation of [fe(o)(n4py) ], we explored the photocatalytic generation of 2 (route b, scheme 1a). irradiation with visible light (led = 447 20 nm) of a solution containing 1 (0.4 mm), 5 mol % [ru(bpy)3]cl2 (0.02 mm), and 10 equiv of na2s2o8 (4 mm) under a n2 atmosphere at 25 c in ch3cn / h2o (1:3 v / v) resulted in immediate changes in the uv / vis absorption spectrum of the reaction mixture (figure 5). on one hand, the instantaneous oxidation of [ru(bpy)3 ] into [ru(bpy)3 ] is evidenced by the immediate disappearance of the absorption band of [ru(bpy)3 ] at 450 nm. in addition, formation of 2 under these photocatalytic conditions was clearly indicated by the progressive growth within 100 s of its characteristic absorption band now centered at 715 nm (= 240 m cm ; no correction was applied for the calculation of the value in this solvent mixture). the 20-nm shift of the max of 2 in ch3cn / h2o 1:3 with respect to ch3cn (figure 2, top) is due to a solvatochromic effect. such dependence of the max on the solvent is also observed for other iron(iv)-oxo compounds, such as 3, [fe(o)(n4py) ] and [fe(o)(bntpen) ], for which a blue shift of the d control experiments showed that compound 2 was not formed in the absence of [ru(bpy)3]cl2 or na2s2o8. uv / vis absorption spectra obtained before and during irradiation (447 nm) of a sample containing 1 (0.4 mm), [ru(bpy)3]cl2 (0.02 mm), and 10 equiv of na2s2o8 (4 mm) in ch3cn / h2o (1:3 v / v). inset : kinetic trace at 715 nm. in the absence of compound 1 and under irradiation, [ru(bpy)3 ] underwent oxidation by na2s2o8 to [ru(bpy)3 ], as manifested by the rapid decrease in the absorption band at = 450 nm and formation of the characteristic weak absorption bands of ru around 650 nm (si figure s9, left). the ability of [ru(bpy)3 ] to achieve the 2-electron oxidation of 1 to 2 was proven by the stoichiometric reaction of 1 with 2 equiv of isolated [ru(bpy)3 ] (route c, scheme 1a), which provided full conversion to 2 (si figure s10, left). the use of another 1e oxidant, such as cerium(iv) ammonium nitrate, also produced compound 2 (si figure s10, right). these experimental data are in accordance with the mechanism depicted in scheme 2, earlier proposed by nam and fukuzumi for the photochemical preparation of the iron(iv)-oxo compound [fe(o)(n4py) ]. na2s2o8 itself oxidizes the starting iron(ii) complex (1) to iron(iii), as clearly observed by the disappearance of the characteristic 414 nm absorption band of 1 when na2s2o8 is added and the appearance of the characteristic absorption bands of [fe(oh)(mepy2tacn) ] (si figure s9 right and table s5). accordingly, the measured redox potential of the fe / fe oh couple was 0.38 v vs sce in ch3cn / h2o 1:3 (si figures s5 and s6). further confirmation of the nature of this iron(iii)-hydroxo complex was gained by mssbauer and epr spectroscopies (si figures s3 and s4, tables s3 and s5). direct electron transfer from the iron(iii) center to [ru(bpy)3 ] gives 2 together with the corresponding one - electron - reduced ru complex. in turn, the sacrificial electron acceptor, na2s2o8, regenerates [ru(bpy)3 ] by one - electron oxidation of the [ru(bpy)3 ] excited state, which is formed upon excitation of [ru(bpy)3 ] at 447 nm (scheme 2). remarkably, only 5 mol % of the ruthenium photosensitizer was needed to achieve the complete transformation of 1 to 2, which is a significantly smaller amount than the 40 mol % [ru(bpy)3 ] used by nam and fukuzumi to generate [fe(o)(n4py) ] photochemically. the use of low amounts of the ruthenium photosensitizer is an especially appealing strategy because it entails the in situ generation of a strong oxidant specifically, [ru(bpy)3]in catalytic amounts. under the conditions described above, photocatalytically generated compound 2 reacted with sulfides, as clearly evidenced by the disappearance of the characteristic absorption band of 2 at 715 nm. the rate of the reaction between 2 and an excess of substrate (5 equiv) was obtained by fitting the absorbance at 715 nm over time to a single - exponential decay function. in the absence of irradiation, the observed rate constant (kobs) for the reaction of photochemically generated 2 (0.4 mm 1, 0.02 mm [ru(bpy)3]cl2, 4 mm na2s2o8 in ch3cn / h2o 1:3) with 5 equiv of 4-methoxythioanisole (phsme) was 1.5 10 s at 25 c. surprisingly, when the same experiment was carried out under irradiation, the decay of 2 occurred much faster, with kobs = 0.22 s (a 150-fold increase with respect to the experiment without irradiation). indeed, this significant enhancement of the oxygenation rate of the substrate by light deserves special consideration. under the photocatalytic conditions described above, compound 2 thus, after generating 2, the irradiation was stopped, and 1 equiv of phsme was added. irradiation triggered a much faster oxidation of the substrate, manifested by the immediate and abrupt decay of the characteristic band at 715 nm, which was completely depleted (figure 6). under continuous irradiation, once phsme had been consumed, compound 2 was regenerated in 87% yield, as observed by the recovery of its characteristic absorption at 715 nm (figure 7). such regeneration can be rationalized by considering that the putative iron(ii) complex, formed after oxo - transfer to the sulfide, is reoxidized by excess na2s2o8 (fe fe oxidation) or by the in - situ - photogenerated [ru(bpy)3 ] (fe fe fe) to give 2 again. this process was repeated several times, although the extent of the regeneration decreased with every cycle (see the intensity of the band at 715 nm after each cycle in figure 6). the reason for this incomplete recovery might be partial decomposition of the iron complex or the photosensitizer, processes usually associated with prolonged irradiation. interestingly, a delay time between full decay of 2 and the onset of its regeneration was observed after the first cycle, and the extent of this delay increased from cycle to cycle. the origin of the delay was the oxidation of the remaining sulfide not consumed in the initial fast reaction with 2. because 2 was increasingly decomposed under photoexcitation from cycle to cycle, the amount of excess sulfide increased accordingly, as did the time needed to accumulate more 2. kinetic trace at 715 nm corresponding to a reaction mixture containing 1 (0.4 mm), 5 mol % [ru(bpy)3]cl2 (0.02 mm), and na2s2o8 (4 mm, 10 equiv) under n2 atmosphere at 25 c. labels on the figure indicate the initial (on) and final (off) points of irradiation (= 447 20 nm) as well as the addition of 1 equiv of phsme. uv / vis spectral changes upon irradiation (447 nm) of a sample of photochemically generated 2 (0 = 0.4 mm, 5 mol % [ru(bpy)3]cl2 (0.02 mm), and 10 equiv of na2s2o8 (4 mm)) after addition of 1 equiv of phsme under a n2 atmosphere at 25 c in ch3cn / h2o 1:3. a shows the instantaneous decay of compound 2 upon irradiation, and step b shows the progressive regeneration of 2 (up to 87%) once the sulfide substrate has been consumed (spectra were recorded every 10 s). the rate enhancement observed upon light irradiation is remarkable and unprecedented and prompted us to explore the origin of the increase in reactivity. because the photocatalytic generation of compound 2 takes place in a complex reaction mixture containing several components (na2s2o8, [ru(bpy)3 ], sulfide, and iron complex), a simplification of the system was necessary to shed some light on the origin of this phenomenon. to gain insight into the origin of the rate enhancement upon light irradiation, we simplified the system by eliminating na2s2o8 from the reaction mixture. this is convenient not only to reduce the number of variables but also because it is well - known that under photoirradiation, [ru(bpy)3 ] acts as a noninnocent oxidative quencher that generates high energy sulfate radicals (e(so4) = 2.0 v) from s2o8. therefore, we studied the capacity of compound 2 (chemically generated by reaction of 1 with phio in ch3cn / h2o 1:3) to oxidize sulfides under a range of conditions : (i) with / without irradiation and/or (ii) in the presence / absence of the photosensitizer (table 1). the rate of the direct reaction of 2 (0.4 mm) with 5 equiv of phsme (2 mm) (kobs = 11 1 10 s) was unaffected by irradiation at 447 nm (entries 1 and 2). in sharp contrast, the presence of 5 mol % of [ru(bpy)3 ] (0.02 mm) accelerates the decay of 2 7-fold under irradiation (entries 3 and 4, si figure s11). control experiments showed that in the absence of the sulfide, the decay of 2 also occurred faster under irradiation in the presence of [ru(bpy)3 ] (entries 5 and 6). the effect of light on the self - decay rate (without the sulfide substrate or photosensitizer) is minor, albeit significant (entries 7 and 8). from this set of experiments, it is clear that the combination of [ru(bpy)3 ] and irradiation was crucial for the enhanced oxidation rate of phsme by 2 ; that is, [ru(bpy)3 ] acts as a photosensitizer, accelerating the process. addition of 5 equiv para - x - phenylmethylsulfide (phsme, 2 mm) with respect to 2 in the reaction mixture. addition of 5 mol % [ru(bpy)3]cl2 (0.02 mm) with respect to 2 in the reaction mixture. kobs values were obtained by fitting the decay of the absorbance at 715 nm over time to a single exponential function. apart from phsme, the photosensitized oxidation of other para - substituted thioanisoles (phsme, x = me, h, and cl) was also considerably faster under irradiation and gave 40% yield of the corresponding sulfoxide in all cases (si table s6). the uv / vis absorption spectrum at the end of the reaction did not show the characteristic band of 1 at 414 nm, indicating that 2 did not revert to the starting iron(ii) after the oxygen - atom transfer reaction. instead, esi - ms spectra evidenced the formation of iron(iii)-hydroxo species with major peaks at m / z 547.11 and 199.09 corresponding to { [fe(oh)(mepy2tacn)](otf) } and { [fe(oh)(mepy2tacn) ] } (si figure s12). addition of 1 equiv of ascorbic acid (with respect to iron) at the end of the reaction further confirmed this result because more than 75% of 1 was regenerated, as ascertained by the formation of the characteristic band of 1 at 414 nm (si figure s13). because photochemical and redox processes associated with [ru(bpy)3 ] and iron(iv)-oxo species are rich and varied, several mechanisms can be postulated to rationalize the observed photoenhanced oxygen - atom transferability of 2 (scheme 3). some reaction pathways can be discarded on the basis of thermodynamic considerations (scheme 4). an electron transfer from 2 or phsme to [ru(bpy)3 ] is not plausible on the basis of the redox potentials of the [ru(bpy)3]/[ru(bpy)3 ] (e = + 0.84 v vs sce), fe(o)/fe(o) (estimated to be e > 1.5 v by dft calculations, unpublished results), and phsme / phsme (e = + 1.13 v vs sce) couples. likewise, electron transfer from [ru(bpy)3 ] to phsme can be discarded if the highly negative reduction potential of this compound is taken into account. thus, the two most plausible mechanisms would be (scheme 3) (i) energy transfer from [ru(bpy)3 ] to 2 to give a highly reactive 2 and (ii) an electron - transfer from [ru(bpy)3 ] to compound 2, resulting in the formation of [ru(bpy)3 ], which would subsequently oxidize phsme to the sulfide radical cation (phsme). the rates of reaction of 2 with a series of phsme (x = ome, me, h, and cl) were studied in the presence of [ru(bpy)3 ] (5 mol %, 0.02 mm) under irradiation at 447 nm to gain further information regarding the mechanism of the photoenhancement of the oxidation rates. as indicated by the small slope of the hammett plot (= 0.09) represented in si figure s14b, the reaction rates are not much influenced by the electron - withdrawing or electron - donating abilities of the para substituents of the sulfide ; thus, the electronic properties of the substrate do not affect the rate - determining step of the process. these data are in agreement with the fact that the steady - state [ru(bpy)3 ] concentration was unchanged during the reaction because the intensity of its characteristic absorption band at 450 nm remained stable during the reaction, thus suggesting that the rate - determining step occurred just after the transformation of the photosensitizer and prior to the involvement of the substrate. in sharp contrast, when the hammett plot was determined from intermolecular competition experiments of p - x - thioanisoles versus thioanisole, by plotting the relative amounts of sulfoxide products formed (si figure s14c), a value of 2.5 was obtained. this result indicates that the electronic properties of the substrate have a very significant influence in the product - determining step, but this step is not rate - determining. plotting these values against the one - electron oxidation potentials (eox) of the sulfides gives a linear correlation with a slope of 5.0 (si figure s15), a value significantly below the typical values reported for oxygen - atom transfer processes (between 2 and 3) but somewhat above those obtained for processes involving an electron transfer mechanism (between 8 and 10). nanosecond time - resolved absorption spectroscopy (nd : yag, 532 nm, 10 ns pulse) was employed to gain insight into the mechanism. pulsed excitation at 532 nm (15 mj / pulse) of deaerated ch3cn / h2o (1:3) solutions of [ru(bpy)3 ] (0.07 mm, absorbance 0.06 at 532 nm) led to the disappearance of the latter, as evidenced by the strong bleaching near 470 nm. this was accompanied by the formation of [ru(bpy)3 ] (mlct state), which exhibits characteristic absorption bands below 400 nm and an emission centered at 620 nm, with a lifetime,, of 920 ns (si figure s16). as expected, the bleaching at 470 nm recovered fully in <3 s (figure 8 curve a), which is associated with the full recovery of [ru(bpy)3 ]. comparatively, the same experiment in the presence of increasing amounts of 2 showed that 2 quenched the emission of [ru(bpy)3 ] at 620 nm with a rate constant, kq, of 5.7 10 m s (stern volmer plot, si figure s17). however, under these conditions, [ru(bpy)3 ] did not completely revert back to the starting [ru(bpy)3 ] compound ; that is, its characteristic absorption at 470 nm was not fully recovered. this prolonged bleaching (no changes were detected, even after 150 s of the laser pulse) (curve b and inset in figure 8, si figure s18) is in accordance with the formation of a new long - lived species with an absorbance in this region lower than that of [ru(bpy)3 ]. this new species could be [ru(bpy)3 ], whose absorption at 470 nm is only 4% that of [ru(bpy)3 ] (si figure s9). interestingly, the 470 nm band was completely recovered when the [ru(bpy)3]/2 mixture was excited in the presence of phsme (3.4 mm) (figure 9a). in this case, formation of a species with an absorption band centered at 580 nm was detected (figure 9b inset). this species can be assigned to a phsme radical cation (phsme) formed by oxidation of phsme with the in - situ - generated [ru(bpy)3 ]. indeed, formation of phsme by in - situ - generated [ru(bpy)3 ] was confirmed by laser excitation at 532 nm of a [ru(bpy)3]/na2s2o8/phsme mixture (si figure s20). taken together, the data suggest that the photoenhanced oxidation of phsme with 2 occurs through the electron - transfer mechanism depicted in scheme 3. to identify the species responsible for the final 1e oxidation of phsme to give the observed sulfoxide product, we monitored the uv / vis spectral changes occurring upon addition of phsme (5 equiv) to a mixture containing 2 and [ru(bpy)3 ] (5 equiv). interestingly, compound 2 was immediately consumed upon substrate addition, even though [ru(bpy)3 ] can generate 2 by oxidation of its iron(ii) or iron(iii) precursors (figure 7). thus, under these conditions, [ru(bpy)3 ] reacts instantaneously with phsme to produce phsme, which, in turn, gets immediately oxidized by 2 to give the corresponding sulfoxide and the iron(iii)-hydroxo compound. this experiment, together with the results from laser - pulse time - resolved absorption spectroscopy, indicates that compound 2 serves as a 1e oxidant of both [ru(bpy)3 ] and phsme so that the oxidation of 1 equiv sulfide to the corresponding sulfoxide requires 2 equiv of 2 (scheme 3). this electron transfer mechanism rationalizes the observed exclusive presence of iron(iii) at the end of the reaction and the low yield of sulfoxide product (40%) observed for phsme (x = ome, me, h, and cl) (see above). transient kinetic trace observed at 470 nm after laser flash photolysis (532 nm) of deaerated solution of [ru(bpy)3 ] (0.07 mm) in ch3cn / h2o (1:3) (a) in the absence and (b) in the presence of 2 (3.4 mm). inset : [ru(bpy)3 ] time profile monitored at 470 nm in the presence of 2 (3.4 mm) over a period of 160 s. (a) transient kinetic traces monitored at 470 nm after laser flash photolysis (532 nm) of a deaerated ch3cn / h2o (1:3) solution of [ru(bpy)3 ] in the presence of 2 (3.4 mm) (black) or 2 (3.4 mm) and phsme (3.4 mm) (red). (b) transient kinetic traces monitored at 550 nm after laser flash photolysis (532 nm) of a deaerated solution of [ru(bpy)3 ] in ch3cn / h2o (1:3) in the presence of 2 (3.4 mm) and phsme (3.4 mm). inset : transient absorption spectrum of a deaerated solution of [ru(bpy)3 ] in ch3cn / h2o (1:3) in the presence of 2 (3.4 mm) and phsme recorded 2 s after laser excitation (532 nm). p - cyanothioanisole (phsme) constitutes a limiting case for the photocatalyzed electron transfer because its redox potential to form the radical cation (1.61 v) is significantly higher than the oxidation potential of [ru(bpy)3 ] (1.26 v), and therefore, the photoenhanced oxidation of this sulfide by an electron transfer mechanism would be unlikely or it would occur with very low efficiency. as expected, time - resolved absorption spectroscopic studies performed in [ru(bpy)3]/2 mixtures in the presence of phsme led neither to the full recovery of the absorption at 470 nm nor to the detection of the phsme radical cation (si figure s21) in the time scale of our laser experiment. these observations indicate that, as expected, the electron transfer from [ru(bpy)3 ] to phsme is not taking place. interestingly, the photoenhanced decay of 2 in the presence of [ru(bpy)3 ] is slightly accelerated with phsme (compare entries 4 and 6 in table 1) and the sulfoxide yield is low (1012%). given the fact that electron transfer with phsme is not operative, an energy transfer mechanism is proposed to explain the moderate rate acceleration and the formation of sulfoxide. this would involve the formation of the 2 excited state by energy transfer from [ru(bpy)3 ] to 2 (scheme 3). formation of 2 could also be at the origin of the accelerated decay of 2 with the photosensitizer in the absence of substrate. however, time - resolved absorption measurements failed to detect 2. under our photochemical conditions, excitation of [ru(bpy)3]/2 mixtures either at 532 nm or at 355 nm did not provide any transient absorption or emission that could be attributed to 2. in conclusion, in this work, we demonstrate that [ru(bpy)3 ] can photochemically enhance the reaction of an s = 1 oxoiron(iv) complex toward phsme (x = och3, ch3, h, and cl). nanosecond time - resolved absorption spectroscopic results strongly support an electron transfer from [ru(bpy)3 ] to 2 to generate [ru(bpy)3 ] and iron(iii)-hydroxo complexes. subsequently, [ru(bpy)3 ] would oxidize the sulfide to its corresponding radical cation, which would react with 2 to form the sulfoxide. at the same time, nanosecond time - resolved absorption data suggest that the photosensitized rate enhancement observed for phsme is unlikely to occur through an electron - transfer mechanism. for this reason, we propose that partial contribution of the energy transfer mechanism from [ru(bpy)3 ] to 2 to give rise to the 2 excited state could be relevant for this substrate, and it could explain the low amounts of oxidized product detected. this excitation would presumably involve population of a low - lying, more reactive s = 2 excited state. this light - induced low - spin / high - spin transition is reasonable because this process is already well - documented for d d metal complexes. ongoing experiments to further clarify this mechanism are currently under examination. further exploration of the photochemical reactivity of the iron(iv)-oxo complexes, the mechanism of activation, and expansion of this phenomenon toward the reactivity of other substrates are currently being explored. reagents were purchased from commercial sources and used as received without any further purification. compounds methyl p - tolyl sulfide, 4-chlorothioanisole, and formaldehyde were purchased from fluorochem, alfa aesar, and scharlab, respectively ; the rest of the compounds were purchased from sigma - aldrich. solvents were purchased from sds and scharlab, purified and dried by passing through an activated alumina purification system (mbraun sps-800), and stored in an anaerobic glovebox under n2. preparation of 1,4-bis(2-pyridylmethyl)-1,4,7-triazacyclononane, me2py2tacn, and [fe(o)(me2py2tacn) ] (3) were carried out as previously described. water (18.2 mcm) was purified with a milli - q millipore gradient ais system. uv / vis / nir spectra were recorded on an agilent 8453 diode array spectrophotometer (1901100 nm range) in 1 cm quartz cells. electrospray ionization mass spectrometry (esi - ms) experiments were performed on a bruker daltonics esquire 6000 spectrometer. elemental analyses were conducted in a carlo erba instrument, model chns 1108. crystals of 1 were used for low temperature (100(2) k) x - ray structure determination. the measurement was carried out on a bruker smart apex ccd diffractometer using graphite - monochromated mo k radiation (= 0.71073) from an x - ray tube. the measurements were made in the range 2.1128.64 for. full - sphere data collection was carried out with and scans. a total of 45423 reflections were collected, of which 14395 [r(int) = 0.0750 ] were unique. programs used : data collection, smart version 5.631 (bruker axs 1997 - 02) ; data reduction, saint + version 6.36a (bruker axs 2001) ; absorption correction, sadabs version 2.10 (bruker axs 2001). structure solution and refinement was performed using shelxtl version 6.14 (bruker axs 20002003). the structure was solved by direct methods and refined by full - matrix least - squares methods on f. the non - hydrogen atoms were refined anisotropically. the h - atoms were placed in geometrically optimized positions and forced to ride on the atom to which they are attached. laser flash photolysis experiments were carried out with the second harmonic (532 nm) of a q - switched nd : yag laser (spectra physics quantaray (indi) ; pulse width 9 ns and 15 mj pulse). the signal from the monochromator / photomultiplier detection system was captured by a tektronix tds640a digitizer and transferred to a pc computer that controlled the experiment and provided suitable processing and data storage capabilities. h and c nmr spectra were recorded on a bruker avance 400 mhz spectrometer as solutions at 25 c and referenced to residual solvent peaks. gc product analyses were performed on an agilent 7820a gas chromatograph equipped with a hp-5 capillary column 30 m 0.32 mm 0.25 m and a flame ionization detector. epr spectra were recorded on an x - band bruker emx spectrometer equipped with an oxford instruments esr-900 continuous - flow helium cryostat and an er-4116 dm bruker cavity. fe mssbauer experiments were performed at 80 k on a zero - field mssbauer spectrometer equipped with a janis svt-400 cryostat as already described. analysis of the data was performed with the program wmoss (web research, edina, mn, usa). fe k - edge x - ray absorption spectra were collected on beamline 9 - 3 of the stanford synchrotron radiation lightsource (ssrl) at the slac national accelerator laboratory with a spear storage ring current of 450 ma at a power of 3.0 gev. the incoming x - rays were unfocused using a si(220) double crystal monochromator, which was detuned to 40% of the maximal flux to attenuate harmonic x - rays. four (4) scans were collected from 6882 to 8000 ev at a temperature (10 k) that was controlled by an oxford instruments cf1208 continuous flow liquid helium cryostat. data were obtained as fluorescence excitation spectra with a 100-element solid - state ge detector array (canberra). in fluorescence mode, photon scattering noise was reduced using a 3 m mn filter and a soller slit. an iron foil was placed in the beam pathway prior to i0 and scanned concomitantly for an energy calibration, with the first inflection point of the edge assigned to 7112.0 ev. photoreduction was monitored by scanning the same spot on the sample twice and comparing the first derivative peaks associated with the edge energy during collection, but none was observed in the present study. the detector channels from the scans were examined, calibrated, averaged, and processed for exafs analysis using exafspak to extract (k). scatterer pair were calculated using feff 8.40 and were utilized by the opt parameters for 2 were calculated using similar coordinates of the available crystal structure of the corresponding fe complex (1). in all analyses, the coordination number of a given shell was a fixed parameter and was varied iteratively in integer steps while the bond lengths (r) and mean - square deviation () were allowed to freely float. the amplitude reduction factor, s0, was fixed at 0.9 while the edge - shift parameter e0 was allowed to float as a single value for all shells. thus, in any given fit, the number of floating parameters was typically equal to (2 number of shells) + 1. pre - edge analysis was performed on data normalized in the process program of the exafspak package, and pre - edge features were fit between 7108 and 7118 ev using the fityk program with pseudo - voigt functions composed of 50:50 gaussian / lorentzian functions. 1,4-bis(2-pyridylmethyl)-1,4,7-triazacyclononane (0.34 g, 1.09 mmol) was dissolved in formaldehyde 37% (3 ml), 98% formic acid (3 ml), and water (2.5 ml), and the resulting yellow solution was refluxed for 30 h. after cooling to room temperature, 3 ml of hcl was added, and the mixture was left stirring for 10 min. the solvent was removed under vacuum, and a small amount of water (10 ml) was added to the resulting residue. the solution was brought to ph 14 by the addition of naoh (4 m). after stirring for 20 min, the aqueous phase was extracted with ch2cl2 (3 50 ml). the combined organic phases were dried over anhydrous mgso4, and the solvent was removed under reduced pressure. the resulting residue was treated with n - hexane (75 ml) and stirred for 12 h. the solvent was decanted and removed under reduced pressure to yield 0.162 g of a colorless oil (0.50 mmol, 46%). h nmr (cdcl3, 300 mhz, 300 k), ppm : 8.50 (d, 2h, pyh), 7.64 (dt, 2h, pyh), 7.49 (d, 2h, pyh), 7.10 (dd, 2h, pyh), 3.82 (s, 4h, ch2-py), 2.902.82 (m, 8h, n the analysis is consistent with the previously reported synthesis of mepy2tacn. to a vial containing mepy2tacn (150 mg, 0.46 mmol) dissolved in thf (1 ml) was added dropwise a thf solution (1 ml) of fe(ch3cn)2(cf3so3)2 (200 mg, 0.46 mmol). after stirring for 3 h, the resulting red solid was filtered, washed with et2o (3 2 ml), and dried under vacuum. recrystallization of the red solid by et2o diffusion into a saturated ch2cl2/ch3cn solution yielded dark red single crystals (198 mg, 60%) suitable for diffraction analysis. h nmr (cd3cn, 400 mhz, 273 k), ppm : 8.90 (d, j = 5.6 hz, 1h, pyh), 7.94 (td, j = 7.6 hz, j = 1.2 hz, 1h, pyh), 7.81 (td, j = 7.6 hz, j = 1.2 hz, 1h, pyh), 7.61 (d, j = 7.6 hz, 1h, pyh), 7.56 (t, j = 6.6 hz, 1h, pyh), 7.52 (d, j = 7.6 hz, 1h, pyh), 7.29 (d, j = 5.2 hz, 1h, pyh), 7.18 (t, j = 6.4 hz, 1h, pyh), 4.72 (d, j = 16 hz, 1h, ch2-py), 4.58 (d, j = 15.6 hz, 1h, ch2py), 4.54 (d, j = 16 hz, 1h, ch2py), 4.06 (d, j = 16.4 hz, 1h, ch2py), 3.52.9 (m, 10h, n ch2), 2.59 (s, 3h, ch3), 1.61 (td, j = 13.2 hz, j = 6.4 hz, 1h, n ch2). c nmr (cd3cn, 100 mhz, 273 k), ppm : 166.07, pyc), 166.03 (1c, pyc), 157.42 (1c, pyc), 154.39 (1c, pyc), 137.57 (1c, pyc), 137.42 (1c, pyc), 125.09 (1c, pyc), 125.06 (1c, pyc), 122.60 (1c, pyc), 122.15 (1c, pyc), 68.07 (1c, py - ch2), 67.01 (1c, py - ch2), 61.05 (1c, n - ch2), 60.18 (2c, n - ch2), 59.01(1c, n - ch2), 58.20 (1c, esi - ms (m / z) : [m ch3cn cf3so3 ] = 530.2 (100%), [m ch3cn 2cf3so3 ] = 190.5 (26%). anal. (%) for c23h30f6fen6o6s21/2h2o (729.43) : c, 37.87 ; h, 4.28 ; n, 11.52 ; s, 8.79. found : c, 37.99 ; h, 4.31 ; h, 11.41 ; s, 8.67. in an anaerobic glovebox, 1 (2.3 mg, 3.9 10 mmol) and phio (14 mg, 6.4 10 mmol) were mixed in ch3cn (2 ml). removal of unreacted phio was achieved by filtration, which afforded a pale green solution of compound 2. the yield of the reaction was estimated according to the amount of fe determined by mssbauer spectroscopy by preparation of a 50% fe - enriched sample of compound 2. h nmr (cd3cn, 400 mhz, 300 k), ppm : 46.46 (s, 1h, pyh), 13.34 (s, 1h, pyh), 11.22 (s, 1h, pyh), 2.05 (s, 1h, pyh), 1.36 (s, 1h, pyh), 13.27 (s, 1h, pyh). esi - ms (m / z) : [m cf3so3 ] = 546.1 (100%), [m 2cf3so3 ] = 198.5 (5%). uv / vis (ch3cn / h2o 1:3) : max = 715 nm, = 240 m cm in an anaerobic glovebox, a solution of 1 (0.72 mg, 1 10 mmol) in ch3cn (625 l) was placed in a uv / vis cuvette. addition of 5 mol % of [ru(bpy)3]cl2 (0.05 mol, 100 l of a 0.5 mm solution in deaerated water), 10 equiv of na2s2o8 (10 mol, 100 l of a 100 mm solution in deaerated water), and deaerated water (1.6 ml) afforded the initial reaction mixture (solvent ratio ch3cn / h2o 1:3, 0.4 mm in 1). irradiation at 447 nm caused immediate changes in the uv / vis spectrum that led to the formation of 2, as evidenced by the appearance of its characteristic band at 715 nm (see figure 5). the required amount of 2 (625 l of a 1.6 mm solution of 2 in ch3cn obtained by direct oxidation of 1 with phio) was diluted in deaerated milli - q water (1.67 ml), then the desired quantity of photosensitizer (dissolved in ch3cn : h2o 1:3) and/or sulfide (phsme, dissolved in ch3cn) was added. finally, the appropriate amounts of ch3cn and h2o were added to reach a ch3cn / h2o ratio of 1:3 and an initial concentration of 2 of 0.4 mm. the progress of the reaction was monitored by uv / vis spectroscopy at 25 c. reaction of 2 with sulfides (phsme) caused a decay of its characteristic absorption band (max = 715 nm). after full decay of this band, an internal standard was added to the solution (trimethoxybenze or biphenyl), and the amount of formed sulfoxide was quantified by h nmr spectroscopy or gas chromatography.	the preparation of [feiv(o)(mepy2tacn)]2 + (2, mepy2tacn = n - methyl - n, n - bis(2-picolyl)-1,4,7-triazacyclononane) by reaction of [feii(mepy2tacn)(solvent)]2 + (1) and phio in ch3cn and its full characterization are described. this compound can also be prepared photochemically from its iron(ii) precursor by irradiation at 447 nm in the presence of catalytic amounts of [ruii(bpy)3]2 + as photosensitizer and a sacrificial electron acceptor (na2s2o8). remarkably, the rate of the reaction of the photochemically prepared compound 2 toward sulfides increases 150-fold under irradiation, and 2 is partially regenerated after the sulfide has been consumed ; hence, the process can be repeated several times. the origin of this rate enhancement has been established by studying the reaction of chemically generated compound 2 with sulfides under different conditions, which demonstrated that both light and [ruii(bpy)3]2 + are necessary for the observed increase in the reaction rate. a combination of nanosecond time - resolved absorption spectroscopy with laser pulse excitation and other mechanistic studies has led to the conclusion that an electron transfer mechanism is the most plausible explanation for the observed rate enhancement. according to this mechanism, the in - situ - generated [ruiii(bpy)3]3 + oxidizes the sulfide to form the corresponding radical cation, which is eventually oxidized by 2 to the corresponding sulfoxide.
from may 2004 to may 2005, a study of acute gastroenteritis in children 0.1). no coinfections with other viruses (rotavirus, astrovirus, and adenovirus) were detected. ten (71%) of the noroviruses detected were identified as belonging to genogroup gii ; the remaining 4 (29%), to gi. gi noroviruses were further classified into 3 genotypes : gi.1 (1 isolate), gi.4 (1 isolate), and gi.3 (2 isolates). em, electron microscopy, nov, norovirus ; rv, rotavirus ; av, astrovirus ; pos, positive ; neg, negative. all norovirus - positive samples were negative for av and rv when tested by reverse transcription pcr. phylogenetic tree of noroviruses based on the 330-bp region (for gi) and 344-bp region (for gii) of the capsid n terminus / shell gene. fourteen novel sequences were included, designated according to isolate code, place, and year (e.g., dr001-madag04) ; 25 sequences of reference norovirus strains (6) were included, designated according to genogroup - genotype, place, country, and year (e.g.,gii-2/melksham - grb1994). comparative strains are gi-1/nv - usa1968 (norwalk, m87661), gi-2/sov - gbr1993 (southampton, l07418), gi-3/dsv - usa1993 (desert shield, u04469), gi-4/chiba - jpn2000 (ab042808), gi-5/musgrov - gbr1989 (musgrove, aj277614), gi-6/hesse - deu1998 (af093797), gi-7/wnchest - gbr1994 (winchester, aj277609), gi-8/boxer - usa2001 (af538679), gii-1/hawai - usa1971 (u07611), gii-2/melksham - gbr1994 (x81879), gii-3/toronto - can1993 (u02030), gii-4/bristol - gbr1993 (x76716), gii-5/hillingd - gbr1990 (hillingdon, aj277607), gii-6/seacrof - gbr1990 (seacroft, aj277620), gii-7/leeds - gbr1990 (aj277608), gii-8/amstdam - nld1990 (amsterdam, af195848), gii-9/vabeach - usa1997 (ay038599), gii-10/erfurt - deu2000 (af427118), gii-11/sw918-jpn1997 (ab074893), gii-12/wortley - gbr1990 (aj277618), gii-13/faytvil - usa1998 (fayetteville, ay113106), gii-14/m7-usa1999 (ay130761), gii-15/j23-usa1999 (ay130762), gii-16/tiffin - usa1999 (ay502010), and gii-17/cse1-usa2002 (ay502009). the median age of children with norovirus infection was 18 months (range 351 months). all infections with genogroup gi noroviruses were found in children < 24 months of age (table 2). noroviruses were detected throughout the year ; however, infections peaked during the wet season in madagascar. november and december were the months of major norovirus prevalence (35.7% each month). seasonality of gi and gii norovirus infections, antananarivo, madagascar, may 2004may 2005. to our knowledge, ours is the first study that has used molecular detection methods to investigate the role of noroviruses in pediatric gastroenteritis in madagascar. we showed that infections with gi and gii noroviruses are relatively common. in a 1-year collection of stool samples, we detected noroviruses by rt - pcr in 6% of children with acute gastroenteritis in antananarivo. this rate establishes norovirus as the second most commonly detected enteric virus in this population, behind rotavirus (38%) and followed by astrovirus (2.5%) (data not shown). the median age of children with norovirus infection (18 months) was higher than previously reported (7) and higher than that of the rotavirus - infected group (median 10 months, range 1 day to 48 months) and that of the astrovirus - infected group (median 10 months, range 520 months) (data not shown). noroviruses were detected throughout the year, but the number peaked in november and december. such seasonality in a tropical country is not really expected, as year - round circulation has been previously documented (15). our findings confirm the finding of previous studies that gii is the predominant norovirus genogroup circulating in communities worldwide. considerable genetic diversity was observed among the norovirus gii isolates, and some were identified as belonging to a potentially novel cluster. the closest reference strain to the potentially novel cluster was the recombinant hu / nov / gii.1/hawaii/1971/us. in contrast, norovirus gi isolates were clustered with prototype strains ; hu / nov / gi.3/dsv395/1990/sa (desert shield) was predominant (2 strains), followed by hu / nov / gi.1/norwalk/1968/us (norwalk) and hu / nov / gi.4/chiba407/1987/jp (1 each). the sample size was small, and we examined samples collected over a 13-month period. longer, longitudinal studies are required to address issues such as norovirus seasonality and temporal genetic variability. in addition, we restricted our analysis to specimens collected from patients at rehydration clinics and hospitals, so prevalence of norovirus infections in the general population may have been underestimated. furthermore, the use of short conserved sequences, although successful for diagnosis of norovirus infection, should be used with caution for classification and phylogenetic analyses. further analysis by full capsid sequencing might be required. nevertheless, continued norovirus surveillance is needed to monitor the spread and persistence of the various genotypes infecting children in madagascar.	of 237 children with acute gastroenteritis in antananarivo, madagascar, during may 2004may 2005, 14 (6%) were infected with norovirus. seasonality (november december peak) was detected. reverse transcription pcr identified gii as the most common genogroup. gis belonged to gi.1, gi.3, and gi.4. noroviruses in madagascar show extensive genetic diversity.
the analysis of cancer predisposition syndromes has been an important approach towards the identification of oncogenes and tumor suppressor genes. some hereditary cancer syndromes, such as li - fraumeni syndrome, are caused by the mutation of critical tumor suppressor genes (tp53) and lead to wide - spread tumorigenesis including many different tumor types. however, other hereditary cancer syndromes appear to have a more limited tumor spectrum.for example, individuals with syndromes such as wagr (wilms tumor, aniridia, genitourinary abnormalities, and mental retardation syndrome) and denys - drash syndrome have mutations in the wt1 gene, and these patients are primarily at risk for wilms tumor [2, 3 ]. the identification of an underlying genetic mutation or predisposition to develop specific cancers is helpful not only to family members with that syndrome, but also to many other individuals who develop cancer without known risk factors. knowledge of how specific tumors arise can be applied to targeted prevention, surveillance, and even therapeutic strategies. ewing 's sarcoma, first described by james ewing in 1921, is the second most common pediatric bone cancer after osteosarcoma. it is an aggressive cancer of children and young adults, with 30%60% survival depending on tumor site and the presence or absence of metastases at diagnosis [4, 5 ]. while osteosarcoma is thought to arise from bone cell progenitors, the cell of origin of ewing 's sarcoma remains unknown. james ewing himself initially described this disease as an endothelioma of bone, and later suggested that it arises from perivascular lymphatic endothelium [7, 8 ]. since that time, other investigators have suggested myriad cells of origin, including hematologic, mesenchymal / fibroblastic [10, 11 ], and neural crest derivatives [12, 13 ]. more recently, emerging evidence has suggested that ewing 's sarcoma arises from a mesenchymal stem or progenitor cell [1416 ]. a definitive answer to the cell of origin question will require additional analyses. while the cell of origin of ewing 's sarcoma is not yet known, the molecular genetics of the tumor are better understood. ewing 's sarcomas are highly associated with a limited set of recurring, somatic chromosomal rearrangements. the most common of these, t(11;22)(q24;q12), is found in approximately 85% of cases, while t(21;22)(q22;q12) is found in 10% of cases [17, 18 ]. the remaining translocations are found in < 5% each [19, 20 ]. these translocations fuse the ewsr1 gene on chromosome 22 with an ets family member, most commonly the fli1 gene on chromosome 11 [17, 1921 ]. this ewing 's sarcoma - specific translocation generates an ews / ets fusion protein [17, 1921 ]. the ewing 's sarcoma fusion proteins contain a strong transcriptional activation domain fused to an ets type dna binding domain and thus function as aberrant transcription factors that dysregulate target genes contributing to oncogenic transformation. a number of genes that are dysregulated by ews / fli have been identified, and their roles in the oncogenic process are under active investigation [2329 ]. the presence of ews / ets translocations is specific to ewing 's sarcoma, and the presence of an ews / ets fusion protein can be used clinically to diagnose patients with ewing 's sarcoma who have small round blue cell tumors. two main cooperating mutations have been identified in ewing 's sarcoma : p53 and rb pathway mutations [3033 ]. mutations in tp53 (encoding the p53 protein) occur with a frequency of 5%20% in ewing 's sarcoma, amplifications of mdm2 occur in 0%10% of cases, and deletions of the cdkn2a locus (encoding overlapping p16 and p14 transcripts) occur in about 15% of cases [30, 32, 34, 35 ]. a similar percentage of ewing 's sarcoma tumors also have alterations in the rb pathway [30, 32, 33 ]. alterations in these pathways may be required to bypass a growth inhibitory effect mediated by the ews / ets fusion protein [36, 37 ]. although alterations in the p53 and/or rb pathways may cooperate with ews / ets fusion proteins to induce ewing 's sarcoma, this disease is not traditionally considered to be a part of the li - fraumeni syndrome and has rarely been reported as a second tumor in patients with heritable retinoblastoma [3842 ]. ewing 's sarcoma does not appear to be a component of other tumor susceptibility syndromes, either. there are no well - documented environmental causes of this disease and only a handful of epidemiological studies have focused on ewing 's sarcoma. while ewing 's sarcoma is not common, with an incidence of about 3 per one million people under 20 years of age, it remains uniformly deadly when untreated. interestingly, ewing 's sarcoma has a strong predilection for caucasians, being far more common in this population than in asians and ten times more common than in those of african descent. a molecular postulate has been proposed for the racial predilection noted : intron 6, near the molecular breakpoint region, is at least fifty percent smaller due to diminished interspersed repeat sequences (alu elements) in about 10 percent of the african population.it is hypothesized that (alu elements are preferential sites for genetic recombinations in cancer. beyond the observation of different rates by ethnicity, ewing 's sarcoma is considered to be nonfamilial, with no genetic lineage predisposition. we reviewed the english literature to find any evidence in the demographics and epidemiology of ewing 's sarcoma to suggest a familial predisposition. we considered cases of consanguinity and any onco - syndromic conditions that might imply a predisposition genotype. additional tumors beyond classic ewing 's sarcoma have been found to have similar histologic and molecular phenotypes, including the specific t(11 ; 22) translocation. ewing 's sarcoma and another small round blue cell malignancy often seen in soft tissues, termed primitive neuroectodermal tumor (pnet), were found to not only have similar histologic features but also to contain the identical translocation in greater than 95% of cases. ewing 's sarcoma family of tumors to encompass ewing 's sarcoma, pnet, as well as atypical ewing 's sarcoma and askin tumor (ewing 's sarcoma of the chest wall). because of the consistent genetic lesion, we will continue to refer to this entire group as ewing 's sarcoma. there are currently no known cancer syndromes of which ewing 's sarcoma tumors are included, and ewing 's sarcoma tumors do not seem to be associated with any other types of tumors either in pediatric or adult oncology. chronologically, ninety percent of cases occur in patients between 5 and 25 years of age. about 25% of cases occur before age 10, while 65% arise between ages 10 and 20 years old. boys and young men are affected more frequently than girls and young women. males also do less well than females. the pelvis is the most common location, followed by the femur, tibia, humerus, and scapula. however, ewing 's sarcoma can be found in any part in the body. several reports have highlighted the general association of ewing 's sarcoma and parental exposure to pesticides, solvents, and farming or agricultural occupation [4751 ]. hernia, both inguinal and umbilical have also been linked to ewing 's sarcoma [47, 48, 52, 53 ]. valery. surmised that ewing 's sarcoma and hernia have common embryologic neuroectodermal pathways. interestingly, these cases arose in farming families perhaps suggesting some unknown environmental influence the link of the two entities. at least one report discounts this association. in a case - control study, winn. matched 208 ewing 's cases with one sibling control and one age matched regional population control. although hernia was seen 6 times more frequently among ewing 's patients compared to regional controls, sibling controls experienced hernias with the same frequency as ewing 's patients. reports of patient height and onset of pubertal growth have been varied with no clear or consistent pattern developing in their association with ewing 's sarcoma [5561 ]. the ethnic epidemiology of ewing 's sarcoma is fascinating as it uniquely follows racial boundaries, similar to wilms ' tumor. ewing 's sarcoma has a strong predilection for caucasians, being far more common than in asians and those of african descent [44, 6265 ]. have noted that intron 6, near the molecular breakpoint region, is at least fifty percent smaller due to diminished interspersed repeat sequences (alu elements) in about 10 percent of the african population. alu elements are a type of sine, or short interspersed element, and are approximately 300 bp in length with approximately 1,000,000 copies in the human genome, accounting for approximately 10% of genetic material. it is hypothesized that alu elements are preferential sites for genetic recombinations in cancer. perhaps in families with ewing 's sarcoma probands that develop remote ewing 's sarcoma in their descendents, increased genomic predominance of alu elements leads to subsequent rechimerization of ews / fli and related translocations. large - scale studies on germline dna from ewing 's sarcoma patients have yet to be performed which could support this hypothesis. most recently, johnson. explored the association between parental age and risk of all childhood cancers.the previous studies had explored the association between advanced maternal and paternal age with congenital syndromes (including several which predispose to cancer) and a handful of other reports had provided preliminary support of an association between older parental age and an increased risk of some childhood cancers [68, 69 ]. johnson. followed up on these investigations and performed a pooled analysis on 17,672 childhood cancer cases diagnosed during 19802004 and 57,9666 controls born during 19702004. cancer and birth registry records from new york, washington, minnesota, texas, and california were linked, and johnson. calculated logistic regression for parental age and specific childhood cancers adjusting for sex, birth weight, gestational age, birth order, plurality, maternal race, birth year, and state. report that older maternal age seemed to increase the risk for most common childhood cancers. interestingly, ewing 's sarcoma was found to be associated with the highest risk of all childhood cancer subtypes in relation to a 5-year increase in both maternal age (odds ratio 1.18 [1.021.35 ]) and paternal age (odds ratio 1.19 [1.061.34 ]). they speculate that the increased risk of cancer in older mothers could be due to age - related increases in de novo epimutations in oocyte genes transmitted to offspring. a similar phenomenon in epimutations could be occurring in the spermatocytes of older fathers. although limited to a single pooled analysis, this large study provides intriguing data to suggest a slight but possible contribution of genetic risk to the development of ewing 's sarcoma. the association between ewing 's sarcoma and other forms of cancer seen in a proband 's pedigree has been reported, some as early as 1952.. noted that 9 of 80 patients (11%) were noted to have close family relatives, usually a grandfather or aunt, with some form of malignant tumor. in their series, eight years later the first reported incidence of ewing 's sarcoma in siblings was reported by huntington.. two sisters, each diagnosed in their teens, eventually died of metastatic disease. none of their other siblings (five boys and two girls) showed any evidence of disease. hutter. reported the case of two siblings, both female. one sister was diagnosed at age 3 and died of metastatic disease shortly thereafter. her sister was diagnosed at age 16 and at the time of reporting was alive and disease free. interestingly, their mother was treated for breast carcinoma, and their maternal grandfather died of carcinoma of the colon. joyce. reported the third case of ewing 's sarcoma in siblings in 1983. the first sibling was diagnosed at age 9 and treated successfully with chemotherapy and radiation. her sister was diagnosed at age 19 and at the time of publication was alive with pulmonary disease that seemed responsive to chemotherapy. a careful history showed no reports of neoplastic disease in the immediate or extended family. although isolated to case reports, these siblings with ewing 's sarcoma also would imply a slight but definitely suggestible contribution of genetics to the risk of developing ewing 's sarcoma. however, given their limited numbers and lack of genomic dna for analysis, environmental contributions also can not be ruled out. it is also interesting to note that these isolated siblings with familial ewing 's sarcoma were all females. finally, several authors have reported on the association of ewing 's sarcoma after diagnoses and treatment for retinoblastoma [75, 76 ].. published on a cohort of 6 ewing 's patients diagnosed after treatment for various cancers including lymphoma, leukemia, wilms tumor, and retinoblastoma., via meta - analysis, found that while ewing 's has been reported after a number of different malignancies. only the predominance of retinoblastoma prior to ewing 's differs dramatically from the low frequency of retinoblastoma among childhood cancers in the general population. in contrast, cancers other than retinoblastoma were proportionate to those in the general population. the mechanisms by which oncogenic ets fusion proteins, which are dna - binding transcription factors, target genes necessary for tumorigenesis are not well understood. analyzed promoters of these target genes and described a significant overrepresentation of highly repetitive ggaa - containing elements (microsatellites). they also reported that ews / fli uses ggaa microsatellites to regulate the expression of target genes, and that the ability to do so depends on the number of consecutive ggaa motifs. speculated that these microsatellite polymorphisms may contribute to differences in individual and population susceptibility to ewing 's sarcoma, and that this may also be true of other diseases mediated by ets transcription factors. most recently, this same group combined transcriptional analysis, whole genome localization data, and rna interference knockdown to identify glutathione s - transferase m4 (gstm4) as a critical ews / fli target gene in ewing 's sarcoma. they found that the recurrent ewing 's sarcoma translocation t(11;22) directly binds and regulates gstm4 expression through the same ggaa - microsatellite described above. microsatellite sizes differ between individuals, and so in addition to possible genetic contribution to ewing 's sarcoma susceptibility, there may be inherited differences in ewing 's sarcoma therapeutic responses. ewing 's sarcoma case - control studies analyzing microsatellite size and frequency are now required to support these findings. the epidemiological evidence supports a slight but possible genetic contribution to the risk of developing ewing 's sarcoma. however, due to its rarity, many of these studies lack statistical power to definitely prove or disprove a genetic susceptibility to this sarcoma. there is no smoking gun to suggest an underlying cancer predisposition in the majority of cases of ewing 's sarcoma. large - scale studies investigating the genetic epidemiology of ewing 's sarcoma are sorely needed to answer the question of genetic disease risk. because of its rarity, a remote familiality may have evaded detection thus far. we believe that an in depth investigation into the genetic epidemiology of ewing 's sarcoma is required to see if a predisposition gene or set of genes might contribute to this deadly disease in some subtle manner. this will only be accomplished through a stringent analysis of existing ewing 's sarcoma registries or large population databases.	ewing 's sarcoma is a highly malignant tumor of children and young adults. the molecular mechanisms that underlie ewing 's sarcoma development are beginning to be understood. for example, most cases of this disease harbor somatic chromosomal translocations that fuse the ewsr1 gene on chromosome 22 with members of the ets family. while some cooperative genetic events have been identified, such as mutations in tp53 or deletions of the cdkn2a locus, these appear to be absent in the vast majority of cases. it is therefore uncertain whether ews / ets translocations are the only consistently present alteration in this tumor, or whether there are other recurrent abnormalities yet to be discovered. one method to discover such mutations is to identify familial cases of ewing 's sarcoma and to then map the susceptibility locus using traditional genetic mapping techniques. although cases of sibling pairs with ewing 's sarcoma exist, familial cases of ewing 's sarcoma have not been reported. while ewing 's sarcoma has been reported as a 2nd malignancy after retinoblastoma, significant associations of ewing 's sarcoma with classic tumor susceptibility syndromes have not been identified. we will review the current evidence, or lack thereof, regarding the potential of a heritable condition predisposing to ewing 's sarcoma.
the frequency of cesarean section (cs) is persistently increasing all over the world. the expanding rate of cs is due to many factors including pregnancy after the age of 35 years and maternal requests. the obstetric practices such as labor induction and epidural anesthesia all have contributed to the rise in the rate of cs rate (1). studies have shown that the rate of complications associated with cs is several - fold that of vaginal delivery (2, 3). although, it occurs but reports of the rates, causes, and risk factors are lacking. gedikbasi. in 2008 reported that there are only three descriptive studies documenting re - laparotomy after cs in the obstetrics literature (4). in view of this scant literature and lack of comparative studies examining the risk and outcome of relaparotmy, the aim of the current study was to investigate risks, treatment options and outcome for relaparotomy after cesarean in a case - controlled study in this prospective study, 2500 cesarean deliveries were performed at mansoura university hospital, egypt from january 1. the decision of relaparotomy and the surgical procedure were conducted by the consultant on duty. in this study, patients were included if they had undergone relaparotomy after cs for any event which is related to the primary procedure. each patient of the study group was randomly matched to 2 cases that had undergone cesarean delivery during the same period. the demographic data we recorded included, maternal age ; parity ; number of previous abortions ; antenatal care converge, numbers of previous scars and gestational age. our variables of interest were indications for cesarean section ; indications for relaparotomy ; the type of cs ; admission to the icu ; blood transfusion ; length of hospital stay as well as fetal and maternal morbidity and mortality. for clarity, macrosomia is defined as birth weight 4 kg or greater, the diagnosis of fetal distress was on the ctg findings. this study was approved by the ethics committee of the college of medicine of mansoura university. the statistical package for the social sciences (spss 17 for windows) was used for recording and statistical analyses of data. the descriptive statistics used included the mean, the frequency distribution and the standard deviation. a chi - square test was used to compare the means of qualitative data, whereas a student 's t - test was used to compare the means of quantitative data. in multivariate analysis the results of the analysis are presented as odds ratio (or) and 95% confidant interval (95% ci). the total number of cs during the study period was 2500. of these ; 26 patients had undergone re - laparotomy. the majority 21(80.8%) were delivered by emergency cs and few 10 (38.5%) of them had antenatal coverage. relaparotomy was done on average time of 5.57691.94264 hours after the primary surgical procedure, but cases with sepsis were re - opened on an average of 3 - 8 days. the leading indication for re - laparotomy was hemorrhage in 24(92.3%) of patients (indicated by hemodynamic instability). intra - operative findings were intra - abdominal bleeding 41.7% (n=10), and hematoma (broad ligament and rectus sheath hematoma) % 29.2 (n=7). uncontrolled postpartum hemorrhage was reported in 29.2% (n=7). seven (26.9%) patients required admission to the intensive care units and the average length of hospital stay was 4.02.87054 days. the majority of patients 53.8 % (n=14) were treated by uterine arteries ligation, while 46.2 % (n=12) were treated by subtotal hysterectomy. the common complications were dic (3 cases, 11.5%), cardiac arrest (2 cases 7.7%) and bladder injury (1 case, 3.8%) demographic and obstetrical characteristics of the study and the control cases are shown in table 1. the study subjects had increased frequency of the number of previous cesarean delivery (p=.007) compared to the control group. demographic and obstetrical characteristics of the study and the control cases the principal indication for cs were, complete placenta previa (9 cases, 34.6%), macrosomia (7 cases, 26.9%), multiple pregnancy (3 cases, 11.5%), eclampsia (2 cases7.7%), fetal distress (2 cases7.7%), failure to progress in labor (2 cases7.7%) and scar dehiscent (1 case, 3.8) as shown in table 2. indications for cesarean section binary logistic regression was used to examine how well some selected obstetrics characteristics can predict relaparotomy by using the control group as a reference category as shown in table 3. stronger predictors of relaparotomy were placenta previa (or=6.898, 95% ci=1.867- 25.4, p=.004), fetal macrosomia (or=6.409, 95% ci=1.444 - 28.44, p=.015). there was no association with the number of previous cesarean delivery, and parity, p value >. in the current study, the incidence of relaparotomy after cs was found to be 1.04%. the majority of previous studies reported a rate of relaparotomy ranging between 0.2%-.7% (5, 6, 7). the high rate of re - laparotomy in our study, could be explained by poor antenatal care coverage (indicated by 61.5% non - user), consequently high rate of emergency cs which has a 4-folds increase in the rate of relaparotomy (p=.005). (6) reported that, of these 66 cases requiring relaparotomy following cesarean delivery, 63 (95.5%) had an emergency cesarean delivery. perhaps, low rates reported by some authors indicate availability of health services, good conduction of the antenatal care, selection of patients at risks for elective cs and early intervention. the principal indication for relaparotomy after cs in the current study, was hemorrhage, both intra - abdominal bleeding and uncontrolled pph accounted for 24 (92.3%). all previous studies have reported that intra - abdominal bleeding was the leading cause for relaparotomy after cs, but with different rates. a figure of 70.9% recently, kessous. (9) reported that bleeding accounted for 70% of indications for relaparotomy. bleeding secondary to uterine atony is preventable by adopting active management of the third stage of labor in women with identifiable risk factors for uterine atony by either rectal misoprostol or oxytocin infusion (10). intra - abdominal bleeding after cs depends on the skill and the experience of the operator and the surgical technique used, such factors would be difficult to discern in the current study, because this essential parameter was not evaluated. sometimes we faced difficulties in identifying the source of bleeding, especially with huge hematoma. in this study, relaparotomy was performed with within 5.5 hours after the primary procedure, agreed with previous reports (11). on the other, relaparotomy performed for sepsis required an average of 6 - 11 days after primary surgical operation. it was found that the important risks factors for infection after cs were the duration of the surgical procedure especially when exceeding 38 min and body mass index of greater than 30and the commonest site for infection is the surgical site (12). in our series both cases had pelvic abscess which was treated successfully with evacuation, drainage and antibiotics. in our study, surgical treatment received for intraabdominal bleeding were either hysterectomy in 46.2 % (n=12) and uterine arteries ligation in53.8 % (n=14). literature evaluating the indications for cs leading to re - laparotomy is scant and insufficient to make a valid comparison about this important issue. in the current study, the most important indications for cesarean delivery leading to relaparotomy were complete placenta previa with an odds ratio of 6.898 and fetal weight greater than 4 kg with an odds ratio of 6.409. (13) who concluded that placenta previa posses a high risk of morbidity and mortality for both fetal and mother among subjects underwent relaparotomy after cs. kessous reported additional risks for re - laparotomy after cs including previous cs, severe preeclampsia, uterine rupture, placental abruption, cervical tear and pph (9). (5) who reported that, placental abruption, duration of primary surgery, and the experience of the chief surgeon were significant risks for relaparotomy after cs. sak (14) in analysis of 113 cases that underwent re - laparotomy concluded that, placental abruption, hellp syndrome and previous cesarean section were the most important risks for relaparotomy. this can be explained by high rate of placenta previa which accounted for 34.6% of the indications for re - laparotomy in our study. (15) reported that the risk for hysterectomy increased by a 14-fold in patients who had 3 and more previous cs and the risk after placenta abruption and multiple pregnancy were 15.28 and 1.85 respectively. unfortunately, there were 3 maternal deaths in our report accounted for 11.5%, which is very high. (16) reported 1 case fatality after relaparotomy of the 35 studied cases. in two cases the indication for cs was placenta previa complicating previous scar resulted in intra - operative bleeding. in the third case cs placenta previa, fetal macrosomia and emergency cesarean delivery were the best predictor of relaparotomy after cs.	abstractaim : to identify risks, indications and outcomes for relaparotomy after cesarean delivery.methods:a prospective case - controlled study conducted at mansoura university hospital, egypt from 2009 to 2012. each case was matched randomly to 2 cases that had delivered by cesarean section during the same period and did not undergo repeated surgical intervention. information 's on indications were obtained to gather information 's on risks factors.results:relaparotomy complicated 1.04 % (n= 26) of the total number of the cesarean section (cs) (n=2500). the principal indications for relaparotomy were internal bleeding (intra - abdominal bleeding in 41.7% (n=10) ; rectus sheath hematoma in 29.2% (n=7) and uncontrolled postpartum hemorrhage (pph) in 29.2 % (n=7) of cases, followed by infections in 7.7% (n=2) of cases. resulting in 11.5 % (n=3) maternal death. predictors for relaparotomy after cesarean delivery from univariate logistic model, placenta previa (or=6.898, 95% ci=1.867- 25.4, p=.004), fetal weight greater than 4 kg (or=6.409, 95% ci=1.444 - 28.44,. 015). previous cesarean section and parity were not a risk for re-laparotomy.conclusion:in this study, the incidence of relaparotomy after cesarean delivery was very high (1.04%). associated with high maternal mortality (11.5%). the main predictors were placenta previa and fetal macrosomia.
	cellular progression to malignancy appears to require a number of distinct steps in which genetic damage in key regulatory genes accumulates. immortalization, or escape from senescence, is considered to be one of the first phenotypic changes. ni2 + treatment of normal human kidney epithelial (nhke) cells in vitro resulted in immortalization of the cells ihke cells). the combined action of ni2 + and v - ha - ras oncogene fully transformed the cells to tumorigenicity in athymic nude mice. sequence analysis of dna from ihke cells revealed point mutation in the p53 gene at codon 238 with t-->c transition. these findings suggest that ni - induced mutation in the p53 gene can be involved in the immortalization of the nhke cells. the results also show that changes in the responses to egf and tgf beta and in the expression of their receptors occur during malignant progression in vitro.imagesfigure 1.figure 2.
beyond its effects on cardiovascular and type 2 diabetes mellitus risk, metabolic syndrome pathogenesis involves massive release of pro - inflammatory factors (c - reactive protein, intercellular adhesion molecules, and monocyte chemoattractant protein-1), adipokines (adiponectin and resistin) and cytokines (tnf - a, il-6). excessive production of pro - inflammatory factors can decisively contribute to cancer initiation and promotion, which could explain the increased risk of hepatocellular carcinoma, and renal cell carcinoma due to obesity and metabolic syndrome. since prevalence of metabolic syndrome is increasing worldwide, it is urgent to study the role of nutritional interventions, as well as possible risk or protective factors, especially those related to the human lifestyle (diet, sleep, physical activity, fat / weight gain). in this manner, dietary intake of milk and calcium has been associated with lower prevalence and incidence of metabolic syndrome. similar protective effect of regular milk intake (at least one cup per day) regarding reduction of metabolic syndrome risk was observed in data from korean nhanes iii. a preliminary study had reported an inverse association between coffee drinking and metabolic syndrome risk. in this respect, the objective of this work was to study metabolic syndrome and its possible associated factors among a brazilian population. this descriptive and transversal case - control study covered 250 people, from 18 to 81 years old, attending at " arnulfo da cunha coutinho " public laboratory from barra do garas, mt, brazil. controls were people who had none of the clinical and laboratory criteria for metabolic syndrome. we compared 74 metabolic syndrome patients with 176-matched controls attended at a public health central unit. incident cases diagnosed according to atp iii guidelines were matched with control group composed of healthy subjects performing routine examinations. following the revised national cholesterol education program (ncep) atp iii guidelines, subjects with three or more of the following criteria were defined as having metabolic syndrome : abdominal obesity (waist circumference > 88 cm in women and > 102 cm in men) ; hypertriglyceridemia (triglycerides > 150 mg / dl ; for conversion to millimoles per liter, multiply by 0.0113) ; low high - density lipoprotein cholesterol fraction (< 40 mg / dl in men and < 50 mg / dl in women ; for conversion to millimoles per liter, multiply by 0.0259) ; high blood pressure (130/85 mmhg) ; high fasting glucose levels (100 mg / dl ; for conversion to millimoles per liter, multiply by 0.0555). blood samples were collected to determinate fasting glycemia, total cholesterol, ldl - cholesterol, hdl, urea, and triglycerides. body weight and waist circumference were measured using a digital body scale tbf-551 model (tanita, japan) and an anthropometric tape (sanny, brazil). in 2013, the brazilian minimum salary was r$678.00 which is equivalent to us$297.00 according to the ministry of work and employment (http://portal.mte.gov.br/sal_min/). before engaging into the research people received an explanation regarding the procedures and they signed a written informed consent. the study was approved by the ethics committee on research of the julio mller university hospital (hujm), from federal university of mato grosso (ufmt) (protocol. the estimation of odds ratio and data analysis were performed using the programs epicalc and epitools. considering that the distribution of sample population is approximately normal, a two - tailed 2-sample z test was used to compare sample proportions, considering at least a 5% significance level (p < 0.05). other socioeconomic and epidemiological characteristics of studied the population are presented in table 1. as expected hyperglycemia and having 40 years old or more had been associated with increased prevalence (or odds) of metabolic syndrome (table 2). having lower educational level compared to highest levels trend to increase metabolic syndrome prevalence, which was not statistical significant. interestingly, daily drinking two to three cups of coffee (p = 0.0005) or until 2 cups of milk (p = 0.0231) were inversely associated with metabolic syndrome odds. sleeping seven to eight hours per night was also associated with decreased odds of metabolic syndrome (p = 0.0001) (table 2). however, the prevalence of metabolic syndrome in central brazil reached 32%. in the current study which was performed in a central - western brazilian city, the prevalence of metabolic syndrome was similar to previous studies in brazil and korea. in a previous study, with a sample of adults from the same city, there was no association between education and metabolic syndrome, whereas lowest family income was correlated with increased odds of that disease. in the present study, a meta - analysis also reported that regular dietary intake of high - fat dairy foods was also inversely associated with obesity. according to the systematic review performed by cappuccio. some authors consider short sleep as being 5 hours, 6 hours, and 7 hours per night. anyway, sleep 7 hours or less is very harmful. many studies have been reported inverse associations between short sleep duration and metabolic syndrome risk. in the current study, exposition to seven to eight hours of sleep was associated with decreased risk of metabolic syndrome. it has been suggested that sleeping 5 hours induced -cell dysfunction and hyperglycemia as well as it provoked insulin resistance, contributing to obesity pathogenesis. sleeping 5 hours or sleep restriction (some consecutive days of shorter sleep, e.g., 5 hours per night) has also been associated inflammation, impairment of growth hormone secretion, delay on muscle glucose regulation and insulin sensitivity, oxidative stress, and endothelium dysfunction both biological mechanisms involved in cardiometabolic disorders. experimental studies with rats demonstrated that caffeine intake improved glucose tolerance, insulin sensitivity, and decreased liver steatosis, body fat, and systolic blood pressure. in an obesity rat model regular coffee drinking improved both blood glucose values and decreased expression of eight inflammatory genes. into the same approach, regular intake of coffee up - regulated mitochondrial citric acid cycle and urea cycle. both studies suggested important plausible biological anti - metabolic syndrome mechanisms. comparing people who did not eat chocolate with those who ate at least two portions of chocolate per day a decreased prevalence of metabolic syndrome was found. a systematic review and meta - analyses study suggested that chocolate consumption was associated with reduced risk of cardiometabolic diseases however, the nhlbi family health study, a transversal epidemiological design, found no association between chocolate intake and metabolic syndrome prevalence. among obese mice feeding a high - fat diet supplemented with high polyphenolic cocoa it was reported a decrease on body weight gain, insulin resistance, inflammation, and liver steatosis with concomitant increase on fecal lipid excretion. in this regard, daily intake of chocolate had been associated with decreased risk of overall cardiovascular disease (19%), coronary artery disease (23%), incident type 2 diabetes mellitus (28%), and cerebrovascular disease (32%) in humans. the present study with a small sample confirmed the data from previous studies with larger populations. notwithstanding, the sample size, the use of a food frequency questionnaire, and use a non - probabilistic sampling, were the limitations of the present work. normal sleep duration (7 - 8 hours), milk and chocolate intake, and coffee drinking were inversely associated with metabolic syndrome prevalence. but more epidemiological and experimental studies are needed.	metabolic syndrome incidence is increasing worldwide then it is important to study the possible risk and protective factors. our previous study suggested an association between coffee consumption and metabolic syndrome. the aim of this study was to address possible associations between dietary lifestyle factors with metabolic syndrome. in a case - control study we compared 74 metabolic syndrome patients with 176-matched controls attended at a public health central unit. incident cases diagnosed according to atp iii criteria were matched with control group composed of healthy subjects performing routine examinations. having lower educational level compared to highest levels tend to increase metabolic syndrome prevalence, which was not statistically significant. similar pattern was observed for marital status. no difference was found regarding gender and metabolic syndrome odds. interestingly, daily drinking two to three cups of coffee (or=0.0646, 95% ci, 0.0139 - 0.3005, p=0.0005) or until 2 cups of milk were inversely associated with metabolic syndrome odds (or=0.5368, 95% ci, 0.3139 - 0.9181, p=0.0231). sleeping seven to eight hours per night was also associated with decreased odds of metabolic syndrome (or=0.0789, 95% ci, 0.0396 - 0.1570, p<0.0001). eating at least two portions of chocolate was also associated with decreased risk of metabolic syndrome (or=0.3475, 95%ci, 0.1865 - 0.6414, p=0.0009). adequate sleeping and dietary intake of some foods materially decreased the metabolic syndrome.
the online version of this article (doi:10.1007/s10886 - 010 - 9787 - 1) contains supplementary material, which is available to authorized users. plants effectively combat herbivorous insects through direct and indirect defenses (kessler and baldwin 2002). direct defense comprises the production and storage of metabolites that negatively influence herbivore performance (wittstock and gershenzon 2002). in contrast, indirect defense encompasses the production of metabolites that benefit the natural enemies of herbivores (dicke. natural enemies such as parasitoids use herbivore - induced plant volatiles (hipvs) to locate their herbivore host and thereby indirectly aid plants in their combat against herbivorous insects. hipvs are complex blends of compounds and mainly comprise green leaf volatiles (glv) (c6 aldehydes, alcohols, and derivatives), terpenoids, and phenolics (par and tumlinson 1997 ; dicke 1999b). green leaf volatiles originate from linolenic and linoleic acid, which are released particularly when cells are damaged (bate and rothstein 1998). terpenoids are synthesized via the mevalonic acid (mva) or methylerythritol phosphate (mep) pathway (dudareva. finally, aromatic compounds, such as methyl salicylate (mesa) and indole, are formed via the shikimic acid pathway (par and tumlinson 1997). the induced volatile production is orchestrated by at least three main signal - transduction pathways : the jasmonic acid (ja), salicylic acid (sa), and ethylene (et) pathways (dicke and van poecke 2002 ; kessler and baldwin 2002). these pathways can be induced differentially by different herbivore species (heidel and baldwin 2004 ; de vos. 2005 ; schmidt. 2005), leading to the emission of a volatile blend that is specific for an herbivore species (vet and dicke 1992 ; ozawa. these herbivore - specific volatile blends can provide foraging natural enemies of herbivores, such as predators and parasitoid wasps, with detectable and reliable information to locate their prey or host respectively (dicke. variation in attraction of carnivorous arthropods towards host or non - host infested plants is ascribed mainly to the presence and relative abundance of attractive compounds within the hipv blend (dalessandro. an intriguing question is which components of the complex hipv blend affect parasitoid attraction most. up to now, several approaches have been applied to study the relative importance of certain hipvs in the attraction of carnivorous arthropods, e.g., by offering synthetic compounds alone or in mixtures, inducing certain subsets of the hipv blend with elicitors, or manipulating signal - transduction or biosynthetic pathways through a molecular genetic approach (for review see dalessandro and turlings 2006 ; snoeren. 2007 ; schroeder and hilker 2008). one hipv component for which biological relevance for carnivorous arthropod attraction has been addressed is methyl salicylate (mesa) (de boer and dicke 2004 ; de boer. 2004 ; james and price 2004 ; zhu and park 2005 ; ishiwari. this methyl ester of the plant hormone salicylic acid (sa) has been reported in the hipv blends emitted by several plant species, e.g., lima bean (dicke. 1990), tomato (ament. 2004), cabbage (geervliet 1997 ; poelman. 2009), and arabidopsis (van poecke. 2001 its role in the attraction of carnivorous arthropods has been addressed by investigating the response towards synthetic mesa in field (james and price 2004 ; zhu and park 2005) and in laboratory studies (dicke. 1990 ; de boer and dicke 2004 ; de boer. 2004 ; ishiwari. however, so far, no studies have addressed the effects of the absence of mesa from an otherwise complete hipv blend on the behavior of carnivorous arthropods. in this study, we focused on the function of mesa within the hipv blend through a molecular ecological approach that involves the elimination of mesa. mesa is synthesized by sa carboxyl methyltransferase (samt), a member of the sabath methyl transferase family, to which jasmonic acid, indole - acetic acid and cinnamic / p - coumaric acid methyltransferases also belong (seo. ; zubieta. 2003 ; kapteyn. 2007). related enzymes that methylate benzoic acid (ba) to give meba also some sabath enzymes can methylate both sa and ba with roughly equal efficiencies, and have been designated as benzoic acid and salicylic acid methyl transferases (bsmts). 2003). while samt or bsmt enzymes have been identified in a number of plant species, including fairy fans (clarkia breweri), snapdragon (antirrhinum majus), petunia (petunia hybrida), arabidopsis, and jasmine (stephanotis floribunda), we selected arabidopsis for further work on the physiological significance of mesa emission because of the availability of molecular genetic tools and because this species is a valuable stepping stone towards other brassicaceous plants for studying the role of hipvs in plant we addressed the role of mesa in the foraging behavior of the parasitoid wasp diadegma semiclausum helln (hymenoptera, ichneumonidae) that attacks caterpillars feeding on brassicaceous plants including arabidopsis. we used wild - type arabidopsis plants and a knock - out mutant that does not have a functional bsmt and, thus, no mesa biosynthesis. we addressed the effects of the mutation on parasitoid- and herbivore behavior, and on headspace composition. plants and insect material an arabidopsis line with an insertion in the atbsmt1 gene was obtained from the torrey mesa institute collection, and the position of the insertion was verified by sequencing (fig. 1, s1). arabidopsis seeds (a. thaliana wild - type columbia (col-0) and atbsmt1-ko in a col-0 background) were germinated in sandy arabidopsis soil (lentse potgrond bv, lent, netherlands), and cultivated in a growth chamber at 21 2c, 50 to 60% relative humidity (rh), and 8:16 hr, l : d photoperiod with 80 to 110 mol m sec ppfd. the soil was heated to 90c for at least 2 hr prior to sowing of the plants. two - wk - old seedlings were transferred to plastic containers (5 cm diam) filled with the same soil type. plants were watered twice a week. to prevent infestation by root - feeding sciarid flies, the soil was treated weekly with the entomopathogenic nematode steinernema feltiae (koppert biological systems, berkel en rodenrijs, the netherlands). fully grown vegetative plants were used for the experiments, i.e., at 68 wk after sowing. 1illustration of the atbsmt t - dna mutant (garlic_776_b10 line) from the torrey mesa institute s t - dna insertion mutant collection. the position of the approximately 200-nt t - dna insertion, as well as a deletion of 328-nt extending downstream from the right border of the t - dna element and including the end part of intron 2 and the beginning part of exon 3 also are shown. furthermore, we determined that transcripts from the mutant gene were still produced in the plant, and the processed mrna had a similar size to that of the wild type (wt) mrna. however, when the mutant transcript was amplified by rt - pcr and sequenced, it was seen that in the absence of the original 3 end of intron 2, a new 3 splice site was used within the remains of exon 3. this led to a protein that is much shorter and does not have samt activity (see also fig. s1)the small cabbage white butterfly, pieris rapae l. (lepidoptera, pieridae), was reared on brussels sprouts plants (brassica oleracea var. l : d ; 20 2c and 70% rh) as described in detail in fatouros. (2005).the parasitoid wasp d. semiclausum was reared on plutella xylostella on brussels sprouts in a climatized room (16:8 hr, l : d ; 20 2c and 70% rh). emerging wasps were provided ad libitum with water and honey, and are referred to as nave wasps, as they had received neither exposure to plant material, nor obtained an oviposition experience. this parasitoid is known to be attracted to the volatiles emitted by p. rapae - infested arabidopsis col-0 plants (loivamki. illustration of the atbsmt t - dna mutant (garlic_776_b10 line) from the torrey mesa institute s t - dna insertion mutant collection. the position of the approximately 200-nt t - dna insertion, as well as a deletion of 328-nt extending downstream from the right border of the t - dna element and including the end part of intron 2 and the beginning part of exon 3 also are shown. furthermore, we determined that transcripts from the mutant gene were still produced in the plant, and the processed mrna had a similar size to that of the wild type (wt) mrna. however, when the mutant transcript was amplified by rt - pcr and sequenced, it was seen that in the absence of the original 3 end of intron 2, a new 3 splice site was used within the remains of exon 3. this led to a protein that is much shorter and does not have samt activity (see also fig. s1) plant treatments plants were infested by equally distributing 20 first - instar p. rapae larvae per plant over the fully expanded leaves. uninfested plants that otherwise received a treatment similar to the infested plants were used as controls. in all experiments, plants from both genotypes that required an infestation were treated 24 hr before the experiments and kept in a climate room (21 2c, 5060% rh ; 8:16 hr, l : d photoperiod, and 80110 mol m sec ppfd). y - tube olfactometer behavioral assays the effects of hipvs from wild - type arabidopsis col-0 and transgenic atbsmt1-ko plants on parasitoid behavior were tested in a closed - system y - tube olfactometer as described by bukovinszky. in short, filtered air was led through activated charcoal and split into two air streams (4 l min) that were led through five - liter glass vessels containing the odor sources consisting on each experimental day of 4 plants each with the feeding caterpillars. the olfactometer was illuminated with 4 high - frequency fluorescent tubes (philips 840, 36 w) from above at an intensity of 60 5 mol m sec ppfd. all experiments were conducted in a climatized room (20 2c).nave, 37-d - old female d. semiclausum were transferred individually from the cage into the y - tube olfactometer on a plant leaf ; this was done by using alternately a col-0 or atbsmt1-ko leaf, that had been infested previously by p. rapae and from which the caterpillars and their products had been removed carefully. this procedure increases the general behavioral response of the parasitoids to plant cues but does not induce a shift of preference (kaiser and carde 1992 ; bleeker. parasitoid behavior and parasitoid choice for one of the two odor sources was observed and scored as described in detail by bukovinszky. parasitoids that did not explore one of the two arms of the olfactometer within 5 min or that did not make a final choice within 10 min after release were considered as non - responding individuals, and were excluded from preference analysis. after every 5 parasitoids tested, the odor sources were interchanged to compensate for any unforeseen asymmetry in the set - up. effect of atbsmt knock - out on parasitoid attraction to caterpillar - infested plants to assess the role of mesa as a cue for parasitoids in planta, we compared parasitoid behavior in response to an hipv blend that lacked mesa vs. a complete hipv blend that included mesa. caterpillar - infested mutant atbsmt1-ko and wild - type col-0 plants were offered as odor sources in the olfactometer to d. semiclausum. experiment 2. supplementing headspace of caterpillar - infested atbsmt1-ko plants with synthetic mesa to investigate further the role of mesa, we supplemented the hipv blend of atbsmt1-ko plants by adding synthetic mesa (merck, 99% pure). the supplemented hipv blend from these atbsmt1-ko plants was tested against the emitted hipv blend from atbsmt1-ko or col-0 plants. to determine if parasitoid behavior to mesa was dose dependent, different doses of mesa (0.2 g, 2 g, 20 g, 200 g) were added downwind to the earlier infested atbsmt1-ko plants. mesa was diluted in n - hexane (sigma - aldrich, 95%). in all experiments, 0.1 ml of the mesa solution was applied on filter paper (15 cm) and positioned in the last section of the olfactometer arm. a piece of filter paper with 0.1 ml hexane was placed at a similar position in the other arm as a control. the solvent was allowed to evaporate for 30 to 60 sec, after which a parasitoid was introduced into the olfactometer. new filter papers with mesa or hexane were used for each parasitoid tested in the olfactometer. caterpillar - feeding areas of consumed leaf - tissue were assessed for the caterpillar - infested col-0 and atbsmt1-ko plants that were used in experiment 1. immediately after finishing an olfactometer bioassay, individual leaves were taped on paper and scanned with a hewlett - packard scan jet 3570c. quantification of consumed leaf - tissue area was performed using winfolia pro 2006a, regent instruments (qubec, canada). headspace collection and volatile analysis dynamic headspace sampling was carried out in a climate room (20 2c, 70% rh ; 8:16 hr, l : d photoperiod and 90 to 110 mol photons m sec ppfd). twenty - four h before sampling, the pots were removed, roots and soil were carefully wrapped in aluminum foil, and 4 plants were placed together in a 2.5 l glass jar. the glass jars then were covered with insect - proof gauze. just before trapping, the gauze was removed and the jars were closed with a viton - lined glass lid having an inlet and outlet. inlet air was filtered by passing through tubes filled with 200 mg tenax ta (20/35 mesh ; grace - alltech, deerfield, mi, usa). air was sucked out of the jar with 100 ml min by passing through a tube filled with 200 mg tenax ta. fresh weights of all rosettes were determined immediately after the experiments. on each experimental day, headspace samples for two or three replicates of each treatment were collected simultaneously.headspace samples were analyzed with a thermo tracegc ultra (thermo fisher scientific, waltham, ma, usa) connected to a thermo tracedsq (thermo fisher scientific, waltham, ma, usa) quadrupole mass spectrometer. before desorption of the volatiles, the tenax cartridges were dry - purged with helium at 30 ml min for 20 min at ambient temperature to remove moisture. samples were desorbed from the cartridges by using a thermal desorption system at 250c for 3 min (model ultra markes llantrisant, uk) with a helium flow at 30 ml min. analytes were focused at 0c on an electronically - cooled sorbent trap filled with tenax and carbograph (unity, markes international ltd, llantrisant, uk) and were then transferred in splitless mode to the analytical column (rtx-5 ms, 30 m, 0.25 mm i.d., 1.0 m film thickness, restek, bellefonte, pa, usa) by rapid heating of the cold trap to 250c. the gc was held at an initial temperature of 40c for 3.5 min, followed by a linear thermal gradient of 10c min to 280c, and held for 2.5 min with a column flow of 1 ml min. mass spectra were acquired by scanning from 45400 m / z with a scan rate of 3 scans sec.compounds were identified by using the deconvolution software amdis (version 2.64, nist, usa) in combination with nist 98 and wiley 7th edition spectral libraries and by comparing their retention indices with those from the literature (adams 1995). for quantification, characteristic quantifier ions were selected for each compound of interest. metalign software (pri - rikilt, wageningen, the netherlands) was used to remove baseline noise, to align the peaks of all chromatograms of the samples, and to integrate peak areas of quantifier ions. the peak areas of all compounds were corrected for the fresh weight of the leaf rosettes. y - tube olfactometer behavioral assays parasitoid preference for infested atbsmt1-ko vs. infested col-0 plants (i.e., experiment 1) were analyzed statistically by using a chi - square test, with the null - hypothesis that parasitoids did not have a preference for any of the two odor sources. second, we analyzed whether the parasitoids exhibited a mesa - dose - dependent response when the hipv blend from atbsmt1-ko plants was supplemented with synthetic mesa (i.e., experiment 2). we constructed an overall generalized linear model (glm) including mesa dose as a covariate, the tested genotype combination as a fixed factor, and the interaction of the terms. as the null - hypothesis, we defined that addition of mesa did not affect attraction of parasitoids. total number of parasitoids that preferred infested atbsmt1-ko plants with the supplemented mesa over the control (infested col-0 or atbsmt1-ko) plants was taken as response variate. total numbers of parasitoids that made a choice per day were used as the binomial total, which were fixed on 10 except for a single case where 9 wasps responded, and we used a logit - link function. we used a chi - square test, with the aforementioned null - hypothesis, to test for a significant preference of each tested mesa dose per genotype combination. parasitoids that did not make a choice were not included in the test.third, we investigated the effects of mesa dose on the proportion of parasitoids that made a choice in the olfactometer experiments. we used the same overall glm model as described above, only now with number of non - responding parasitoids as the response variate (spss 15.0, chicago, il, usa).a mann - whitney - u test was used to test whether the consumed leaf area of col-0 and atbsmt1-ko plants (experiment 1) was different (spss 15.0, chicago, il, usa). headspace collection the fresh weight of the corrected peak area for a volatile compound quantifier ion was log10 transformed, and for each hipv, the following mixed model was used to screen for hipv compound differentiation per genotype : log10(vijk)gi+tj+g : tij+rk+ijk, where v = area of quantifier ions per gram fresh weight ; g = genotype ; t = treatment ; r = replicate ; =residual ; i=1,2 ; j=1,2 ; and k=1,,5. both g and t were used as fixed effects and r as a random effect. subsequently, two - tailed t - tests followed by a benjamini and hochberg false discovery rate (bh - fdr) multiple comparison correction were conducted per compound for the genotypes [significance : q < 0.05 ; benjamini and hochberg 1995 ]. y - tube olfactometer behavioral assays parasitoid preference for infested atbsmt1-ko vs. infested col-0 plants (i.e., experiment 1) were analyzed statistically by using a chi - square test, with the null - hypothesis that parasitoids did not have a preference for any of the two odor sources. second, we analyzed whether the parasitoids exhibited a mesa - dose - dependent response when the hipv blend from atbsmt1-ko plants was supplemented with synthetic mesa (i.e., experiment 2). we constructed an overall generalized linear model (glm) including mesa dose as a covariate, the tested genotype combination as a fixed factor, and the interaction of the terms. as the null - hypothesis, we defined that addition of mesa did not affect attraction of parasitoids. total number of parasitoids that preferred infested atbsmt1-ko plants with the supplemented mesa over the control (infested col-0 or atbsmt1-ko) plants was taken as response variate. total numbers of parasitoids that made a choice per day were used as the binomial total, which were fixed on 10 except for a single case where 9 wasps responded, and we used a logit - link function. we used a chi - square test, with the aforementioned null - hypothesis, to test for a significant preference of each tested mesa dose per genotype combination. parasitoids that did not make a choice were not included in the test.third, we investigated the effects of mesa dose on the proportion of parasitoids that made a choice in the olfactometer experiments. we used the same overall glm model as described above, only now with number of non - responding parasitoids as the response variate (spss 15.0, chicago, il, usa).a mann - whitney - u test was used to test whether the consumed leaf area of col-0 and atbsmt1-ko plants (experiment 1) was different (spss 15.0, chicago, il, usa). headspace collection the fresh weight of the corrected peak area for a volatile compound quantifier ion was log10 transformed, and for each hipv, the following mixed model was used to screen for hipv compound differentiation per genotype : log10(vijk)gi+tj+g : tij+rk+ijk, where v = area of quantifier ions per gram fresh weight ; g = genotype ; t = treatment ; r = replicate ; =residual ; i=1,2 ; j=1,2 ; and k=1,,5. both g and t were used as fixed effects and r as a random effect. subsequently, two - tailed t - tests followed by a benjamini and hochberg false discovery rate (bh - fdr) multiple comparison correction were conducted per compound for the genotypes [significance : q < 0.05 ; benjamini and hochberg 1995 ]. headspace volatile analysis to evaluate the effects of the bsmt knock - out mutation, we analyzed the headspace of uninfested and p. rapae - infested plants of wild - type col-0 and atbsmt1-ko mutant plants. for the analysis, we selected compounds in the hipv blend that are known to influence the behavior of carnivorous arthropods, e.g., methyl salicylate, hydrocarbon alcohols, and terpenoids, and compounds predicted to be affected by the ko mutation, i.e. methyl salicylate and methyl benzoate (meba) (fig. 2) (turlings and fritzsche 1999 ; dicke. 1990 ; chen. 2emission of volatile compounds of col-0 wild - type and atbsmt - ko arabidopsis plants, either uninfested or infested with 20 pieris rapae caterpillars, expressed as peak area (arbitrary units ; mean se ; n = 5) for the identifying ion per g fw. compounds : 1=1-pentanol (m / z 70) ; 2= (z)-2-penten-1-ol (m / z 57) ; 3= (z)-3-hexen-1-ol (m / z 67) ; 4= -pinene (m / z 93) ; 5= 1-octen-3-ol (m / z 57) ; 6= - myrcene (m / z 93) ; 7= (z)-3-hexen-1-ol acetate (m / z 67) ; 8= (e)- - ocimene (m / z 93) ; 9= linalool (m / z 93) ; 10= methyl benzoate (m / z 136) 11= 1-nonanol (m / z 56) ; 12= methyl salicylate (m / z 120) ; 13= ethyl salicylate (m / z 120) ; 14= indole (m / z 117) ; 15= (e, e)--farnesene (m / z 93) ; 16= (e, e)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (tmtt) (m / z 69). bars marked with indicate a treatment for a genotype significantly emitting more volatiles than its opposite uninfested or infested genotype. bars marked with arrows represent compounds emitted in significantly different amounts by the two genotypesinfested col-0 and atbsmt1-ko plants differed significantly in the emission of mesa, ethyl salicylate (etsa) and meba (q < 0.001) ; these compounds were induced in col-0 but not in atbsmt1-ko. thus, the headspace analysis for infested atbsmt1-ko and infested col-0 showed that the production of meba (no. these compounds are emitted from atbsmt1-ko at 14, 59, and 17 times lower emission rates, respectively (fig. 2).several other compounds1-pentanol, linalool, indole, (e, e)--farnesene, and (e, e)-4,8,12trimethyltrideca-1,3,7,11tetraene (tmtt)similarly were induced in infested col-0 and atbsmt1-ko plants when compared to uninfested plants. the green leaf volatile (z)-3-hexen-1-ol was induced significantly in only the mutant (q = 0.005) but not in the wild - type. uninfested col-0 and atbsmt1-ko plants did not differ in the emission of volatiles, except for tmtt, which was emitted in somewhat larger amounts by uninfested col-0 plants than by uninfested atbsmt1-ko plants (q = 0.011). finally, uninfested col-0 plants emitted more 1-octen-3-ol and 1-nonanol than infested plants (fig. 2). emission of volatile compounds of col-0 wild - type and atbsmt - ko arabidopsis plants, either uninfested or infested with 20 pieris rapae caterpillars, expressed as peak area (arbitrary units ; mean se ; n = 5) for the identifying ion per g fw. compounds : 1=1-pentanol (m / z 70) ; 2= (z)-2-penten-1-ol (m / z 57) ; 3= (z)-3-hexen-1-ol (m / z 67) ; 4= -pinene (m / z 93) ; 5= 1-octen-3-ol (m / z 57) ; 6= - myrcene (m / z 93) ; 7= (z)-3-hexen-1-ol acetate (m / z 67) ; 8= (e)- - ocimene (m / z 93) ; 9= linalool (m / z 93) ; 10= methyl benzoate (m / z 136) 11= 1-nonanol (m / z 56) ; 12= methyl salicylate (m / z 120) ; 13= ethyl salicylate (m / z 120) ; 14= indole (m / z 117) ; 15= (e, e)--farnesene (m / z 93) ; 16= (e, e)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (tmtt) (m / z 69). bars marked with indicate a treatment for a genotype significantly emitting more volatiles than its opposite uninfested or infested genotype. bars marked with arrows represent compounds emitted in significantly different amounts by the two genotypes experiment 1. effect of atbsmt knock - out on parasitoid attraction to caterpillar - infested plants diadegma semiclausum females are attracted to the headspace of p. rapae - infested arabidopsis col-0 plants (loivamki. 2008) in which mesa is an induced volatile compound (van poecke. 2001). to investigate whether changes in this headspace, as a result of a knock - out mutation in the atbsmt1 gene, affected parasitoid attraction, we investigated the behavioral responses of d. semiclausum towards plant volatiles induced by p. rapae herbivory in a y - tube olfactometer. females of d. semiclausum preferred the volatiles emitted by herbivore - infested atbsmt1-ko plants over those emitted by infested col-0 plants (p < 0.05 ; fig. 3behavioral responses of nave diadegma semiclausum females to volatiles of two sets of arabidopsis thaliana plants (col-0 vs. atbsmt1-ko), as assessed in the y - tube olfactometer. all plants were infested (inf.) with 20 pieris rapae caterpillars and in some cases the headspace was supplemented with synthetic methyl salicylate (mesa), added downwind from the plants. added mesa - doses (g) are indicated to the right of the bars in the left bar plot. each bar represents the percentage of choices for each of the two odor sources as determined in five replicate experiments ; on each replicate day 10 parasitoids were tested per odor source (x test, p values). generalized linear model analysis for experiments b and c, demonstrated that mesa dosage (p < 0.001) and not the offered genotype (p = 0.167) explained parasitoid behavior. parasitoid mesa dosage responses between two tested genotype combinations did not differ (p = 0.270). the right bar plots indicate the percentage of no choice in each experiment ; total number of tested parasitoids are given in these barsthe amount of leaf - tissue consumed by the caterpillars did not differ between col-0 and atbsmt1-ko plants (mean se : col-0 10.41 0.91 mm, atbsmt1-ko 10.88 0.80 mm ; mann whitney u test : u = 259.00 ; p = 0.90, n = 23). thus, the difference in attraction can not be explained by a difference in the amount of feeding by the caterpillars. behavioral responses of nave diadegma semiclausum females to volatiles of two sets of arabidopsis thaliana plants (col-0 vs. atbsmt1-ko), as assessed in the y - tube olfactometer. all plants were infested (inf.) with 20 pieris rapae caterpillars and in some cases the headspace was supplemented with synthetic methyl salicylate (mesa), added downwind from the plants. added mesa - doses (g) are indicated to the right of the bars in the left bar plot. each bar represents the percentage of choices for each of the two odor sources as determined in five replicate experiments ; on each replicate day 10 parasitoids were tested per odor source (x test, p values). generalized linear model analysis for experiments b and c, demonstrated that mesa dosage (p < 0.001) and not the offered genotype (p = 0.167) explained parasitoid behavior. parasitoid mesa dosage responses between two tested genotype combinations did not differ (p = 0.270). the right bar plots indicate the percentage of no choice in each experiment ; total number of tested parasitoids are given in these bars experiment 2. supplementing headspace of caterpillar - infested atbsmt1-ko plants with synthetic mesa to assess whether the absence of mesa in the hipv blend can explain the effect on parasitoid behavior, the volatile blend of infested atbsmt1-ko plants was supplemented by adding synthetic mesa downwind of the plant. different mesa doses were used to test for dose - dependent effects of mesa presence. the addition of mesa eliminated the preference for the knock - out plants, as seen in experiment 1, and with increasing mesa dose this effect was stronger. parasitoid preference was not influenced by the different genotype combinations, but solely by the mesa dose used (glm : genotype combination p = 0.167, mesa dose p < 0.001, r = 0.28). analysis of parasitoid choices for each tested mesa dose within a tested genotype combination demonstrated that parasitoid preferences slightly varied among the mesa doses used. yet, for both genotype combinations tested, atbsmt1-ko hipv complementation with 0.2 g mesa did not result in discrimination between the two odor sources and a complementation with 200 g mesa resulted in significant preference for the odor without supplemented mesa (fig. when parasitoids were offered a choice between volatiles from infested atbsmt1-ko plants supplemented with 2 or 20 g mesa vs. infested col-0 hipvs, they preferred the latter. in contrast, no discrimination between odors from infested atbsmt1-ko plants supplemented with 2 or 20 g mesa vs. infested atbsmt1-ko plants was observed.the addition of mesa negatively influenced the proportion of parasitoids that made a choice for one of the two odor sources. analysis of the number of wasps that did not make a choice for one of the two odor sources, showed no effect of the offered genotype combination, but only an effect of the mesa dose used (glm : genotype combination p = 0.75, mesa dose p = 0.010, r = 0.15). experiment 1. effect of atbsmt knock - out on parasitoid attraction to caterpillar - infested plants diadegma semiclausum females are attracted to the headspace of p. rapae - infested arabidopsis col-0 plants (loivamki. 2008) in which mesa is an induced volatile compound (van poecke. 2001). to investigate whether changes in this headspace, as a result of a knock - out mutation in the atbsmt1 gene, affected parasitoid attraction, we investigated the behavioral responses of d. semiclausum towards plant volatiles induced by p. rapae herbivory in a y - tube olfactometer. females of d. semiclausum preferred the volatiles emitted by herbivore - infested atbsmt1-ko plants over those emitted by infested col-0 plants (p < 0.05 ; fig. 3behavioral responses of nave diadegma semiclausum females to volatiles of two sets of arabidopsis thaliana plants (col-0 vs. atbsmt1-ko), as assessed in the y - tube olfactometer. all plants were infested (inf.) with 20 pieris rapae caterpillars and in some cases the headspace was supplemented with synthetic methyl salicylate (mesa), added downwind from the plants. added mesa - doses (g) are indicated to the right of the bars in the left bar plot. each bar represents the percentage of choices for each of the two odor sources as determined in five replicate experiments ; on each replicate day 10 parasitoids were tested per odor source (x test, p values). generalized linear model analysis for experiments b and c, demonstrated that mesa dosage (p < 0.001) and not the offered genotype (p = 0.167) explained parasitoid behavior. parasitoid mesa dosage responses between two tested genotype combinations did not differ (p = 0.270). the right bar plots indicate the percentage of no choice in each experiment ; total number of tested parasitoids are given in these barsthe amount of leaf - tissue consumed by the caterpillars did not differ between col-0 and atbsmt1-ko plants (mean se : col-0 10.41 0.91 mm, atbsmt1-ko 10.88 0.80 mm ; mann whitney u test : u = 259.00 ; p = 0.90, n = 23). thus, the difference in attraction can not be explained by a difference in the amount of feeding by the caterpillars. behavioral responses of nave diadegma semiclausum females to volatiles of two sets of arabidopsis thaliana plants (col-0 vs. atbsmt1-ko), as assessed in the y - tube olfactometer. all plants were infested (inf.) with 20 pieris rapae caterpillars and in some cases the headspace was supplemented with synthetic methyl salicylate (mesa), added downwind from the plants. added mesa - doses (g) are indicated to the right of the bars in the left bar plot. each bar represents the percentage of choices for each of the two odor sources as determined in five replicate experiments ; on each replicate day 10 parasitoids were tested per odor source (x test, p values). generalized linear model analysis for experiments b and c, demonstrated that mesa dosage (p < 0.001) and not the offered genotype (p = 0.167) explained parasitoid behavior. parasitoid mesa dosage responses between two tested genotype combinations did not differ (p = 0.270). the right bar plots indicate the percentage of no choice in each experiment ; total number of tested parasitoids are given in these bars experiment 2. supplementing headspace of caterpillar - infested atbsmt1-ko plants with synthetic mesa to assess whether the absence of mesa in the hipv blend can explain the effect on parasitoid behavior, the volatile blend of infested atbsmt1-ko plants was supplemented by adding synthetic mesa downwind of the plant. different mesa doses were used to test for dose - dependent effects of mesa presence. the addition of mesa eliminated the preference for the knock - out plants, as seen in experiment 1, and with increasing mesa dose this effect was stronger. parasitoid preference was not influenced by the different genotype combinations, but solely by the mesa dose used (glm : genotype combination p = 0.167, mesa dose p < 0.001, r = 0.28). analysis of parasitoid choices for each tested mesa dose within a tested genotype combination demonstrated that parasitoid preferences slightly varied among the mesa doses used. yet, for both genotype combinations tested, atbsmt1-ko hipv complementation with 0.2 g mesa did not result in discrimination between the two odor sources and a complementation with 200 g mesa resulted in significant preference for the odor without supplemented mesa (fig. when parasitoids were offered a choice between volatiles from infested atbsmt1-ko plants supplemented with 2 or 20 g mesa vs. infested col-0 hipvs, they preferred the latter. in contrast, no discrimination between odors from infested atbsmt1-ko plants supplemented with 2 or 20 g mesa vs. infested atbsmt1-ko plants was observed.the addition of mesa negatively influenced the proportion of parasitoids that made a choice for one of the two odor sources. analysis of the number of wasps that did not make a choice for one of the two odor sources, showed no effect of the offered genotype combination, but only an effect of the mesa dose used (glm : genotype combination p = 0.75, mesa dose p = 0.010, r = 0.15). variation in the attraction of carnivorous arthropods to hipvs often is ascribed to the relative contribution of attractive compounds within the complex herbivore - induced blend (van den boom. plant species can differ in the relative emission rates of individual attractive compounds in response to feeding by distinct herbivores. the commonly induced attractive compounds comprise green leaf volatiles (glv), terpenoids, phenyl propanoids, and benzenoids. several studies already have shown in planta the importance of some glv and terpenoids in attracting carnivorous arthropods (kappers., empirical evidence for the quantitative importance of single compounds in the total hipv blend of infested plants is still rare (but see de boer and dicke 2004). the parasitoid d. semiclausum is attracted to the headspace of p. rapae - infested col-0 plants (loivamki. 2008), in which mesa is an induced volatile (van poecke. 2001). here, we studied the effect of mesa on preference for volatile blends in the parasitoid d. semiclausum. the newly - available arabidopsis knock - out mutant for benzoic acid and salicylic acid carboxyl methyltransferase (atbsmt1-ko) (fig. 1, fig. s1), allowed us to study in planta the ecological effects of an hipv mixture lacking wild type levels of mesa. our data show clearly that caterpillar - infested atbsmt1-ko plants attract more parasitoids than infested wild - type col-0 plants (see fig. thus, although the total hipv blend from wildtype plants strongly attracts d. semiclausum parasitoids (loivamki. 2008), a genotype that does not emit mesa in response to caterpillar infestation is even more attractive to the parasitoids. these data indicate that mesa does not contribute to the attraction of nave d. semiclausum females but instead results in a reduced attractiveness. this negative effect of mesa on d. semiclausum attraction was not anticipated, as mesa is commonly induced after herbivory in many plant species, e.g., in lima bean (dicke. ; chen. 2003). moreover, the salicylic acid - deficient arabidopsis mutant nahg is less attractive to cotesia rubecula parasitoids upon infestation by p. rapae, which suggests an attractive role of mesa for this parasitoid species (van poecke and dicke 2002). furthermore, synthetic mesa has also been demonstrated to attract other carnivore species, such as predatory mites, lacewings, and mirid bugs (dicke. 1990 ; de boer and dicke 2004 ; james and price 2004). however, in our approach we tested the role of mesa within the total hipv - blend, by using atbsmt1-ko plants with a hampered mesa production. by additionally testing the response of the wasps to an hipv blend from atbsmt1-ko plants that we had supplemented with synthetic mesa against hipvs from col-0 plants and atbsmt1-ko plants moreover, the repellent effect of mesa was dose - dependent, and this was reflected in the response level of the parasitoids, as fewer wasps move upwind to make a choice between odor sources with higher mesa - doses. additionally, the ko - mutation did not affect the feeding rate of the caterpillars. thus, the change in volatile emission and parasitoid attraction can not be attributed to effects of the ko - mutation. for example, an hipv fraction of maize, containing (z)-3-hexen-1-ol acetate, linalool, and (3e)-4,8-dimethyl-1,3,7-nonatriene (dmnt) was found attractive compared to other tested fractions of the hipv blend of maize to the parasitoid cotesia marginiventris (turlings and fritzsche 1999). for the same plant - herbivore system, nave microplitis rufiventris parasitoids preferred hipv blends lacking the induced compound indole (dalessandro. 2006). compounds that are repellent also may affect or override the attractiveness of other compounds in the headspace. isoprenoids have, for example, been found to interfere with host - finding, as demonstrated for transgenic arabidopsis plants emitting isoprene (loivamki. therefore, to determine whether a hampered enzyme activity for samt and bamt affected the headspace composition in other respects, we also analyzed the headspace of infested knock - out mutant and wild - type plants. we observed that the headspace of caterpillar - infested col-0 and atbsmt1-ko plants differed only in the emission of mesa, meba, and etsa. yet, all other investigated hipvs were emitted at similar rates by these infested plants (fig. 2). the emission rates of the compounds whose rates were affected, were up to 60 times lower for atbsmt1-ko plants compared to wild - type plants (fig. 2). this agrees with the reported activity of the enzyme encoded by the bsmt1 gene (chen. the very low emission of mesa that remains may be ascribed to activity of another sabath enzyme that shows low levels of activity towards sa and ba (chen. 2003). eliminating a functional bsmt1 gene also decreased the emission rate of etsa. 2004) and is perceived by insect chemoreceptors (ramachandran. 1990 ; reinecke. whether meba and etsa also affect the attraction of d. semiclausum wasps remains to be investigated. we investigated the role of mesa by eliminating it from the total hipv blend rather than by investigating its role as an isolated compound or as an addition to an artificial blend (see e.g., de boer and dicke 2004). we thus demonstrated that it has a repellent effect on the behavior of nave d. semiclausum parasitoids. this finding provides another view on the fitness effects of individual components of herbivore - induced plant volatile blends that do not lure nave carnivores but rather repel them. an alternative function of hipv emitted compounds can be to repel herbivores (dicke 1986 ; bernasconi. 1998 ; kessler and baldwin 2001 ; bruce. 2008 ; piesik. 2008), which also has been demonstrated for mesa (hardie. 1994 ; james and price 2004 ; prinsloo. 2007 ; mesa may activate disease resistance and the expression of defense related genes in neighboring plants and in healthy tissue of infected plants (shulaev. therefore, the emission of mesa also could benefit the plant through protecting it at herbivory - derived wounds against infectious pathogens, as has been reported for several glvs (brown. plant signaling role for mesa (ozawa. 2000 ; james and price 2004), as well as indications for a priming effect of mesa on plant defense (turlings and ton 2006). a few studies indicate that parasitoids can learn to respond to individual compounds following a learning experience with an odor mixture (meiners. this suggests that an innate response of d. semiclausum to avoid mesa - emitting plants could potentially turn into attraction after a learning experience in the presence of a host. in summary, our study provides evidence for an hipv component that makes a plant less attractive to a carnivorous insect. this should be seen in the context of the different selection pressures on a plant s emission of volatiles in a multitrophic context. understanding these selection pressures will provide insight into the role of induced volatiles in the biology of plants (dicke and baldwin 2010).	the indirect defense mechanisms of plants comprise the production of herbivore - induced plant volatiles that can attract natural enemies of plant attackers. one of the often emitted compounds after herbivory is methyl salicylate (mesa). here, we studied the importance of this caterpillar - induced compound in the attraction of the parasitoid wasp diadegma semiclausum by using a mutant arabidopsis line. pieris rapae infested atbsmt1-ko mutant arabidopsis plants, compromised in the biosynthesis of mesa, were more attractive to parasitoids than infested wild - type plants. this suggests that the presence of mesa has negative effects on parasitoid host - finding behavior when exposed to wild - type production of herbivore - induced arabidopsis volatiles. furthermore, in line with this, we recorded a positive correlation between mesa dose and repellence of d. semiclausum when supplementing the headspace of caterpillar - infested atbsmt1-ko plants with synthetic mesa.electronic supplementary materialthe online version of this article (doi:10.1007/s10886 - 010 - 9787 - 1) contains supplementary material, which is available to authorized users.
it is the most common nonobstetric indication for antepartum hospitalization, and its associated risk factors, diagnosis, and management in the antepartum period are well described [14 ]. sepsis and septic shock occur secondary to pyelonephritis more frequently than secondary to any other infectious process during pregnancy. acute respiratory distress syndrome complicates approximately 18.5% of pyelonephritis cases [6, 7 ]. while the implications of pyelonephritis in the antepartum period are well described, there is little data about outcomes and complications when pyelonephritis occurs at the time of delivery [13, 811 ]. as these studies describe outcomes of pregnancies complicated by pyelonephritis, none of these describe specifically outcomes of patients who deliver during the admission during which they were diagnosed with pyelonephritis. the objective of this study was to expand on these studies, using a large nationwide data set, to estimate the medical, infectious, and obstetric complications associated with pyelonephritis at delivery. the nationwide inpatient sample (nis) from the healthcare cost and utilization project of the agency for healthcare research and quality (ahrq) for years 20082010 was queried for all delivery discharges. the nis is the largest all - payer inpatient database in the united states and contains data from approximately 8 million hospital admissions from over 1,000 hospitals in 42 states (2008) to 45 states (2010). the hospitals in the nis are stratified based on ownership, bed size, teaching status, urban / rural location, and region. from each stratum, the nis contains approximately 20% of united states (us) hospitals, and the sampling frame comprises 90% of all us hospital discharges. the information included in the nis is similar to that in a typical discharge summary with safeguards to protect the privacy of individual patients, physicians, and hospitals. weighting estimates are included for each hospitalization that allows for national estimates to be made. using the nis for each of the years 20082010 a delivery admission was defined as a hospitalization that included a delivery code (international classification of diseases, 9th revision, clinical modification [icd-9-cm ] codes 74 for cesarean section ; 72, 73, and 75 for vaginal delivery ; v27 or 650659 for nonspecific delivery codes) [13, 14 ]. drg codes 765 and 766 were utilized to identify cesarean deliveries and codes 767, 768, 774, and 775 for vaginal deliveries. the icd-9-cm codes used to identify discharges with pyelonephritis were 590.0590.9. to identify comorbidities, both the icd-9-cm code for a particular condition in pregnancy and the general icd-9-cm codes were used (supplemental table 1 of the supplementary material available online at http://dx.doi.org/10.1155/2013/124102 for list of icd-9-cm codes used). logistic regression analyses were used to compute odds ratios with 95% confidence intervals for age, race / ethnicity, medical conditions and events, and pregnancy - related complications among women with pyelonephritis compared to women without pyelonephritis. a multivariable logistic regression model was created controlling for age, insurance status, income quartile of residing zip code, and the studied preexisting medical conditions to identify the odds of various medical and obstetric complications among women with pyelonephritis compared to women without pyelonephritis. race / ethnicity was not included in the final model due to the large number of entries in the nis with missing race / ethnicity data. statistical significance for all analyses analyses were performed using sas version 9.3 (sas institute inc., cary, nc) and graphpad prism 6.0 for macintosh (graphpad software, san diego, ca). this study protocol was reviewed and approved by the duke university medical center institutional review board as exempt research. during the period from 2008 through 2010, there were estimated 12,628,746 deliveries in the nis, of those, 26,397 patient records included a diagnosis of pyelonephritis at a delivery discharge. the overall rate of pyelonephritis during delivery admission for the years 2008 through 2010 was 2.1 per 1000 deliveries. women with pyelonephritis during the delivery admission were more likely to be african american or hispanic, were younger, less likely to have private insurance, and were more likely to reside in a zip code whose median income was in the lowest quartile (table 1). race / ethnicity data was missing for 13.8% of women with pyelonephritis and for 15.5% of women without pyelonephritis. women with pyelonephritis during the delivery admission were more likely to have cardiomyopathy, asthma, diabetes, systemic lupus erythematosus, anemia, thrombocytopenia, sickle cell disease / thalassemia, or chronic renal failure compared to women without pyelonephritis. women with pyelonephritis during the delivery admission were also more likely to use drugs, alcohol, or tobacco compared to women without pyelonephritis. women with pyelonephritis had increased odds of acute heart failure, deep vein thrombosis, pulmonary edema, and acute renal failure compared to women without pyelonephritis. women with pyelonephritis at a delivery admission had increased odds of pneumonia, acute respiratory distress syndrome, and sepsis compared to women without pyelonephritis. in addition, women with pyelonephritis were more likely to have a transfusion and require mechanical ventilation compared to women without pyelonephritis (table 3). women with pyelonephritis were more likely to be carrying a multiple gestation or have their pregnancy complicated by preterm labor or chorioamnionitis compared to women without pyelonephritis. cesarean delivery, gestational diabetes, preeclampsia / eclampsia / gestational hypertension, premature rupture of the membranes, and postpartum hemorrhage were all less common among women with pyelonephritis compared to women without pyelonephritis at the time of delivery admission (table 4). table 5 outlines the adjusted analysis for various medical and obstetric complications at delivery while controlling for age, insurance status, income of residing zip code, and pre - existing medical conditions in women who had pyelonephritis at the time of delivery. most significantly, sepsis was 108 times more likely to occur among women with pyelonephritis compared to women without pyelonephritis. other infectious complications remained significantly associated with pyelonephritis, including pneumonia, acute renal failure, pulmonary edema, and acute respiratory distress syndrome in our adjusted model. multiple gestation, chorioamnionitis, and preterm labor were also more common in the pyelonephritis population. pyelonephritis is the most common nonobstetric indication for antepartum hospitalization and is a common source of maternal and neonatal morbidity. here we reconfirm several well - known preexisting medical conditions that have been associated with pyelonephritis during pregnancy and further demonstrate that women with pyelonephritis at delivery admission are at increased risk for severe infectious complications, such as pneumonia, ards, and sepsis, and are also at increased risk for transfusion, requiring mechanical ventilation, acute heart failure, pulmonary edema, and acute renal failure. as our study utilized data from a large database representing the entire united states, the conclusions and findings from our analysis largely validate findings from previously noted smaller, single - center studies. it is well known that women who develop pyelonephritis during pregnancy are at increased risk of sepsis and ards and require icu admission compared to nonpregnant women who develop pyelonephritis [13, 5, 15 ]. using the nis, it became evident that these women are at markedly increased risk for these and other complications. women with pyelonephritis during the delivery are over 100 times more likely to have sepsis than women who do not have pyelonephritis. pneumonia was 18 times more likely in women with pyelonephritis, and ards and pulmonary edema were both 11 times more likely to occur in this population. there was no difference in the risk for pulmonary emboli or deep vein thrombosis, suggesting an inflammatory mechanism for lung injury not a coagulation - related mechanism. these findings illustrate that women with pyelonephritis at delivery are at risk for significant morbidity. utilized the nis from a single year, 2006, to identify demographic data among women admitted at any time during pregnancy with pyelonephritis. similar to our study, they found that women less than 20 and women of african american or hispanic race / ethnicity were at higher risk for pyelonephritis in pregnancy, though they did not compare these demographics to women without pyelonephritis. they found that women with pyelonephritis had associated risk for sepsis (2.0%), anemia (22.4%), diabetes (3.7%), and preterm labor (3.8%), but again, they did not determine the rate of these outcomes among women without pyelonephritis. in our study, by analyzing data only at admissions for delivery, rather than at antepartum admission, we were able to estimate outcomes for individuals, rather than hospitalizations, as women could possibly have more than one antepartum hospitalization during a pregnancy but only a single hospitalization per woman per pregnancy. additionally, by examining discharge data, obstetric outcomes data could be captured, which would not be available using antepartum discharges. lastly, we were able to calculate ors for associated complications among women with pyelonephritis since we also determined the rates of these events in pregnant women at delivery without pyelonephritis. our findings rely on a large discharge database, and therefore we are unable to determine if the associated preexisting medical conditions caused pyelonephritis, or if pyelonephritis was causative of the observed medical and obstetric events that were more common among women with pyelonephritis. next, there is no way to ascertain if coded preexisting medical conditions were active at the time of delivery. furthermore, preexisting conditions or medical / obstetric events occurring at delivery would have been missed if the variable was not coded. finally, neonatal discharge records are not linked to maternal discharge records within the nis ; thus, we are unable to provide information on neonatal outcomes among those neonates born to women with pyelonephritis. yasmeen. recently assessed the accuracy and reliability of obstetric discharge databases and found them to provide helpful information. thus, despite the limitations of the nis database, the study design allows the examination or relatively rare disorders using a large numbers of pregnancies. in doing so, odds ratios and maternal morbidity rates for medical conditions, pregnancy - related conditions, and pregnancy outcomes that would have otherwise been difficult to quantify can be estimated. our multivariable logistic regression model attempted to identify an independent association of pyelonephritis with various medical and obstetric outcomes. in doing so, we controlled for clinically relevant preexisting medical conditions and social factors that are known to be commonly associated with pyelonephritis. in using a database such as the nis, it is impossible to account for all potential confounders, and as such we acknowledge this limitation. in summary, this dataset reaffirms many of the known risk factors for pyelonephritis, including minority race, young age, lower socioeconomic status, nongestational diabetes, and sickle cell disease, but also uniquely examines preexisting medical conditions and medical and obstetric complications among women with pyelonephritis occurring at delivery. many women with pyelonephritis at delivery were found to have other associated infectious complications such as sepsis, pneumonia, and acute respiratory distress syndrome. it is unknown if pyelonephritis was directly causative of these outcomes, and future research is warranted.	objective. pyelonephritis is a common cause of antepartum admission and maternal morbidity. medical complications associated with pyelonephritis during delivery are not well described ; thus the objective of this study was to estimate medical, infectious, and obstetric complications associated with pyelonephritis during the delivery admission. study design. we conducted a retrospective cohort study using the nationwide inpatient sample (nis) for the years 20082010. the nis was queried for all delivery - related discharges. during the delivery admission, the icd-9-cm codes for pyelonephritis were used to identify cases and were compared to women without pyelonephritis. a multivariable logistic regression model was constructed for various medical, infectious, and obstetric complications among women with pyelonephritis compared to women without, while controlling for preexisting medical conditions and demographics. results. during the years 20082010, there were 26,397 records with a diagnosis of pyelonephritis during the delivery admission, for a rate of 2.1 per 1000 deliveries. women with pyelonephritis had increased associated risks for transfusion, need for mechanical ventilation, acute heart failure, pneumonia, pulmonary edema, acute respiratory distress syndrome, sepsis, acute renal failure, preterm labor, and chorioamnionitis, while controlling for preexisting medical conditions. conclusions. pyelonephritis at delivery admissions is associated with significant medical and infectious morbidity.
biomarkers of alzheimer s disease (ad) are important for both early diagnoses and evaluating treatment effects. three decades of research indicate that proton magnetic resonance spectroscopy (mrs) is a potential biochemical imaging marker in ad. the focus of this review is to discuss the role of proton mrs in alzheimer s disease. mrs allows regional measurement of metabolites including myo - inositol (mi), choline (cho), n - acetyl aspartate (naa), and creatine (cr). cr is typically used as an internal reference to control for variability in measurement because it remains unchanged in ad.13 other metabolites that can be measured with proton mrs with advanced mrs sequences and post - processing methods include gamma - aminobutyric acid (gaba) and glutathione which may not be available in conventional clinical scanners and are not the focus of this review. mrs may serve to identify patients with ad before clinical symptom onset as well as help distinguish ad from other neurodegenerative disorders. in 1992, klunk demonstrated a decrease in the neuronal metabolite naa on mrs on autopsy brain samples of patients with ad compared to controls. the lower naa level correlated with the amount of plaque and tangle pathology.4 naa is considered a neuronal marker and is synthesized in mitochondria.5 supporting the notion that naa levels correspond to neuronal integrity, reduced naa levels in cortical tissue from patients with ad demonstrated a correlation between naa concentration and neuronal density.6 decreased naa seen in head trauma, seizure, or coronary artery bypass surgery can resolve after recovery.5,79 further, naa levels decreased in ad show transient improvement with acetylcholinesterase inhibitor treatment.10 the neurofibrillary tangles pathology of ad follow a typical progression from limbic to neocortical areas as ad advances.11 similarly, changes in naa follow a regional pattern as disease advances. for example, ad patients show a regional decrease in naa / cr ratio in the superior temporal lobe and posterior cingulate voxels compared to controls, but mild cognitive impairment (mci) patients do not show a decline in naa / cr as ad - related neurodegeneration has not yet extended to these regions.12 medial occipital lobe ad pathology involvement typically occurs at the final stage of the disease.11 therefore, it is not surprising that there is no regional decrease in naa / cr in mild ad in the occipital lobe while more advanced cases demonstrate decrease naa / cr ratio.1214 eventually, naa changes become widespread and have been shown to involve the parietal, temporal, and frontal lobe.12,1519 decreased naa / cr ratios are non - specific and can be seen in other types of dementia including normal pressure hydrocephalus and have even been reported in cognitive decline associated with acquired immunodeficiency syndrome (aids).20,21 in 1993, it was demonstrated that in addition to decreased naa levels, ad patients have elevated myo - inositol to creatine (mi / cr) levels.13 while naa is a neuronal marker, mi is associated with glia and elevated levels with glial proliferation.22,23 the finding of decreased naa and elevated mi in ad has been confirmed in several studies.12,16,24 the role of choline metabolite in ad is more controversial. a number of studies demonstrated increased choline in ad.2527 other studies have demonstrated no change in choline concentration in ad compared to controls.2831 one study reported a decrease of choline / h20 in the medial temporal lobes of ad patients.32 brain choline is concentrated in phospholipids. the choline peak in mrs represents cytosolic glycerophosphocholine and phosphocholine which are breakdown products of phosphatidylcholine.33 therefore the larger choline peak may be due to increased membrane turnover. also, it has been proposed that catabolism of the phospholipid membrane bilayer allows ad subjects to produce choline to compensate for declining acetylcholine.34 administration of xanomeline, an m1 selective muscarinic cholinergic agonist, to ad patients resulted in a significant decline in cho / cr ratios, perhaps representing a reduction in compensatory mechanisms to produce acetylcholine through phospholipid breakdown.35 cho / cr increases in amnestic mci if it progresses to ad but the ratio decreases if cognition remains stable.36 decreased glutamate (glu) or glutamate + glutamine / cr ratio has also been found in the grey matter of ad patients, but not the white matter.3740 furthermore, increased glu and the ratio of glu to cr measured from the hippocampus by mrs after galantamine treatment were associated with increased cognitive performance.41 mrs studies in transgenic mice with ad mutations have shed light on the pathological correlates of metabolite changes seen in ad. transgenic mice with ad mutations demonstrate similar decreased naa and increased mi as seen in human ad patients.6,42 in addition, lower naa and glutamate levels correlate with amyloid beta (a) plaque load in the frontal cortex of mice with ps2app mutation.43 further, the mrs metabolite changes consistent with ad precede overt cognitive dysfunctions in early - stage ad.44 the temporal progression of metabolite abnormalities in ad are characterized by an increased mi / cr followed by a decrease in naa / cr and an increase in cho / cr.12 a recent study of pathologic correlates of mrs metabolite changes in cases of varying ad pathology demonstrated that antemortem naa / cr and mi / cr levels correlate with the pathologic severity of ad, and that the strongest predictor of ad pathology was a naa / mi ratio.45 longitudinal studies have demonstrated that naa / cr and naa / mi decrease over time compared to controls.36,46,47 naa / cr and mi / cr ratios correlate with cognitive testing in alzheimer s disease.16,17,24,4851 in one study, naa / cr in the medial temporal lobe, primary motor and sensory cortices correlated with mini - mental state examination and the cognitive part of the alzheimer disease assessment scale scores.16 naa / cr, mi / cr, naa / mi have also been shown to correlate with verbal memory testing (auditory verbal learning test) and general cognition (dementia rating scale).50 several studies have investigated the ability of mrs to distinguish ad patients from controls with varying results depending on the anatomic area analyzed and acquisition parameters. the sensitivity was as high as 90% in the temporoparietal region and as low as 57% in the parietal lobe grey matter. the specificity was as a high as 95% in the medial occipital lobe and as low as 73% in the posterior cingulate.14,19,5254 furthermore, adding hippocampal volume to mrs, improves the ability to distinguish ad.19,5356 a few studies with relatively small sample sizes have investigated mrs as a biomarker for treatment response in ad. naa / cr improved after acetylcholinesterase inhibitor treatment in ad.10,28 another trial showed decreases in cho / cr and mi / cr in the hippocampus in absence of clinical improvement in ad subjects, however, this study showed continued decrease in naa / cr in contrast to the studies mentioned above.39 mrs also correlates with psychiatric symptoms in ad patients. ad subjects with psychosis have significantly reduced cortical naa compared to ad subjects without psychosis.57 psychiatric and behavioral symptoms in ad including delusional thinking correlated with a decrease in naa / cr and an increase in mi / cr in the anterior cingulate.58 naa / cr levels in mci are mildly reduced but decline as patients with mci progress to ad.36 further, lower naa / cr in mci patients predicts progression to ad.36,59,60 cho / cr and mi / cr levels are also elevated in the posterior cingulate in mci although higher levels of these metabolites are detected in ad.12 the cho / cr ratio is also useful in determining progression from mci to ad. in mci patients, a decline in cho / cr predicted stability versus an increase, which predicted conversion to ad. the changes in metabolite concentration on mrs correlate as strongly as ventricular volume in predicting cognitive decline.36 mrs is also useful in distinguishing subtypes of mci. amnestic mci patients have smaller hippocampi with elevated mi / cr ratios compared to patients with non - amnestic mci in line with the observation that amnestic mci patients are more likely to progress to ad than non - amnestic mci.61 the main differential diagnoses of alzheimer s dementia are other types of dementia including dementia with lewy bodies (dlb), frontotemporal dementia (ftd), and vascular dementia (vad). the mrs signature of ad is decreased naa / cr and elevated cho / cr and mi / cr metabolites.25 several studies have investigated the metabolite patterns among different types of dementia in order to identify patterns of metabolite changes unique to each dementia. many patients with dementia have significant overlap in underlying pathology.62 in patients with vad, the location of the metabolite change is important for distinguishing vad from ad. for example, naa levels in vad are decreased in a similar way to those in ad patients and the decrease is greater than ad in the white matter.6365 unlike ad, cortical mi is normal in vad.66,67 further, mi / cr is higher in ad compared to vad.25 therefore, mi and grey matter naa may serve as useful markers to distinguish ad from vad. similar to ad, ftd is associated with elevated mi / cr and decreased naa / cr.68,69 despite the similarities, regional differences in metabolites may help distinguish the two dementias. compared to early ad, patients with ftd have higher mi / cr and lower naa / cr in the frontal cortex.68,70 however, others have noted no difference between ftd and ad with similar levels of metabolite abnormalities outside the frontal lobes.25 similar to the other dementias, significant overlap exists between dlb and ad. compared to other dementia subtypes, dlb has a normal naa / cr ratio in the posterior cingulate.25 the normal naa / cr is possibly related to the relative preservation of neurons in dlb compared to ad.71 in the hippocampus of dlb patients, the naa has been reported to be decreased although it is unclear if this decrease represents concomitant ad given the overlap in pathologies mentioned above.72 additionally, naa is similarly decreased in the white matter of patients with dlb.73 therefore, naa levels in the posterior cingulate may help distinguish ad from dlb. the relative preservation of the cingulate is in agreement with fluorodeoxyglucose (fdg) positron emission tomography (pet) which demonstrates a relative preservation of glucose metabolism in the cingulate of dlb when compared to ad.74 similar to ad, dlb patients have an elevated cho / cr compared to controls.25 this finding is intriguing because both ad and dlb are characterized by a cholinergic deficit.75 since cho / cr levels decrease with cholinergic agonist treatment in ad,35 the decrease raises the possibility that cho / cr can be used as a therapeutic biomarker in ad and dlb. in summary, while significant overlap exists between dementias, ad has a unique metabolite pattern compared to other dementias when regional differences are taken into account. the american academy of neurology practice parameter recommends against routine imaging with quantitative mri techniques in the evaluation of dementia because of insufficient evidence.76 nonetheless, as a research tool, mrs can provide valuable information in the differential diagnoses of dementia. ad biomarkers, such as amyloid pet imaging and cerebrospinal fluid a, provide useful information about whether ad is the pathology underlying a given dementia. in addition, mrs allows for identification of a metabolite signature of different dementia subtypes which can provide complementary data to the clinical impression. to date, no mrs study has used metabolite signature in conjunction with regional differences to determine the underlying pathology. in 2011, the national institute on aging and alzheimer s association revised criteria for preclinical, mci, and alzheimer s disease.7780 the pathology that contributes to ad begins to accumulate years before clinical symptoms. therefore, identifying the population at high risk of developing symptoms has become important. principal ad imaging biomarkers include a imaging with pet, fdg - pet, and structural mri. in cognitively normal older adults, mi / cr and cho / cr correlate with a load in amyloid pet imaging as demonstrated in two example cases with high and low a load in their brains in figure 1.81 according to one model of biomarkers in ad, amyloid pathology accumulates before evidence of neurodegeneration.82 amyloid pet imaging serves as a surrogate for brain amyloid load and fdg - pet and structural mri serve as markers of neurodegeneration. while these imaging markers are well validated measures of amyloid and neurofibrillary tangle pathology, they do not measure microglial activation. a recent 13-carbon mrs and 1h mrs linked glial and microglial activity to mi elevation in ad.83 this raises the possibility that elevated mi represents inflammation which is an early event in the evolution of ad pathology.2 while hippocampal atrophy predicts ad pathology and is an imaging marker of neurodegeneration in the national institute on aging alzheimer s association (criteria for preclinical ad),84,85 up to 11% of ad cases are hippocampal sparing, with corresponding preserved hippocampal volumes on mri.86,87 subjects with at least one type of hippocampal sparing ad (posterior cortical atrophy), have been demonstrated to have decreased naa / cr in the posterior cingulate.88 therefore, mrs may serve a role as a critical marker of ad pathology in a significant minority of ad cases where the hippocampus is relatively preserved. other imaging markers that may provide important information in atypical ad cases include resting state functional mri, and diffusion tensor imaging (dti). mrs can serve as a predictor of the degree of ad pathology in clinical trial design. for example, mi / cr is elevated in mci and mild ad even with normal naa / cr.12,24,89 in addition, isolated mi / cr elevation is associated with earlier stage ad pathology compared to elevated mi and decrease in naa / cr which is associated with a later stage ad pathology. isolated elevation in mi / cr can be seen prior to structural mri changes in individuals with familial dementia prior to symptom onset.90 therefore, mrs could potentially serve as an adjunct to help select patients for intervention trials based on degree of ad pathology. in addition to serving as a potential marker of glial activity, mrs can be used along with other biomarkers as a tool to predict cognitive decline. in a group of cognitively normal individuals, elevated cho / cr in the white matter predicts progression to dementia.91 the metabolite formula changes in preclinical familial ad families with amyloid precursor protein, presenilin 1 or 2 mutations. asymptomatic mutation carriers demonstrated elevated mi / cr and decreased naa / cr with reduction in naa / mi correlating with nearness to age of onset.92 in addition to serving as a marker of preclinical disease, mrs has utility in monitoring disease progression. while much progress has been made in understanding the role of mrs in alzheimer s disease, mrs is still not routinely used clinically in the assessment of dementia. reasons for ineffective translation of technology to clinical practice or patient - oriented research are twofold : (1) lack of standardization for multi - site applications and normative data ; and (2) insufficient understanding of the pathologic basis of 1h mrs metabolite changes.94 although metabolite abnormalities in ad have been demonstrated in different samples and in pathologically confirmed cases, the pathological substrates for these metabolite abnormalities are not fully understood. future studies are needed to elucidate the pathological significance of these metabolite changes in ad. as we learn more about the pathophysiologic underpinnings of the metabolite abnormalities, the routine use of mrs as a biomarker will become more prevalent.	aging is the primary risk factor for dementia. with increasing life expectancy and aging populations worldwide, dementia is becoming one of the significant public health problems of the century. the most common pathology underlying dementia in older adults is alzheimer s disease. proton magnetic resonance spectroscopy (mrs) may provide a window into the biochemical changes associated with the loss of neuronal integrity and other neurodegenerative pathology that involve the brain before the manifestations of cognitive impairment in patients who are at risk for alzheimer s disease. this review focuses on proton mrs studies in normal aging, mild cognitive impairment, and dementia, and how proton mrs metabolite levels may be potential biomarkers for early diagnosis of dementia - related pathologic changes in the brain.
dysfunction of the hypothalamic - pituitary - thyroid neuroendocrine axis has been implicated in the pathogenesis of major depressive and bipolar disorders. moreover, symptoms such as fatigue, tiredness, mental slowing, concentration and memory impairments, weight gain and depression are common to both overt hypothyroidism and to unipolar depressive and bipolar disorders. taking this into account, and as the relationship between lithium and hypothyroidism furthermore, there is some support for adjunctive treatment with thyroxine in treatment - resistant depression and in rapid - cycling bipolar disorder. extracts of the medicinal herb, withania somnifera (called ashwagandha in sanskrit, asw) have been used for centuries in ayurvedic medical practice in india as an modern data point to its immunomodulation, brain antioxidant, neuroprotective, anti - inflammatory, and memory - enhancing properties. we tested a standardized asw extract (sensoril) for its pro - cognitive properties in patients with bipolar disorder. however, in view of a case - report of thyrotoxicosis associated with asw, and animal data suggesting that asw induces increases in thyroid hormones, we monitored serum levels of thyroid stimulating hormone (tsh), tri - iodothyronine (t3), and free thyroxine (t4) as safety parameters. the assessment of these thyroid indices in the bipolar study forms the basis of this report. the parent clinical trial, which has been described previously was registered at clinicaltrials.gov (identifier : nct00761761). the study protocol obtained ethical approval by the internal review board and written informed consent was obtained from all participants. in brief, sixty subjects with bipolar disorder participated in an 8 week, randomized, double - blind, placebo - controlled trial. the intervention group received 500mg / day of standardized asw (sensoril) extract in addition to their existing medication regimen, which included medications for treatment of bipolar disorder, comorbid psychiatric conditions, and other medical conditions. thyroid - stimulating hormone (htsh) was measured in serum using a third generation, two - site immunoenzymatic (sandwich) assay with a reference range of 0.300 - 5.000 total t3 was measured in serum using a competitive binding immunoenzymatic assay with a reference range of 0.60 to 1.81 ng / ml. free thyroxine in serum (t4) was measured using a direct, labeled antibody, and competitive immunoassay. during the tenure of the study, the t4 reference range initially was 0.76 to 2.12 ng / dl, but was changed to 0.80 to 1.80 ng / dl. the t4 values of two patients were impacted by this change in that they had one reference range at entry and a different one at study exit, and so the results of those two subjects were excluded. subjects were diagnosed with bipolar i, bipolar ii, or bipolar disorder not otherwise specified (dsm - iv), and their young mania rating scale and montgomery - asberg depression rating scale scores were 10 for 4 weeks prior to study entry, with stable doses of their mood - stabilizing medication 4 weeks. eligible subjects were randomized using a 1:1 randomization to asw or placebo, starting at 250 mg / day orally, for 1 week and increasing to 250 mg twice daily beginning the second week until the end of the 8-week study. subjects whose thyroid indices fell outside the reference ranges either at study entry or upon completion of the study were mainly reviewed for this report. twelve (20%) of the 60 randomized subjects in the original study had values outside the reference range of one of the three thyroid indices however, the t4 results of two patients (receiving placebo) were excluded as these values were confounded by a change in the laboratory reference ranges as noted earlier. details of demography, diagnoses, and medications of the ten patients are provided in table 1, and results of their thyroid indices are noted in table 2. patients with bipolar disorder comprised a group of adult men and women treated with mood stabilizers (lithium, valproate, and lamotrigine), anti - psychotic agents, and anti - depressant and anxiolytic agents, mostly in combinations. these psychotropic medications had been used for months to years in most subjects, and their mood ratings are reflective of a stable patient cohort at study entry and at exit. only one of the group of 10 patients had pre - existing hypothyroidism, which was treated with levothyroxine. demographics, illness, medications and mood ratings in an ashwagandha trial thyroid indices in a clinical trial using ashwagandha two subjects had tsh measurements in the subclinical hypothyroid range [table 2 ]. one subject assigned to placebo received 150 micrograms / day of levothyroxine for hypothyroidism had a normal tsh value at study entry but was in the subclinical hypothyroid range (6.96 miu / l) at exit. he experienced corresponding decreases in t4 and t3 and was counseled to review these changes with his primary care physician for further actions. another subject who received asw had an elevated tsh at baseline (5.7 miu / l), which normalized upon study completion with a corresponding 23.7% increase in the free t4 level from baseline. three patients had elevated t3 values at study entry [table 2 ], and two of them normalized upon completion, one having received asw and the other placebo. a placebo - assigned subject continued to have abnormal t3 results at the beginning and at the end of the study. among those with abnormal free t4 values [table 2 ], three placebo - assigned subjects had normal t4 values at baseline but moved below the lower reference limit (hypothyroid) at study exit, and a fourth subject who also received placebo had low t4 values at both time - points. one asw treated patient had a low free t4 value at study entry, which normalized at study exit. the percentage increases in free t4 levels in the three asw - treated patients were 7%, 12.1%, and 23.7%. among the placebo - assigned patients, with one exception, free t4 values decreased over a 2-month period, ranging from 3.9% to 23.3% with corresponding decreases in the t3 levels. two patients (one placebo and one asw) received lithium treatment, which in itself has been shown to effect thyroid indices. the asw - treated patient had an elevated t3 that normalized, and interestingly the t4 levels increased by 12.1%, while tsh remained normal. the placebo - assigned patient had a normal t4 at baseline, but it dipped into the hypothyroid range at study exit ; t3 decreased as well, but tsh remained normal. for the rest of the 60 patients whose thyroid indices were reported in the normal range, the three thyroid measures were analysed for baseline to end - point differences between the two treatment groups using the non - parametric wilcoxon rank sum test. few patients were impacted by a change in the laboratory reference range for the t4 test during the study period, and some had missing values for all three indices either at the beginning or at the end of the study, therefore the total number of patients varied slightly for each test. however, none of the results were statistically significantly different for any of the thyroid measures reported as normal between the treatment groups. however, and interestingly, over a 2-month period, asw treated patients (n = 18) showed numerically higher values of free t4 (mean increase from baseline : 0.13 0.24 ng / dl) compared to those receiving placebo, n = 19, (mean increase : 0.02 0.18 ng / ml) even though this difference was not statistically significant. since the parent study was not designed to primarily examine thyroid function in bipolar patients treated with asw, the sample size is small and unequally divided between patients who received placebo (n = 7) or asw (n = 3). as noted earlier, a case report of asw - associated thyrotoxicosis, as well as animal studies showing increases in t4 levels in mice treated with asw, prompted us to assess thyroid measures as a safety goal in the study. increases in free t4 in the three asw- treated bipolar patients are consistent with the data in mice administered asw, which also experienced significant increases in t4. moreover, tsh levels in the subclinical hypothyroid range (5.7 miu / l) normalized in one asw - treated patients, whereas the other two asw - treated subjects had normal tsh levels throughout. t4 levels decreased by nearly 4% to 29% during an 8-week period among six of seven subjects assigned to placebo, and one subject who received placebo had a normal tsh level at entry but moved into the subclinical hypothyroid range (6.96 miu / l) at study exit. t3 levels decreased in all subjects regardless of treatment assignment (within the normal range with one exception). interestingly ; in one set of animal experiments, asw had no statistically significant impact on the enzyme iodothyronine5'-mono - deiodinase, which is primarily responsible for the extra - thyroidal conversion of t4 to t3. the authors opined that asw 's actions are mainly driven by stimulation of the thyroid gland to release t4, though the precise mechanism(s) remains unknown. > 10 miu / l) is treated with levothyroxine in the general population, and also in patients with mood disorders. however, as in the general population, the use of levothyroxine in those mood disorder patients in the subclinical hypothyroid range (i.e. tsh levels from 4.5 to 10 miu / l and lacking thyroid antibodies and/or other risk factors) remains somewhat unclear and is not without risk or controversy. studies have suggested the use of adjunctive thyroxine in treatment - resistant depression or supra physiological doses of thyroxine in rapid cycling bipolar disorder but such treatments have not gained wide acceptance. possibly, these changes in thyroid measures occurred randomly, or were caused by an interaction of asw with the existing medication, or were an independent effect of asw on the thyroid gland / receptor. interestingly, however, all three asw - treated patients showed a rise in free t4 (although small changes) similar to the results in animal experiments but replicative studies are required to confirm these early data. at best, future prospective studies that are hypothesis driven with an adequate sample size to demonstrate a thyroid promoting effect for asw are required. moreover, confirmatory and repeat laboratory testing of thyroid indices in future work will provide greater confidence of the results obtained in this study. therefore, even though a single case of thyrotoxicosis associated with asw has been reported in the literature, vigilance may be well advised when asw is utilized in clinical practice. this highlights the importance of thyroid function testing before starting asw among ayurvedic or other healthcare practitioners and emphasizes that repeat testing may be required if there is clinical suspicion of hyperthyroidism during asw treatment. the current report of asw elevating t4, along with its anti - inflammatory, neuroprotective, anti - oxidant, and anti - depressant properties, suggest that asw can be considered for treatment of sub - clinical hypothyroidism in mood disorders. prospective controlled studies among patients with laboratory confirmed subclinical hypothyroidism (i.e. tsh and free t4 along with thyroid antibodies), and other risk factors may provide answers to whether asw extracts are more specifically beneficial in persons with unipolar or bipolar disorders with treatment resistant depression or rapid cycling.	laboratory indices of thyroid function (tsh, free t4, and t3) were measured in a randomized clinical trial in which ashwagandha (asw) was used to improve cognitive function in patients with bipolar disorder. this was done in light of a case - report of asw - associated thyrotoxicosis, and data from mice administered asw that showed significant increases in thyroxine levels. ten (of the original 60) patients showed abnormal results in one of the thyroid measures either at the beginning or end of the 8-week study. one asw- treated patient had subclinical hypothyroidism (tsh - 5.7 miu / l) at baseline that normalized, and all three asw treated patients experienced t4 increases from baseline (7%, 12%, and 24%). six of 7 placebo - assigned patients showed decreases in t4 from baseline (4% to 23%), and one patient 's tsh moved from the normal to subclinical hypothyroid range (6.96 miu / l). as thyroid indices were done for safety, and not the primary goal of the original study, only 16.7% had abnormal thyroid indices, and as there was no sub - stratification for treatment assignment by thyroid status, unequal numbers of subjects received asw (n = 3) or placebo (n = 7). in spite of these limitations, the subtle laboratory changes noted in thyroid indices in an 8-week study suggest that asw may increase thyroxine levels, and therefore vigilance regarding hyperthyroidism may be warranted. nonetheless, the thyroid enhancing properties of asw may also represent a clinical opportunity for the treatment of subclinical hypothyroidism, and these results suggest the need for further study of the effects of asw on thyroid indices, especially in those with bipolar and unipolar mood disorders.
the coordination chemistry of anions is a major field of research in supramolecular chemistry because of the key roles played by anions in chemistry, biology, medicine, catalysis, and environment. halides are common inorganic anions performing many important functions in environment and life. for example, fluoride is used in toothpaste and in city water to prevent tooth decay ; however, the high concentration of fluoride is harmful causing dental fluorosis. an excess amount of chloride and fluoride in water has been implicated in high incidences of lymphoma. in biology, chloride has an important role, which is transported to different organs including kidney and pancreas through cystic fibrosis transmembrane conductance regulator (cftr), while the disruption of chloride - transport may cause a fatal genetic disease known as cystic fibrosis. the presence of bromide in water could be harmful, since it can be converted during water purification process into bromate, which is suspected to be a genotoxic carcinogen. the presence of iodide in drinking water causes an unpleasant odor due to the formation of iodoform with natural organic matter. in human, iodide is known to block the release of thyroid hormone, and is used to treat patients with hyperthyroidism. therefore, there is an increasing interest in understanding interactions of halides with synthetic receptors both by experimental and theoretical approaches. because the ionic size in the halide series increases from f to i, both the charge densities and the basicity decrease from f to i. thus, their binding and selectivity to synthetic receptors depends on a number of factors including sizes, binding sites, charges and geometries of host molecules. historically, polyamine - based receptors are the first and most extensively investigated synthetic receptors for the recognition of halides in solution as well as in solid states. in particular, monocyclic polyamines that are known to be conformationally flexible, bind halides by both sides of their macrocycles, forming ditopic complexes in different fashions. for instances, n6 and metacyclophanes were reported to form chloride, bromide, and iodide complexes in a 1:2 binding mode. however, a larger m - xylyl - based macrocycle containing propylene chains was shown to adopt a chairlike conformation, with six bromides located outside the macrocyclic cavity via nhbr interactions. a series of experimental and theoretical studies revealed that hydrogen bonding and electrostatic interactions are the primary binding forces in such receptors for the stabilization of anion complexes. in such complexes the binding patterns and selectivity can be influenced by the variation of spacers as well as linking amine groups. recently, we developed a structurally simple monocyclic polyamine (l) incorporated with n - methyl-2,2-diaminodiethylamine as an amine linker (scheme 1), coordinating two bromides in its tetraprotonated form. upon further investigation on chloride anions, the receptor was shown to encapsulate two chlorides in the macrocyclic cavity. we were further interested in exploring this receptor for halides in solution and solid states and corroborate the data with results from theoretical calculations. herein is the full report of the halide binding studies of l using h nmr titrations, x - ray crystallography, and dft calculations. the chemicals used for this work were purchased from aldrich as reagent grades and used as received. nuclear magnetic resonance (nmr) spectra were recorded at 25 c on a varian unity inova 500 ft - nmr. chemical shifts for nmr were expressed in parts per million (ppm), and calibrated against trimethylsilane (tms) or sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (tsp) as an external reference used in a sealed capillary tube. mass spectral data were obtained at esi - ms positive mode on a finnigan lcqduo. the synthesis of l was carried out following the procedure described earlier. in a typical reaction, n - methyl-2,2-diaminodiethylamine (0.74 g, 6.3 mmol) and terephthaldehyde (0.85 g, 6.3 mmol) were dissolved separately in ch3oh (250 ml). the solutions were simultaneously added in ch3oh (400 ml) at 0 c over 6 h. the resulting mixture was further stirred overnight at room temperature. after evaporating the solvent under reduced pressure, the oily product was redissolved in ch3oh (100 ml) and nabh4 (1.2 g, 31.7 mmol) was added to reduce the product into an amine. the solvent was evaporated, and the residue was dissolved in water (100 ml). the aqueous phase was extracted by ch2cl2 (3 100 ml), and the organic layers were dried by the addition of mgso4 (2.0 g). the crude oily product was purified by column chromatography on a neutral - alumina column (2% ch3oh in ch2cl2) to yield l as a white powder. h nmr (500 mhz, cdcl3, tms) : 7.19 (s, 8h, arh), 3.75 (s, 8h, arch2), 2.77 (t, j = 5.5 hz, 8h, nhch2), 2.54 (t, j = 5.5 hz, 8h, nhch2ch2), 2.16 (s, 6h, ch3). c nmr (125 mhz, cdcl3,) : 138.9 (ar c), 128.0 (ar ch), 56.5 (nhch2), 53.9 (nhch2), 47.0 (nhch2ch2), 42.2 (ch3). esi - ms : m / z (+) 439.5 [mh ]. calcd for c26h42n6 : c, 71.19 ; h, 9.65 ; n, 19.16. found : c, 71.28 ; h, 9.66 ; n, 19.19. the protonated ligand was prepared by reacting l (70 mg, 0.16 mmol) with 8-fold p - toluenesulfonic acid (243.48 mg, 1.28 10 mol) in methanol (5 ml). the addition of diethyl ether resulted in a white microcrystalline product that was filtered and washed by diethyl ether. nmr (500 mhz, cdcl3, tms) : 7.65 (s, 8h, arh), 7.40 (ts arh), 7.35 (ts arh), 4.24 (s, 8h, arch2), 3.55(8h, nhch2), 3.03 (8h, nhch2ch2), 2.42 (s, 6h, ch3) 2.37 (s, 3h, ts ch3). c nmr (125 mhz, cdcl3,) : 145.35 (ts ar c), 142.25 (ar c), 134.99 (ts ar c) 133.10 (ar ch), 132.30 (ts ar ch), 128.20 (ts ar ch), 56.47 (nhch2), 53.34 (nhch2), 48.05 (nhch2ch2), 41.17 (ch3), 23.33 (ts ch3) anal. calcd for c68h90n6o18s6 : c, 55.49 ; h, 6.16 ; n, 5.71. found : c, 55.41 ; h, 6.17 ; n, 5.73. the free amine l (30 mg, 0.068 mmol) was mixed with 6 n hcl (0.1 ml) in methanol (2 ml) to give a white precipitate. the salt was redissolved in water / methanol mixture (1:1, v / v ; 1 ml), and x - ray quality crystals were grown from this solution by slow evaporation after 5 days. calcd for c26h52.67cl6n6o2.34 : c, 44.64 ; h, 7.59 ; n, 12.01. found : c, 44.67 ; h, 7.56 ; n, 12.03. the free amine l (30 mg, 0.068 mmol) was mixed with 48% aqueous hbr (0.1 ml) in methanol (2 ml) to give a white precipitate. the salt was redissolved in water / methanol mixture (1:1, v / v ; 1 ml), and x - ray quality crystals were grown from this solution by slow evaporation after 3 days. yield : 37 mg, 0.049 mmol, 72%. calcd for : c26h46br4n6 : c, 40.97 ; h, 6.08 ; n, 11.02. found : c, 40.91 ; h, 6.06 ; n, 11.05. the free amine l (30 mg, 0.068 mmol) was mixed with 47% aqueous hi (0.1 ml) in methanol (2 ml) to give a white precipitate. the salt was redissolved in water / methanol mixture (1:1, v / v ; 1 ml), and x - ray quality crystals were grown from this solution by slow evaporation after 3 days. calcd for c28h54i4n6o2 : c, 33.15 ; h, 5.37 ; n, 8.28. found : c, 33.12 ; h, 5.39 ; n, 8.25. binding constants were obtained by h nmr (500 mhz bruker) titrations of h6l6tso with the anions (a = f, cl, br and i) as their sodium salts in d2o. initial concentrations were [l]0 = 2 mm, and [a]0 = 20 mm. sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3,-d4 acid (tsp) in d2o was used as an external reference in a sealed capillary tube. each titration was performed by 15 measurements at room temperature, and repeated three times. the association constants (k) were calculated by fitting of several independent nmr signals using eqnmr. error limit in k was less than 10% which was based on the curve fitting analysis for each anion. the crystallographic data and details of data collection for the crystals (1 - 3) are given in table 1. intensity data for 1 and 2 were collected using nonius kappaccd diffractometer and graphite - monochromated mok radiation (= 0.71073 at 90.0 k), while that for 3 was collected using a diffractometer with a bruker apex ccd area detector and graphite - monochromated mok radiation (= 0.71073). the data were corrected for absorption by the semiempirical method giving minimum and maximum transmission factors of 0.878 and 0.914 for 1, 0.300 and 0.356 for 2 and 0.383 and 0.778 for 3. the structures were solved by direct methods and refined by full - matrix least - squares methods on f. the position of hydrogens bonded to carbons were refined by a riding model, while those of hydrogens bonded to nitrogens were located on a difference map, and their positions were refined independently. hydrogen atom displacement parameters were set to 1.2 times the isotropic equivalent displacement parameters of the bonded atoms. the density functional theory (dft) with the hybrid meta exchange - correlation functional m06 - 2x was applied in all the calculations. the standard valence triple- basis set, augmented with d - type polarization functions for heavy elements and p - type polarization functions for hydrogen, namely 6 - 311g(d, p), and lanl2dz basis set for iodide, were used. the solvent (water) effects were evaluated by the polarizable continuum model (pcm) self - consistent reaction field of tomasi and co - workers. the geometries of all the models have been fully optimized in both the gas phase and in the solvent by analytical gradient techniques. the local minimum energy structures are found by ascertaining that all of the harmonic frequencies are real. the atoms in molecule (aim) theory was also applied to characterize the hydrogen bonds for the complexes. the relative binding energies (ezpe) and the gibbs free energy changes (g) were calculated with zero - point correction both in the gas phase and in the pcm model. the calculations include both 1:1 binding (h6l(x) ]) and 1:2 ([h6l(x)2 ]) binding of the motif [h6l ] using the respective halides (x = f, cl, br, i) in both gas and solvent phases. the synthesis of l was readily accomplished from the schiff base condensation of dialdehyde and diamine, followed by the reduction with sodium borohydride. the chloride (1), bromide (2), and iodide (3) complexes were prepared by mixing of the free ligand with respective inorganic acids in water / methanol mixture. all the salts yield good quality crystals from slow evaporation of the solution. however, attempts to prepare crystals of the free ligand and that of fluoride complex were unsuccessful. all the isolated crystals were fairly stable at room temperature, and characterized by single crystal x - ray diffraction. the binding affinities of [h6l ] for halides (f, cl, br and i) were evaluated by h nmr titrations using their sodium salts dissolved in d2o at ph = 2.1. the ph for the solution was adjusted with tsoh and naod. as shown in figure 1, the addition of halide anions to [h6l ] in d2o resulted in a downfield shift of nch3 (h1) and ch3nch2 (h2) protons. partial h nmr spectra of h6l(tso)6 in the presence of 5 equiv of various halides in d2o at ph = 2.1. h nmr titrations of h6l(ts)6 (2 mm) with the increasing amount of naf (r = [naf]0/[ligand]0) in d2o at ph = 2.1. the highest shift of the proton signals was observed for fluoride, as compared with other anions. for the larger halides (chloride, bromide and iodide), figure 2 displays the stacking of h nmr titration spectra of the ligand obtained after the increasing amount of fluoride anion (010 equiv), showing a gradual change of proton resonances at room temperature. the changes in the chemical shift of the aliphatic protons h1 and h2 as a function of the fluoride concentration are displayed in figure 3. the titrations spectra for other halides are included in the supporting information (figures s4, s6, and s8). the changes in the chemical shift of the ligand as recorded with an increasing amount of anionic solution provided the best fit for a 1:2 binding model for each anion (figures s5, s7, and s9). the binding data is listed in table 1, showing that the binding process involves the formation of both a 1:1 (ligand : anion) complex and a 1:2 (ligand : anion) complex for each anion. however, it is obvious that a 1:2 complex is much stronger than a 1:1 complex, supporting both crystallographic and theoretical data (discussed later). the ligand form 1:2 complexes with halides with the binding constants (in log k2) of 2.82, 2.70, 2.28, and 2.20 for fluoride, chloride, bromide, and iodide, respectively. the overall binding constants (in log 2) are 4.05, 4.00, 3.69, and 3.66 for fluoride, chloride, bromide, and iodide, respectively. in the case of 1:2 complexes, the binding trend follows the order : fluoride > chloride > bromide > iodide, reflecting that the binding strength roughly correlates with the relative basicity of halides. further, this binding order is also an indication that the larger anions may experience a stronger electrostatic repulsion in a single cavity. it is assumed that each anion is held by hydrogen bonding interactions in an axial pocket formed by one n and two nh groups. in contrast, the binding trend in a 1:1 complexation follows the order of fluoride (log k1 = 1.23) chloride > bromide > iodide, indicating that the binding strength depends on the relative size and basicity of halides. three anion complexes including chloride, bromide and iodide have been isolated and their structures have been characterized crystallographically, showing the formation of 1:2 complexes for all three anions. in the chloride complex, two chlorides are found fully encapsulated in the cavity being held strongly by hydrogen bonding interactions. in contrast, larger bromide or iodide anions interact with both sides of the macrocycle instead of being full encapsulation. dft calculations performed on [h6l ] at m062x/6 - 311 g (d, p) level to evaluate halide binding in gas and pmc models suggest that after binding with a halide anion by l, the distance between the apical nitrogens (n2 and n5) in both 1:1 and 1:2 binding models is elongated by 0.4 to 1.6 compared with [h6l ]. whereas, the distance between the two planes of the two benzene rings is shortened by 0.1 to 1.9. in both gas phase and solvent, the binding strength increases in the order : fluoride > chloride > bromide > iodide for either 1:1 or the 1:2 binding mode, supporting the experimental data obtained from the h nmr studies. dft calculations further indicate that a 1:2 binding is energetically more favorable than a 1:1 binding of the ligand.	a p - xylyl - based macrocycle l has been synthesized and its binding properties with halides have been investigated by 1h nmr titrations, single crystal x - ray diffraction analysis, and density functional theory (dft) calculations. as investigated by 1h nmr titrations, the ligand preferentially binds a halide in a 1:2 binding mode, with the association constants (in log k2) of 2.82, 2.70, 2.28, and 2.20 for fluoride, chloride, bromide, and iodide, respectively. the overall binding trend was found to be in the order of fluoride > chloride > bromide > iodide, reflecting that the binding strength correlates with the relative basicity and size of the respective halide. crystallographic studies indicate that the ligand forms 1:2 complexes with chloride, bromide and iodide. in the chloride complex, the ligand is hexaprotonated and each chloride is held via three nhcl bonds. the ligand is tetraprotonated for the other complexes, where each halide is h - bonded to two secondary ammonium nh+ groups via nhx bonds. the results of dft calculations performed on [h6l]6 + at m062x/6 - 311 g (d, p) level in both gas and solvent phases, suggest that the ligand binds halides with the binding energy in the order of f > cl > br > i, supporting the experimental data obtained from 1h nmr studies. results from dft calculations further indicate that a 1:2 binding is energetically more favorable than a 1:1 binding of the ligand.
involvement of esophagus is common in systemic sclerosis (ssc) [1, 2 ]. it consists mainly of esophageal motor disorders (emds) that are responsible for all clinical manifestations as well as gastroesophageal reflux disease (gerd) and its complications [24 ]. the factors which affect occurrence of esophageal involvement in ssc are discussed [57 ]. the aim of this study was to evaluate clinical, endoscopic, and manometric features of esophageal involvement in patients with scleroderma through a large cohort of patients with ssc. the recruitment was prospective, at a single center, and concerned 194 consecutive patients with ssc diagnosed according to the american college of rheumatology criteria, over a period of 20 years (april 1988december 2008). the patient 's mean age was 40.4 13.5 years (1173 years), gender was female in 170 cases (87.6%), and average duration of the disease was 6.8 7,5 years (6 months32 years). according to leroy 's classification of generalized scleroderma, a limited cutaneous form was observed in 158 cases (81.4%) and a diffuse form in 36 cases (18.5%). all the patients underwent a standardized medical card, an upper gastrointestinal endoscopy, and a standard esophageal manometry during the same visit, before initiating any antisecretory treatment. data collected included patient 's age, gender, clinical characteristic of the disease (duration, skin extension), and presence or absence of symptoms of gerd (heartburn and/or acid regurgitation) and/or dysphagia. each symptom was graded on a scale from 0 to 3 by intensity (0 = absent, 1 = mild, could be ignored by the patient, 2 = moderate, could not be ignored, but had no effect on daily life activities, 3 = severe or incapacitating, affecting daily life activities) and by frequency (0 = absent or less than one per month ; 1 = less than 1 per week ; 2 = several times per week ; 3 = every day). symptoms where then categorized as mild for a total score less than, or equal to six, moderate for a total score of seven to twelve and severe for a total score greater than twelve, or when one symptom was considered incapacitating every day (score = 9). upper gastrointestinal endoscopy was used to identify and stage reflux esophagitis according to the los angeles classification. manometry was performed with a four - channel infusion probe (distilled water infusion rate : 0.5 ml / mn). the procedure was used after discontinuation of medication that might affect esophageal motility at least 72 h before exam and following a 12-hour fast. normal manometric values were established in our laboratory using a control population of 24 healthy subjects matched for age and gender (gender ratio of 0.2, mean age of 41.7 10.7 years, age range : 2960 years). student 's t - test, chi, and a reduced standard deviation (sd) test were used as appropriate to compare means and percentages. esophageal symptoms were found in 118 patients (60.8%) with gerd symptoms observed in 94 cases (48.4%) and dysphagia in 91 cases (46.9%). the clinical complaint was considered mild in 74 cases (38.1%), moderate in 27 cases (13.9%), and severe in 17 cases (8.7%). it was mild or moderate (stage a or b la) in 47 cases (24.2%), severe (stage c or d la) in 7 cases (3.6%), and complicated by stenosis and/or barrett esophagus in 19 cases (9.8%). there was a hypotensive lower esophageal sphincter (les) pressure in 118 patients (60.8%) and emds in 157 cases (80.9%) ; both disorders were present in 108 cases (55.6%). emds were severe, of aperistalsis type, in 118 cases (60.8%), and a low pressure wave amplitude was observed in 36 cases (18.5%) and an uncoordinated peristalsis in 25 cases (12.9%). emds were extended to lower two - thirds of the esophagus in 137 cases (70.6%). presence of esophageal symptoms was not related with duration of the ssc, but it was significantly more frequent in diffuse form of the disease and in those with esophageal mucosal lesions and/or emds (table 1). presence of reflux esophagitis was not linked with subtype or duration of the disease, but it was statistically more frequent in the symptomatic population and was also more observed in forms with emds. nevertheless, it remained silent in 13 cases (17.8%) (table 2). emds were common in all forms of the disease, regardless of age, gender, degree of skin involvement, or duration of the disease. however, they were significantly more often present in cases with clinical complaints and/or reflux esophagitis, but they remained asymptomatic in 48 cases (30.5%) (table 3). overall incidence of esophageal symptoms during ssc has been estimated to be between 42% and 79% [2, 3, 1216 ]. the complaint is related to signs of reflux in 1471% of patients and dysphagia in 2482% of cases [2, 5, 15, 17, 18 ], both symptoms being present in 29%66% of cases [15, 19 ]. in our series, overall incidence of symptoms was 61%, signs of reflux and dysphagia were, respectively, near 48% and 47% ; both were present in more than 34% of cases. presence of esophageal complaint was not related to the duration of the disease in the literature [5, 7 ] and in this work. thus, for akesson and wollheim, koshino., and bassotti., presence of symptoms was not related to the subtype of ssc, but in our series, they were significantly more frequent in diffuse form of the disease. esophageal complaint was also more frequent and more severe in case of presence of reflux esophagitis in our experience as well as in literature [15, 20 ]. even if, for most authors, there is no correlation between presence of esophageal symptoms and presence of emds [13, 21 ], a link was, however, established between their severity and presence of emds [6, 7, 22 ]. in this study, symptoms were significantly more frequent in presence of esophageal dysmotility and are, therefore, a simple warning sign that should prompt search of emds by manometry. in fact, it is variously reported between 3.2% and 60% [15, 16, 18, 20, 23, 24 ]. the highest rates were reported by zamost. and hendel and are probably due to the fact that these patients on a large catch of anti - inflammatory drugs. the lowest, prevalence (3.2%), reported by poirier in a surgical series concerned forms complicated by stenosis. in an autopsy series of 58 patients with ssc, d'angelo found erosive esophagitis in 40% of cases. complicated forms of reflux esophagitis were also more frequent in patients with ssc than in the general population. reflux esophageal stenosis is reported in 340% of cases depending on the series [13, 15, 2628 ], while barrett esophagus in 6.837% [13, 15, 2628 ] or even 71% of cases and, more rarely, ulcer of esophagus. adenocarcinoma of esophagus would be also more frequent in ssc, it rate reaches 7% for katzka. and but, segel relates only one case in a large series of 680 patients with ssc followed for 11 years. in our series, reflux esophagitis was observed in nearly 38% of cases and was complicated by stenosis and/or barrett esophagus in 10% of cases. esophageal dysmotility of ssc is characterized by a les hypotonia and emds respecting the cervical segment of esophagus. in the recent series, overall frequency of manometric abnormalities les hypotonia is present in more than 50% of cases and is, in general, associated with emds. there is a low pressure wave amplitude in 48%81% of cases [1, 18 ], an uncoordinated peristalsis in 40%91% [18, 34, 35 ], and an aperistalsis in 23%52% [18, 32, 33 ] of patients. for some authors, there is no parallelism between frequency or severity of emds and subtype of ssc [2, 32, 3638 ]. for others, they would be more severe in diffuse form of scleroderma [5, 6, 39 ], yet in other works, emds are both more frequent and more severe in the diffuse ssc [14, 16, 17, 34 ]. in our series, degree of skin involvement did not significantly influence frequency or severity of manometric disorders. indeed, they occur within one year after diagnosis of the disease in the work of hostein. and hamel - roy., even at the stage of isolated raynaud 's phenomenon, before the onset of skin involvement [21, 38 ]. the subsequent evolution is more controversial. frequency and severity of the disorder increase with disease duration [23, 32 ] for some authors, rather not for others [16, 19 ]. for most authors, presence of esophageal symptoms is not a good indicator of those of emds [2, 6, 14, 20, 35, 41 ]. thus, in lock 's series, 25% of emds remained asymptomatic, and, conversely, 50% of symptomatic patients had a normal manometry. the existence of symptoms had a negative predictive value of 50% and a positive predictive value of 62%. for this author, this means a weak association and justifies manometry in the assessment of any ssc. in other series, severe emds remained asymptomatic in 21%40% of cases and the presence of symptomatic patients with normal manometry is also often reported [15, 17, 39 ].. however, there seems to be a link between severity of symptoms and existence of esophageal dysmotility [6, 7, 41 ]. in our experience, although manometry was significantly more often and more severely impaired in patients with esophageal complaint, emds remained asymptomatic in 48 cases (30.5%). emds are also a major predictor factor of occurrence of reflux esophagitis [4, 16, 17, 24, 37 ]. peristalsis is never or rarely normal in this case as well for us than in published series [15, 20 ]. esophageal manometry is useful in all forms of the disease, more particularly in asymptomatic population and without endoscopic lesions. at an early stage, it allows detection of emds which are a strong marker of the disease and at definite form of the disease, and it assesses risk of occurrence of reflux esophagitis. then, manometry could be a screening procedure in scleroderma.	aim. to evaluate characteristics of esophageal involvement in scleroderma. methods. the study was prospective and concerned 194 patients with a definite systemic sclerosis. gastroesophageal endoscopy and esophageal manometry were performed in all the cases. results. symptoms were present in 118 cases (60.8%) ; they were signs of gerd or dysphagia, respectively, in 94 (48.4%) and 91 patients (46.9%). reflux esophagitis was found in 73 cases (37.6%) ; it was mild or moderate in 47 cases (24.2%) and severe or complicated in the remaining cases. manometry revealed a lower esophageal sphincter incompetence and esophageal motor disorders, respectively, in 118 (60.8%) and 157 cases (80.9%). presence of these late was not related to age, duration, or skin extension of the disease, but with clinical complaint and/or mucosal damage. conclusion. esophageal involvement is frequent during scleroderma. manometry is the most sensible examination and could be a screening procedure.
with the growing aging population, the number of osteoporotic elderly hip fracture patients has been gradually increasing. bone cement prosthesis replacement has become a very effective method to treat these fractures. due to the continual friction between the joint prosthesis, aseptic loosening induced by wear particles therefore, how to prevent bone loss and the aseptic loosening after joint prosthesis replacement has become a research focus. as a class of synthetic analogs of pyrophosphate, bisphosphonate is a potent new drug to inhibit bone resorption. experiment researches had shown that the drug could inhibit bone loss after joint prosthesis arthroplasty, continuously increasing bone densities around the prostheses, inhibiting the release of osteolytic factors, inhibiting osteolysis induced by wear particles, promoting the proliferation and differentiation of osteoblasts, enhancing osteoblast activity, inhibiting apoptosis of osteoblasts and bone absorption of osteoclasts, and accelerating apoptosis of osteoclasts. it is supposed that the drug may be an ideal drug to prevent and cure aseptic loosening after prosthesis replacement. if these drugs are taken orally for a long time, they have a lot of side effects, such as low bioavailability, high treatment costs, and upper gastrointestinal ulcers [12, 13 ]. to avoid these side effects, the topical use added in acrylic bone cement may be a better way of administration. alendronate is a third generation of bisphosphonate and a regular drug used in the treatment of osteoporosis and osteoporotic fractures. in the form of powder, it has some advantages of being mixed easily in bone cement powders, high temperature resistance and remaining drug efficacy in bone cement, and so forth. literatures reported that acrylic bone cement compounded with alendronate had a favorable biocompatibility, and certain contents of alendronate showed no detrimental effects on the fatigue life of composite acrylic bone cement. bone - bone cement and metal - bone cement interfaces are common sites of aseptic loosening after bone cement joint replacement. however, there is no report about whether these interfaces of composite bone cement being affected or not when alendronate is added. to this end, we used composite acrylic bone cement with different dose of alendronate and made the research mentioned above. the aim was to investigate the change of interface strengths, bone densities, and interface microstructure after alendronate was added. pure alendronate powder (merck, usa) and cemex xl bone cement (tecres spa, verona, italy) were used as received ; stainless steel cylinders (diameter 10 mm length 37 mm), hollow polypropylene tube (inside diameter 16 mm outside diameter 20 mm height 20 mm), the axial positioning ring (figure 1(a)) (inside diameter 10 mm outside diameter 20 mm thickness 10 mm), the metal rod positioning system (figure 1(b)), and the universal tester (type instron 8032) were obtained from institute of biological materials, central south university. the bone density scanner was supplied by the endocrine laboratory of the second xiang'ya hospital. the study design and experimental procedures were approved by our institution 's animal care and use committee. according to the amount of alendronate added, all drug samples were divided into 6 groups, g0g5 (i.e., 0, 10, 50, 100, 500, and 1000 mg alendronate were added in 50 g bone cement powder, resp.). bone cement was mixed with different dose of alendronate according to the dose regimes above. the positioning rings were adjusted to their outside diameter overlapping with the outside diameter of the pipes. after bone cement had solidified, the positioning rings were removed, and bone cement - metal interface specimens were prepared (figure 1(c)). specimens were placed on the instron 8032 universal tester, and ten specimens per group, five specimens before immersion, and five specimens 4 weeks after immersion were tested. the metal cylinders were pushed out at the speed of 5.0 mm / min, and the maximum force launched (f) was measured. the metal - bone cement interface shear strengths (e) were calculated by the following formula and its units were mpa. : f stand for metal cylinder 's maximum force launched, in the unit of newton (n), d for metal cylinder 's diameter (10 mm), and h for the height of metal off the bone cement interface (20 mm). six specimens examined by push - out test were chosen (one specimen before immersion and one specimen 4 weeks after immersion for g0, g3, and g5). one part of samples was coated with gold and these interfaces were observed by electron microscopy. new zealand rabbits were operated under intraperitoneal anesthesia (1% sodium pentobarbital, 1.52.0 ml / kg). after the success of anesthesia, the surgical area was shaved and cleansed well with 5% benzalkonium bromide and draped the operation area. during the surgery, an incision about 1.5 cm was made to expose the distal femur by the lateral patellar approach. 3.5 mm drill was used to prepare bone holes and it orientated from the femoral attachment point of the lateral collateral ligament to the femoral medial condyle. when the medial skin of knee was lifted, the incision about 1.0 cm was extended to expose the medial condyle. the wound and bone tunnel were repeatedly washed with hydrogen peroxide and saline, and hemostasis was achieved with fine gauze. when the reaction was full, the mixture was filled into a volume of 20 ml injector and injected into the bone tunnels in the bilateral distal femurs. moderate pressure was applied to both ends of the tunnel until the bone cement solidified. penicillin (800,000 u) was injected by intramuscular injection every day after surgery for 7 days. during the observation period, the rabbits were fed under a standard diet and raised in separate cages. six rabbits were sacrificed on the 1st day, and the other six rabbits on 60th day after surgery. the left specimens were used to test the bone - bone cement interface shear strengths, while the right ones were used to scan bone densities surrounding the interfaces. bilateral femur condyles of the rabbits were trimmed to bone - bone cement interface samples with 9.0 mm lengths with scalpel. then the specimens were loaded on an instron 8032 universal tester, with a loading speed of 5 mm / min. the maximum load was recorded, and the interface shear strengths (e), in the unit of mpa, were calculated by the following equation : (2)e = fdl. in the equation : f stand for maximum load force launched, in the unit of newton (n), d for bone cement cylinder 's diameter (3.5 mm), and l for the length of bone - bone cement interface (9.0 mm). specimens were trimmed to bone - bone cement interface with 3.0 mm thickness with scalpel. then cut unnecessary bone and make these samples of a standard size (length 6.0 mm width 6.0 mm thickness 3.0 mm) and at the same time make sure the bone cement cylinders locate in the center of the specimens. specimens were scanned using a bone density scanner and the bone densities were calculated by the tester 's software. each set of data were expressed as mean standard deviation (sd) and one - way analysis of variance was performed. if there was a significant difference, pairwise comparison was carried out between groups using scheffe post hoc test. paired t - tests were used for the shear strengths and the bone densities at bone - bone cement interfaces between the 1st day and 60th day after surgery, and metal - bone cement interface strengths between before immersion and 4 weeks after immersion. table 1 showed that on the 1st day after surgery, bone - bone cement interface shear strengths showed no significant differences in all groups (p > 0.05). however, on the 60th day after surgery, they showed significant differences (p 0.05), but in the other groups their values increased significantly (p 0.05). table 2 showed that on the 1st day after surgery, bone densities surrounding bone - bone cement interfaces showed no significant differences in all groups (p > 0.05). however, on the 60th day after surgery, they showed significant differences (p 0.05), but in the other groups their values increased significantly (p 0.05). table 3 showed that before immersion, metal - bone cement interface strengths in all groups showed no significant difference (p > 0.05), while 4 weeks after immersion, with the increasing dose of alendronate, the shear strengths decreased gradually, and in g5 the decrease showed significant difference (p < 0.05). compared with that before immersion, the metal - bone cement interface strengths significantly decreased in all groups 4 weeks after immersion (p < 0.05). figure 2 showed that the porosity of bone cement specimens was similar before immersion, or 4 weeks after immersion in g0, g3, or g5. but compared with before immersion, the porosity in the same group increased obviously 4 weeks after immersion. the interfaces between the femoral component and bone cement were known to be a weak area of bone - bone cement prosthesis complex. previously, harris and jasty found that the main mechanism of aseptic loosening on the femoral side was the debonding of the femoral component - bone cement by analyzing the prosthesis removed. finite element analysis showed that shear stress was a major stress factor for joint prosthesis failure. interface shear strengths were influenced by a variety of factors, including surface roughness of the femoral stem component, preheating or precoating of the stem component, precooling of bone cement monomer, the type of bone cement, the type of prosthesis metal, and the load rate. as a part of this study, we investigated the effects of alendronate on metal - bone cement interface shear strengths. the results showed that before immersion, there is no significant difference in the metal - cement interface strengths in all groups, while 4 weeks after immersion, with the increasing dose of alendronate, the shear strengths decreased gradually, and in group g5 the decrease showed significant difference. meanwhile, electron microscopy showed that no significant difference was found with regard to the interface porosity before immersion or 4 weeks after immersion in groups g0, g3, and g5. these results indicated that the decrease of shear strength of metal - bone cement, was more attributed to decrease of the bone cement bonding capacity than the interface porosity. these studies also found that, compared with that before immersion, 4 weeks after immersion the metal - bone cement interface strengths decreased significantly in the same group. therefore, it was proposed that the main factor decreasing bone cement bonding capacity might be related to immersion in saline. bone - bone cement interfaces were another common site of aseptic loosening after joint prosthesis replacement. according to published reports, aseptic loosening about approximately 5079% was found 15 years after total hip arthroplasty for young active patients, and 16% of these patients needed revision arthroplasty. because of the impossible bonding between hydrophobic bone cement and hydrophilic bone tissues, bone cement was used as fillers instead of binders. instead, interfaces between bone cement and bone tissue become stable fixation by a mechanical intercourse locking. some studies showed that the shear strengths of bone - bone cement interfaces could be increased by enhancing microlocking between bone cement and bone, and precoating with a layer of an amphiphilic substance on the bone surface. there are many factors that can influence the interface shear strengths, including bone porosity, trabecular orientation, continuous pressures on the cement, preparation of bone surface, and viscosity of bone cement. moran. found that shear strengths of bone - bone cement interfaces were not influenced by gentamicin (0.5 g, 1.0 g, 2.0 g, or 4.0 g) added in 40 g bone cement powders. moreover, the shear strengths in bone cement were higher than that at bone - bone cement interfaces. therefore, it is obvious that shear strengths of bone - bone cement interface are a key factor for joint prosthesis service life. this study found that on the 1st day after surgery shear strengths of bone - bone cement interface in all groups showed no significant difference. compared with the 1st day, the interface strengths decreased significantly after 60 days after surgery in g0 and g1, but no obvious changes were shown in g2g5. to investigate the reason of shear strengths ' changes, we scanned the bone densities surrounding the bone - bone cement interfaces on the 1st and 60th day after surgery. the results showed that there were similar changes between bone densities and shear strengths at bone - bone cement interfaces. according to the phenomenon above, we inferred that the bone densities might be an important factor to decide the shear strengths at bone - bone cement interfaces. meanwhile, in g0 and g1, bone densities had significant reduction after 60 days after operation. it might be related to surgical trauma, thermal damage from bone cement, and activities reduction of rabbits. however, in g2g5, bone densities at bone - bone cement interfaces showed no significant change. this might result from mineralization capacity enhancement of osteoblast and function inhibition of osteoclast, which was caused by the topical release of alendronate and offset of the negative effects on bone densities. the advantages and disadvantages of this study also deserved discussion : (1) in this study, we used distal femurs of new zealand rabbits to prepare the bone - bone cement interfaces. compared with diseased femoral heads used by moran, the advantages included convenience of obtaining specimens, and an increase in sample volume, and avoiding negative impact from structure differences in diseased bone. these specimens obtained were smaller and more difficult to prepare bone - bone cement interfaces. when alive specimens subjects were used, the bleeding at the interfaces could affect the study results. (2) as artificial femoral stem substitute, stainless steel cylinder had different morphology and surface friction coefficient, which resulted in different shear strength values. however, we had already homogenized these factors that might affect the interface strengths in our experiments, therefore the study results were reliable. in conclusion, these results showed that a certain amount of alendronate in bone cement had a remarkable effect on interface strengths of composite acrylic bone cement and interfacial bone densities. a dose of 50500 mg alendronate in 50 g bone cement powder could prevent the decrease of interface strengths at bone - bone cement interfaces, and it had a similar effect on bone densities around these interfaces. however, the same doses of alendronate showed no effect on the interface strengths of metal - bone cement interfaces before immersion and 4 weeks after immersion. while the addition dose was 100 mg, bone cement showed the best strengths. the results of this study indicated that alendronate - loaded bone cement could be made, but alendronate amount must be controlled to below a certain level which had no effect on the shear strengths at metal - bone cement - bone interfaces.	objective. aim to study how the content of alendronate affected shear strengths at bone - bone cement - metal interfaces. methods. all samples were divided into 6 groups, g0g5. on the 1st and 60th day after surgery, bone - bone cement interface shear strengths and bone densities were examined. interface strengths of metal - bone cement specimens were studied before immersion and 4 weeks after immersion. results. on the 60th day, bone - bone cement interface shear strengths and bone densities showed significant differences (p < 0.05), and compared with g0, g2g5 values increased significantly (p < 0.05), and the peak value was met in g3. compared with the 1st day, on the 60th postoperative day both factors decreased significantly in g0 and g1 (p < 0.05). four weeks after immersion, with the increasing dose of alendronate, the shear strengths decreased gradually and in g5 decreased significantly (p < 0.05). compared with before immersion, the metal - bone cement interface strengths decreased significantly 4 weeks after immersion (p < 0.05). conclusions. 50500 mg alendronate in 50 g cement powders could prevent the decrease of shear strengths at bone - bone cement interfaces and had no effect on metal - bone cement interface strengths. while the addition dose was 100 mg, bone cement showed the best strengths.
approximately 20 to 30% of the patients with epilepsy, ultimately develop refractory seizures that do not respond adequately to pharmacological treatment (1). these patients have only a small chance of obtaining seizure control with one of the established anticonvulsant drugs. one of the groups of drugs that have come into focus recently is the group of diuretics (2). although the first reports of anticonvulsant effects of the loop diuretic furosemide date back nearly thirty years (3, 4), it was discovered only a decade ago that furosemide in high concentrations shows anticonvulsant properties in vitro (5). in the following, anticonvulsant effects of diuretics, particularly of furosemide, were shown using a variety of in vitro models of epilepsy (5 - 8). at the same time, there was epidemiological evidence that diuretic use could protect from the risk of new - onset seizures in a community - based sample of elderly patients (9). most investigators have focused on furosemide, most likely because of its high diuretic potency and the availability of solutions for intravenous or intraperitoneal injections. this is in contrast to the fact that in the population - based study as well as in the maximum electroshock model, the protective effect of thiazide diuretics was stronger than of furosemide (9). in addition, it has been shown that hct as well as other benzothiazide derivates potentiate the effect of low - dose phenytoin on the seizure threshold in the maximum electroshock model. the same study also showed that there is a tendency to elevate the seizure threshold by hct alone (10). the mechanisms of anticonvulsant effect of hct remain to be elucidated. whereas, there is increasing evidence that furosemide has direct effect on neurons and glia via the electroneutral na - k-2cl- co - transporters (nkcc) that modulate intracellular chloride concentration and cell volume (11, 12). thiazides do not have influence on the nkcc or the electroneutral k - cl - cotransporters (kcc), either. carbonic anhydrase (ca) regulates the diffusion of hco3- and protons across the cell membrane (2, 15) and its inhibition increases the hyperpolarizing effect of long - lasting gaba - a currents (16). thus, the most favoured hypothesis is that the epidemiological effect of seizure protection by hct intake may at least partially be an effect of ca inhibition (2). no in vitro experiments investigating the anticonvulsant properties and mechanisms of thiazide diuretics in epilepsy models have been published so far. the aim of the present study was to evaluate whether hydrochlorothiazide (hct), probably the most common used thiazide diuretic, plays anticonvulsant role in brain tissues in vitro. furthermore, we investigated if hct could pass blood - brain - barrier (bbb) to clear if its effect on cns is a neuronal modulatory action or a systemic effect. the experiments were performed on rat hippocampal slices. they have been performed according to national guidelines for animal research. after introduction of deep pentobarbital anesthesia, the animal was decapitated and the brain was removed. then the slices were preincubated in artificial cerebrospinal fluid (acsf) at 28c for 1 the preincubation acsf contained (in mmol / l) : nacl 124, kcl4, cacl2 1.0, nah2po4 1.24, mgso4 1.3, nahco3 26, and glucose 10. after preincubation, cacl2 was elevated to 2.0 mmol / l and the slices were transferred into an interphase - type recording chamber. in the experimental chamber, the temperature was increased to 32c. during the experiments, the ph, the bath temperature and the flow rate (1.5 to 2 ml / min) remained constant. for the preparation of the zero - magnesium solution, we omitted mgso4. hydrochlorothiazide (hct) solution was prepared by adding dimethylsulfoxide (0.5 ml per mmol / l of the crystalline hct substance) to achieve water solubility. extracellular field potential recordings were recorded with glass microelectrodes (resistance 2 - 5 m) in the stratum radiatum of the ca1 and ca3 hippocampal subregions connected with dc amplifiers. extracellular recordings were obtained using a custom made differential amplifier (with band - pass filters at 0.5 - 10 khz, sampling rate 0.3 - 100 hz). the reference electrode and field potentials were recorded by an ink - writer (capable to record between 10 v) as well as by a digital oscilloscope. traces were digitized by nicolet 460 digitizing oscilloscope (nicolet instruments) and analyzed with accompanying software. a continuous single pulse of electrical stimulation was applied through a bipolar platinum electrode attached to the schaffer collaterals of hippocampal slices. evoked field excitatory post - synaptic potentials (fepsp) were recorded in hippocampal ca1 region (stratum radiatum). the fepsp was elicited by adjusting the intensity of stimulation to ~50% of the maximum response (0.1 - 0.5 ma). the amplitude of fepsp 1 m sec after the onset was measured for data analysis. in ltp experiments, tetanic stimulation (100 hz, for 1 sec, at baseline stimulation intensity) was operationally defined as the mean change in fepsp amplitude for five stimuli given 30 min after tetanic stimulation compared with the mean fepsp to five test pulses given immediately before the stimulation. tetanic stimulation was applied 60 min after application of hct (2 mmol / l). the protocol for the zero - magnesium model experiments consisted of four periods : 1) superfusion with acsf for 30 min to test for spontaneously occurring epileptiform field potentials (efp ; control period). 2) superfusion with zero - magnesium acsf until there was a stable efp frequency and amplitude for at least 20 min (baseline period). 3) addition of hct (2 mmol / l, n=5 ; 0.2 mmol / l, n=5 ; 0.02 mmol / l, n=5) to the magnesium - depleted acsf for 60 min (hct period). 4) superfusion with zero - magnesium acsf for 60 min (wash - out period). the efp amplitude was measured peak - to - peak and efp duration was determined as the width at half - maximal amplitude. owing to the small group sizes that make normal distribution and variance homogeneity unlikely, we chose median and 25/75 percentiles for cohort description. the data were processed and analyzed using commercially available software (spss version 12.1, chicago, ill. interval - and ordinal - scaled data were compared using the wilcoxon test, and nominal - scaled data with the chi - square test or fisher 's exact test (2x2 tables). four animals were injected intraperitoneally (ip) with hct (2 mmol / l, 5 ml), two animals only received saline. two hr later, the animals were sacrificed with high doses of isoflurane followed by decapitation. the brain tissue was stored at -20 c for 12 to 48 hr until hplc analysis. hplc was performed using a method developed by zendelovska (17) for determination of hct in human plasma. the system included a lichrospher 100 rp8 column (125x5, 5) with the mobile phase consisting of 0.025 m phosphate buffer (adjusted to a ph of 5 with triethylamine) and acetonitile at a relation of 85/15, a pump, an uv - detector with variable wavelength and interface and dedicated software (lachrom series, merck - hitachi, darmstadt) running on a personal computer with intel pentium iii processor. the clear supernatant was transferred into an eppendorf cap, and 40 l were injected into the hplc system. the flow rate was 1ml / min, and the detection wave length was 230 nm. the experiments were performed on rat hippocampal slices. they have been performed according to national guidelines for animal research. after introduction of deep pentobarbital anesthesia, the animal was decapitated and the brain was removed. then the slices were preincubated in artificial cerebrospinal fluid (acsf) at 28c for 1 the preincubation acsf contained (in mmol / l) : nacl 124, kcl4, cacl2 1.0, nah2po4 1.24, mgso4 1.3, nahco3 26, and glucose 10. after preincubation, cacl2 was elevated to 2.0 mmol / l and the slices were transferred into an interphase - type recording chamber. in the experimental chamber, the temperature was increased to 32c. during the experiments, the ph, the bath temperature and the flow rate (1.5 to 2 ml / min) remained constant. for the preparation of the zero - magnesium solution, we omitted mgso4. hydrochlorothiazide (hct) solution was prepared by adding dimethylsulfoxide (0.5 ml per mmol / l of the crystalline hct substance) to achieve water solubility. extracellular field potential recordings were recorded with glass microelectrodes (resistance 2 - 5 m) in the stratum radiatum of the ca1 and ca3 hippocampal subregions connected with dc amplifiers. extracellular recordings were obtained using a custom made differential amplifier (with band - pass filters at 0.5 - 10 khz, sampling rate 0.3 - 100 hz). the reference electrode and field potentials were recorded by an ink - writer (capable to record between 10 v) as well as by a digital oscilloscope. traces were digitized by nicolet 460 digitizing oscilloscope (nicolet instruments) and analyzed with accompanying software. a continuous single pulse of electrical stimulation was applied through a bipolar platinum electrode attached to the schaffer collaterals of hippocampal slices. evoked field excitatory post - synaptic potentials (fepsp) were recorded in hippocampal ca1 region (stratum radiatum). the fepsp was elicited by adjusting the intensity of stimulation to ~50% of the maximum response (0.1 - 0.5 ma). the amplitude of fepsp 1 m sec after the onset was measured for data analysis. in ltp experiments, tetanic stimulation (100 hz, for 1 sec, at baseline stimulation intensity) was operationally defined as the mean change in fepsp amplitude for five stimuli given 30 min after tetanic stimulation compared with the mean fepsp to five test pulses given immediately before the stimulation. tetanic stimulation was applied 60 min after application of hct (2 mmol / l). the protocol for the zero - magnesium model experiments consisted of four periods : 1) superfusion with acsf for 30 min to test for spontaneously occurring epileptiform field potentials (efp ; control period). 2) superfusion with zero - magnesium acsf until there was a stable efp frequency and amplitude for at least 20 min (baseline period). 3) addition of hct (2 mmol / l, n=5 ; 0.2 mmol / l, n=5 ; 0.02 mmol / l, n=5) to the magnesium - depleted acsf for 60 min (hct period). 4) superfusion with zero - magnesium acsf for 60 min (wash - out period). the efp amplitude was measured peak - to - peak and efp duration was determined as the width at half - maximal amplitude. owing to the small group sizes that make normal distribution and variance homogeneity unlikely, we chose median and 25/75 percentiles for cohort description. the data were processed and analyzed using commercially available software (spss version 12.1, chicago, ill. interval - and ordinal - scaled data were compared using the wilcoxon test, and nominal - scaled data with the chi - square test or fisher 's exact test (2x2 tables). four animals were injected intraperitoneally (ip) with hct (2 mmol / l, 5 ml), two animals only received saline. two hr later, the animals were sacrificed with high doses of isoflurane followed by decapitation. the brain tissue was stored at -20 c for 12 to 48 hr until hplc analysis. hplc was performed using a method developed by zendelovska (17) for determination of hct in human plasma. the system included a lichrospher 100 rp8 column (125x5, 5) with the mobile phase consisting of 0.025 m phosphate buffer (adjusted to a ph of 5 with triethylamine) and acetonitile at a relation of 85/15, a pump, an uv - detector with variable wavelength and interface and dedicated software (lachrom series, merck - hitachi, darmstadt) running on a personal computer with intel pentium iii processor. the clear supernatant was transferred into an eppendorf cap, and 40 l were injected into the hplc system. the flow rate was 1ml / min, and the detection wave length was 230 nm. application of mg - free acsf resulted in efp after a median latency of 41 min (2 mmol / l hct), 47 min (0.2 mmol / l hct) and 43 min (0.02 mmol / l hct), respectively. after reaching a stable level over 15 min, median baseline repetition rate was 13/min (2 mmol / l hct), 11/min (0.2 mmol / l hct) and 10/min (0.02 mmol / l hct) respectively. addition of hct to the bath resulted marked increase of repetition rate, amplitude and duration of the epileptiform field potentials (figure 1). after 1 hr, the increase of the repetition rate was highest with 2 mmol / l hct (median 26/min, 25 - 75 quartiles 21.5/min-38/min), slightly lower with 0.2 mmol / l hct (median 21/min, 25 - 75 quartiles 18.5 - 24.5/min) and with 0.02 mmol hct (median 15/min, 25 - 75 quartiles 14,5 - 20.5/min). the inter - group differences were statistically significant, as were the differences between baseline and 1 hr exposition within each group (p<0.05). the amplitude increased from a median of 0.18 mv to a median of 0.55 mv in the 2 mmol group, from a median of 0.24 mv to a median of 0.37 mv in the 0.2 mmol / l group, and from a median of 0.21 mv to a median of 0.23 mv in the 0.02 mmol / l group. only the within - group differences of the 2 mmol / l and the 0.2 mmol / l groups were significant (p<0.05). discharge duration also increased from 120 msec to 340 msec (median) in the 2 mm group, from 190 msec to 230 msec (median) in the 0.2 mmol / l group, and from 270 msec to 320 msec (median) in the 0.02 mmol / l group. after 60 min of wash - out, repetition rate, discharge amplitude and discharge duration gradually returned to baseline levels (figure 2). a conditioning tetanic stimulation was delivered to the schaffer collaterals pathway followed by pulses with stimulation parameters identical to control values. the evoked fepsp was stable for at least 30 min (with mean amplitude of 0.3 0.03 mv) before application of tetanic stimulation (less than 10% variation ; figure 3). administration of tetanic stimulation produced a rapid, stable, and lasting enhancement of the amplitude of the fepsp in all tested preparations (n=6, 1471.6% control ; figure 3 b). the potentiation rose within 2 to 3 min and stabilized within 5 - 10 min after the train of stimulations. mmol / l ; n = 8) 60 min before tetanic stimulation did not change ltp induction in all tested slices (146 1.4% control, figure 3b). enhancement of ltp after application of hct did not significantly differ from control slices (p<0.05). in hplc, when brain homogenate of hct - treated rats was injected, hplc showed no significant peak at the hct retention time (figure 4). there was also no peak when the brain of rats injected with saline (control) was analyzed. application of mg - free acsf resulted in efp after a median latency of 41 min (2 mmol / l hct), 47 min (0.2 mmol / l hct) and 43 min (0.02 mmol / l hct), respectively. after reaching a stable level over 15 min, median baseline repetition rate was 13/min (2 mmol / l hct), 11/min (0.2 mmol / l hct) and 10/min (0.02 mmol / l hct) respectively. addition of hct to the bath resulted marked increase of repetition rate, amplitude and duration of the epileptiform field potentials (figure 1). after 1 hr, the increase of the repetition rate was highest with 2 mmol / l hct (median 26/min, 25 - 75 quartiles 21.5/min-38/min), slightly lower with 0.2 mmol / l hct (median 21/min, 25 - 75 quartiles 18.5 - 24.5/min) and with 0.02 mmol hct (median 15/min, 25 - 75 quartiles 14,5 - 20.5/min). the inter - group differences were statistically significant, as were the differences between baseline and 1 hr exposition within each group (p<0.05). the amplitude increased from a median of 0.18 mv to a median of 0.55 mv in the 2 mmol group, from a median of 0.24 mv to a median of 0.37 mv in the 0.2 mmol / l group, and from a median of 0.21 mv to a median of 0.23 mv in the 0.02 mmol / l group. only the within - group differences of the 2 mmol / l and the 0.2 mmol / l groups were significant (p<0.05). discharge duration also increased from 120 msec to 340 msec (median) in the 2 mm group, from 190 msec to 230 msec (median) in the 0.2 mmol / l group, and from 270 msec to 320 msec (median) in the 0.02 mmol / l group. after 60 min of wash - out, repetition rate, discharge amplitude and discharge duration gradually returned to baseline levels (figure 2). a conditioning tetanic stimulation was delivered to the schaffer collaterals pathway followed by pulses with stimulation parameters identical to control values. the evoked fepsp was stable for at least 30 min (with mean amplitude of 0.3 0.03 mv) before application of tetanic stimulation (less than 10% variation ; figure 3). administration of tetanic stimulation produced a rapid, stable, and lasting enhancement of the amplitude of the fepsp in all tested preparations (n=6, 1471.6% control ; figure 3 b). the potentiation rose within 2 to 3 min and stabilized within 5 - 10 min after the train of stimulations. application of hct (2 mmol / l ; n = 8) 60 min before tetanic stimulation did not change ltp induction in all tested slices (146 1.4% control, figure 3b). enhancement of ltp after application of hct did not significantly differ from control slices (p<0.05). in hplc, the peak of hct appeared at a retention time of approximately 3.5 min. when brain homogenate of hct - treated rats was injected, hplc showed no significant peak at the hct retention time (figure 4). there was also no peak when the brain of rats injected with saline (control) was analyzed. in contrast to our expectations, hct increased repetition rate, amplitude and duration of the epileptiform discharges elicited by omission of mg from the bath solution in a dose - dependent manner. hct also increased the amplitude of electrically evoked field potentials, but did not have any influence on the ltp induced by tetanic stimulations applied to schaffer collaterals. when intraperitoneally injected, hplc analysis did not show evidence of hct in the brain even at high doses. our recordings used the zero - mg model of epileptiform activity (18 - 20). this model initiates robust epileptiform activity in both normal and lesioned cortex by augmenting the nmda - component of epsp, without pharmacological blockade of synaptic receptors (i.e., gabaa receptors in the bicuculline model), and represents a model for pharmacoresistant status epilepticus. this model represents a well - controlled analysis of drug effects independent of systemic influences (18 - 20). this model quite closely imitates in vivo conditions because recurrent and lateral inhibition remains intact. in addition, low - magnesium activity induces post - synaptic calcium influx, resulting in activation of the same calcium dependent second messenger mechanisms that underlie activity - dependent neuronal plasticity (20). it has been found that diazoxide, another benzothiazide diuretic, is a potent and reversible gating modifier of ampa - type glutamate receptor channels (22) that inhibits the rapid desensitization of the response to a constant agonist concentration. the most potent agent of the group of thiazide desensitizers is cyclothiazide (ctz) (23, 24) that recently has been proposed as an in vitro epilepsy model (25). although there is chemical similarity between different benzothiazide diuretics, the effect on quisqualat - induced ampa - desensitation has been shown to be quite variable. in concentrations of 500umol, hct also potentiated the steady - state component of quisqualat - activated currents (23), but to a much smaller amount than cyclothiazide. it remains undetermined whether hct also - like ctz - increases pre - synaptic glutamate release (26) or directly inhibits gaba - a receptors (27). ltp is a phenomenon in which a constant pre - synaptic high stimulation of excitatory amino acids neuronal pathways results in prolonged enhanced post - synaptic responses. it is well established that nmda receptors are a molecular detector of the coincidence of both the pre - synaptic release of glutamate and a post - synaptic depolarization at the origin of ltp induction (28)., it has been shown that the hct (500 m) potently inhibits non - nmda receptors desensitization in cultured hippocampal neurons (23). cyclothiazide (300 m), a thiazide diuretic and an allosteric inhibitor of non - nmda receptor desensitization, induced potentiated low mg- induced seizure - like events recorded from the ca3 pyramidal layer of juvenile rat hippocampal slices (29). in line with our results, cyclothiazide administered intracerebral ventricle induced epileptiform activities from an initial of multiple evoked population spikes, progressed to spontaneous spikes and finally to highly synchronized burst activities in hippocampal ca1 neurons (30, 31). in contrast to in vivo studies, systemic administration of thiazides, including hydrochlorothiazide (100 mg), enhanced the anticonvulsant action of carbamazepine (32) and gabapentin (33) in the maximal electroshock seizure model in mice. in our experiments, we could not find evidence that hct does permeate bbb in the rat. this is in agreement with studies of dogs where dosages as high as 500 mg / kg administered twice daily did not result in measurable quantities (<3 mg unfortunately, there is no data available in the literature regarding the bbb permeability for hct in humans. in addition, there is no evidence for central nervous effects of hct even in the setting of overdose intoxication (35). one could argue that hct may influence ion transport at the bbb without actually permeating it. however, hct at a relatively high concentration of 1.5 mmol / l does not have any effect on the blood to brain sodium transport in vitro (36). the anticonvulsant effect of hct in vivo most likely is mediated via mechanisms based on effects outside the brain. dashputra and colleagues found that there is no correlation between the effect on the carbonic anhydrase and the potentiation of the anticonvulsant effect of phenytoin in the maximal electroshock model in rats (10). systemic acidosis seems equally unlikely to be related to the protective effect of hct and other benzothiazides because acetazolamide raises the seizure threshold before there has been time for substantial bicarbonate loss (37). it is very unlikely that the protective effect of hct in humans is due to enhancing the action of standard anticonvulsants because the population under study did not suffer from epilepsy (9, 38). first, we did not investigate the effect of hct in other in vitro models of epilepsy. mmol / l to 2 mmol / l. we intended to look across a large window of possible concentrations. however, hct concentration in plasma (or brain) of humans when exposed to hct in clinical practice may be much lower (39). thus, we may have missed relevant effects of concentrations below our lowest concentration, or between concentrations. however, the effects we have observed seem rather robust, which makes it unlikely that with larger groups we would have seen different results. the protective effect of hct and the other diuretics is rather mild in the epidemiological studies. moreover, in the animal studies presented by hesdorffer (9), hct (as well as furosemide) was applied in doses that are 100 to 200 times higher than the doses used in clinical practice of hypertension treatment (hct 165 mg / kg in rats vs. hct 0.3 - 1 mg / kg in humans). therefore, the use of diuretics as alternative convulsant agents can not be recommended yet. although hct seems to have anticonvulsive properties on in vivo experimental models, exposure of hippocampal slices to hct resulted in an enhancement of the epileptiform burst discharges. in addition, hct at the same concentration did not cross the bbb in rats. thus, the anticonvulsive effect of hct most likely is not through direct neuronal effects.	objective(s):protective effects of diuretics, particularly of hydrochlorothiazide (hct), for the development of epilepsy have been described in vivo. however, its mechanism of action is unknown. materials and methods : extracellular field potentials were recorded from the ca1- and ca3-subfields of the hippocampus of rats. epileptiform discharges were induced by omission of mg2 + from the artificial cerebrospinal fluid (acsf). hct was added to the acsf at a concentration of 2 mmol / l, 0.2 mmol / l or 0.02 mmol / l. frequency, amplitude and duration of the epileptiform discharges were evaluated. long - term potentiation (ltp) was induced with and without the presence of hct (n=6 ; 2 mmol / l). in addition, rats were injected with hct (n=4) or saline (n=2), and the brain tissue was analyzed using hplc. results : application of 0.02, 0.2, and 2 mmol / l hct accelerated the frequency of discharges by 50%, 91%, and 100%, respectively. the amplitude of burst discharges also increased by 9%, 54%, and 300%, and the duration of epileptiform discharges increased by 10%, 30% and 120%. all parameters returned close to the basal levels after 60min washout of the substance. hct increased the electrical evoked potentials but did not affect the ltp in hippocampal tissues. there was no evidence of hct in the rat brain after intraperitoneal injection. conclusion : exposure of hippocampal slices to hct enhanced epileptiform activity in a dose - dependent manner. in addition, hct does not seem to cross the blood brain barrier in rats. thus, the anticonvulsive effect of hct most likely is not through direct neuronal effect.
thus, of most importance is improvement in prophylaxis, early diagnosis and treatment (1). the blind person is usually not the one who can easily weave himself into the fabric of a crowd. unlike many other exceptional people we often do not realize that a person has impaired hearing until we talk to him. likewise, the vast majority of mentally - challenged individuals are indistinguishable from others on the basis of physical appearance. a visually impaired person, however, has a variety of symbols like cane, thick or darkened glasses, a guide dog etc. a legally blind person is said to be one (i) who has visual acuity of 20/200 or less in the better eye even with correction, (ii) or whose field of vision is so restricted that it subtends an angle of 20 or less in the better eye after correction. visual acuity is the ability of the eye to clearly see distant objects, which is determined with the help at snellen s chart (2). low vision is defined in terms of reduction in the clarity of vision, whereas partially sightedness is defined in terms of the distance through the snellen s chart for educational purposes (3). the visual acuity of 20/200 refers to the fact that what a blind person can see at 20 feet a person with normal vision sees at 200 feet (normal visual acuity is 20/20). if maladjustment does occur in a blind individual, it is more than likely due to the fact that society has treated him differently. in other words, it is society s reaction to the visually impaired person that determines his adjustment. a blind child suffers from behavioral deficiency because of extreme neglect or over - protection (3)., all these supposed concomitants of blindness, are experienced by the blind person with the full force of identification. the above feelings may create anxiety, frustration and maladjustment among visually impaired individuals (4). the number of disabled, which was 22 million in 2001, rose to 26.8 million in 2011. rural areas have more disabled people than urban areas. according to the 2011 indian census, 20.3% of the disabled are movement disabled followed by hearing impaired (18.9%) and visually impaired (18.8%). poverty is the most probable cause of disability in india because disabled people are more likely to suffer from malnutrition, live in crowded conditions, have limited access to medical care, be poorly educated, have not been immunized, had lacked adequate care during pregnancy and birth, and have mothers who had undergone multiple pregnancies (5). the inability of blind people to control their environment by sight has still another effect. they are frequently disturbed by a fear of being observed by others. a blind person, who can not determine whether he is being observed, or when the observation begins or ends, feels that he must control his movements and his behavior, which produces a state of tension and self - consciousness and anxiety towards the society (6). it was observed that the effects of deafness i.e. auditory deprivation, which further leads to poor communication, are paramount. this can affect social, psychological and many other aspects of life. in spite of their impairment and communication difficulties, they are affected by adjustment problems in their personal and social life (7). visually impaired people had a higher prevalence of depression compared with people with good vision. of visually impaired older individuals, 13.5% were depressed compared with 4.6% of people with good vision (8). people with visual impairment are more likely to experience problems with functioning, which in turn leads to depression (9). psychosocial development and social support of adolescents with and without visual impairments was assessed. the results showed that the psychological wellbeing of the two groups of adolescents did not differ. however, visual impairments tended to cause stress, especially in girls, and many adolescents with visual impairments reported problems in their relationships with friends (10). visual impairment had a greater impact on social relationships according to its severity and onset (11). it was reported that self - concept and social adjustment of adolescents with visual impairments were similar in different educational settings (12). however, small samples, different scales, and gender and cultural differences make it hard to draw conclusions from these studies (13). in this study, an attempt was made to examine the levels of anxiety, frustration, aggression and adjustment and their influence on psycho - physiological disorders among visually impaired adolescent students. the study also aimed at understanding the influences of variables such as gender and age on the occurrence of psychological and psycho - physiological disorders. in order to meet out the above objectives, 150 visually impaired adolescent students studying in schools, in and around coimbatore city (tamil nadu, india) were included in this study. five different tools were used and the data were statistically analyzed. among the visually impaired adolescent students approximately 1550 were doing their regular education in special schools or integrated mainstream education in coimbatore city. for the purpose of this study, the respondents were visually impaired adolescent students selected from five schools, which included both government and private schools in coimbatore city. among the visually impaired students, 71 were males and 79 were females. since purposive sampling was done, exclusion and inclusion criteria only accepted adolescent students aged between 13 and 19 years old. low / partial vision (visual acuity between 20/70 and 20/200) and total visual impairment was considered as an inclusion criteria for the study. only adolescents without hearing impairment (but with visual impairment) were considered for the study. permissions were obtained from the coimbatore district collectorate (tamil nadu state) and head masters of the individual institutions, after explaining the benefits of this particular study. the questionnaires were administered individually to each participant separately by reading the questions for them and marking their responses. the age of the respondents ranged from 13 to 19 years. among 150 visually impaired students, most of them were at the mid - adolescence stage (60.0%, 68.7%) ; followed by early adolescence (32.7%, 24.7%) and late adolescence (7.0%, 6.7%) stage of male and female students, respectively. the five tools used in this study were as follows, the personal information profile questionnaire, taylor s manifest anxiety scale (mas) (14), frustration test (ft) (15), aggression scale (as) (16) and adolescent adjustment inventory (aai) (17). the reliability coefficient values were calculated based on the data obtained from the subjects, based on the translated test and finally compared with the values given in standardized instruments. twenty - five students were administered to take the test after three weeks to calculate reliability. discriminate validity was determined by the agreement or association among variables in a tool and was calculated by spearman s correlation coefficient value between major variables. it was judged on the basis of whether an indicator corresponds to theoretical expectations in terms of relationships with other variables in a tool (18). there were four variables in these tools, anxiety, frustration, aggression and adjustment. all the values were within an acceptable range due to not having any values less than 0.3 (19). this questionnaire was used to collect data regarding the individual aspects of the students, namely, gender, age, class, number of siblings, nature and reason of impairment, type of birth, residential area, monthly income, economic status of the family, parent s marriage details and number of psycho - physiological disorders. the major impairment categories consisted of 1) due to congenital reasons, 2) due to sickness and 3) due to accident. in 1953, taylor published his results of an experiment in which the performance of adults in a classical conditioning situation was found to be related to the scores on a questionnaire relating to anxiety. the taylor s manifest anxiety scale (14), one of the earliest self - report inventories, has been used extensively in experimental results. t (for true) or f (for false) answers for each of the 50 statements. a score that was higher than average (14 or 15) indicated high degree of anxiety. low scores indicated low level of anxiety and scores much higher than the average suggested that the person experienced an unusually high degree of anxiety. taylor reported retest correlations of 0.89, 0.82 and 0.81 over intervals of three weeks, five months and nine to seventeen months, respectively. the split half and test - retest reliability coefficients were found to be 0.92 and 0.87, respectively. frustration test studies the effect of frustration upon the quality of a person s behavior as a whole. scores above 35 in each of the categories are matters of concern because of high frustration level. this scale consists of 40 items, out of which each of the four modes of frustration has 10 items. the split half test - retest reliability coefficients were found to be : regression (0.78), fixation (0.92), aggression (0.87) and as a whole (0.88). an act whose goal response is injury to an organism or organism - surrogate (20). frustration produces instigations to a number of different types of responses, one of which is instigation to some form of aggression. each of the items has five alternate answers (multiple choice) graded on a five - point scale on the positive dimension and a zero point on the negative dimension. this test is valid for the measurement of aggression of 11 to 24-years - olds. average, 46 to 60 as low and scores of 45 and below as the split half and test - retest reliability coefficients were found to be 0.82 and 0.78, respectively. the adolescent adjustment inventory (17) is one of the well - standardized inventories for adolescents. high scores indicate mal - adjustment and low scores indicate that the subject is well adjusted. the odd - even reliability coefficients were 0.84 and 0.95 for personal and social adjustment, respectively, after applying spearman s brown correction. the collected primary data were codified and the data were fed into a computer database using statistical package for social sciences (spss for windows ; version 10.0.1, spss inc.). chi - square test, t test, one - way analysis of variance (anova) and pearson s correlations were conducted to testify the hypothesized tests. among the visually impaired adolescent students approximately 1550 were doing their regular education in special schools or integrated mainstream education in coimbatore city. for the purpose of this study, the respondents were visually impaired adolescent students selected from five schools, which included both government and private schools in coimbatore city. among the visually impaired students, 71 were males and 79 were females. since purposive sampling was done, exclusion and inclusion criteria only accepted adolescent students aged between 13 and 19 years old. low / partial vision (visual acuity between 20/70 and 20/200) and total visual impairment was considered as an inclusion criteria for the study. only adolescents without hearing impairment (but with visual impairment) were considered for the study. permissions were obtained from the coimbatore district collectorate (tamil nadu state) and head masters of the individual institutions, after explaining the benefits of this particular study. the questionnaires were administered individually to each participant separately by reading the questions for them and marking their responses. the age of the respondents ranged from 13 to 19 years. among 150 visually impaired students, most of them were at the mid - adolescence stage (60.0%, 68.7%) ; followed by early adolescence (32.7%, 24.7%) and late adolescence (7.0%, 6.7%) stage of male and female students, respectively. the five tools used in this study were as follows, the personal information profile questionnaire, taylor s manifest anxiety scale (mas) (14), frustration test (ft) (15), aggression scale (as) (16) and adolescent adjustment inventory (aai) (17). the reliability coefficient values were calculated based on the data obtained from the subjects, based on the translated test and finally compared with the values given in standardized instruments. twenty - five students were administered to take the test after three weeks to calculate reliability. discriminate validity was determined by the agreement or association among variables in a tool and was calculated by spearman s correlation coefficient value between major variables. it was judged on the basis of whether an indicator corresponds to theoretical expectations in terms of relationships with other variables in a tool (18). there were four variables in these tools, anxiety, frustration, aggression and adjustment. all the values were within an acceptable range due to not having any values less than 0.3 (19). this questionnaire was used to collect data regarding the individual aspects of the students, namely, gender, age, class, number of siblings, nature and reason of impairment, type of birth, residential area, monthly income, economic status of the family, parent s marriage details and number of psycho - physiological disorders. the major impairment categories consisted of 1) due to congenital reasons, 2) due to sickness and 3) due to accident. in 1953, taylor published his results of an experiment in which the performance of adults in a classical conditioning situation was found to be related to the scores on a questionnaire relating to anxiety. the taylor s manifest anxiety scale (14), one of the earliest self - report inventories, has been used extensively in experimental results. t (for true) or f (for false) answers for each of the 50 statements. a score that was higher than average (14 or 15) indicated high degree of anxiety. low scores indicated low level of anxiety and scores much higher than the average suggested that the person experienced an unusually high degree of anxiety. taylor reported retest correlations of 0.89, 0.82 and 0.81 over intervals of three weeks, five months and nine to seventeen months, respectively. the split half and test - retest reliability coefficients were found to be 0.92 and 0.87, respectively. frustration test studies the effect of frustration upon the quality of a person s behavior as a whole. scores above 35 in each of the categories are matters of concern because of high frustration level. this scale consists of 40 items, out of which each of the four modes of frustration has 10 items. the split half test - retest reliability coefficients were found to be : regression (0.78), fixation (0.92), aggression (0.87) and as a whole (0.88). aggression has been defined as an act whose goal response is injury to an organism or organism - surrogate (20). frustration produces instigations to a number of different types of responses, one of which is instigation to some form of aggression. each of the items has five alternate answers (multiple choice) graded on a five - point scale on the positive dimension and a zero point on the negative dimension. this test is valid for the measurement of aggression of 11 to 24-years - olds. average, 46 to 60 as low and scores of 45 and below as clean. the split half and test - retest reliability coefficients were found to be 0.82 and 0.78, respectively. the adolescent adjustment inventory (17) is one of the well - standardized inventories for adolescents. high scores indicate mal - adjustment and low scores indicate that the subject is well adjusted. the odd - even reliability coefficients were 0.84 and 0.95 for personal and social adjustment, respectively, after applying spearman s brown correction. the collected primary data were codified and the data were fed into a computer database using statistical package for social sciences (spss for windows ; version 10.0.1, spss inc.). chi - square test, t test, one - way analysis of variance (anova) and pearson s correlations were conducted to testify the hypothesized tests. descriptive statistical analysis was performed with scores obtained from the 150 visually impaired adolescent students. it was found that anxiety had a significant influence on adjustment (social and personal) level among visually impaired students (= 185.66, df = 4, p = 0 at p < 0.01). overall, the data revealed the prevalence of moderate levels of both adjustment and anxiety among the 150 visually impaired students (table 2). therefore, the hypothesis stating that anxiety level of the visually impaired students will make a significant difference in their social and personal adjustment scores was accepted. frustration had a significant influence on adjustment (social and personal) level among visually impaired students (= 167.23, df = 4, p = 0 at p < 0.01) (table 3). overall, the data revealed the predominance of moderate levels of both adjustment and frustration among the 150 visually impaired students. therefore, the hypothesis stating that frustration level of the visually impaired students will make a significant difference in their social and personal adjustment scores was retained. it was found that aggression had a significant influence on adjustment (social and personal) level among the visually impaired students (= 57.66, df = 4, p = 0 at p < 0.01) (table 4). overall, the data revealed moderate levels of adjustment and low levels of aggression among the respondents. hence, the hypothesis, stating that aggression level of the visually impaired students will make significant differences in their social and personal adjustment scores, was accepted. gender had a significant effect on anxiety level of the visually impaired students (value = 36.125, df = 2, p = 0 at p < 0.01) (table 5). the majority of the female respondents were moderately anxious, when compared to the male respondents. gender had no significant influence on the frustration level of the students (value = 7.142, df = 2, p = 0.03 at p < 0.05). gender significantly affected aggression level of the visually impaired students (value = 77.75, df = 2, p = 0 at p < 0.01). the majority of the female respondents (n = 70) were less aggressive, when compared to the male respondents (n= 12). gender significantly affected the adjustment (social and personal) score of the visually impaired students (value = 14.19, df = 2, p = 0 at p < 0.01). among the 150 visually impaired respondents, most of the female (n = 61) and male (n = 43) respondents possessed moderate adjustment scores. overall, the data revealed the prevalence of moderate levels of adjustment (social and personal) in both male and female respondents. there was a significant correlation between age and adjustment level of the visually impaired students (r = 0.023, n = 150, p = 0.779, two tailed). there was no significant correlation between age and anxiety level of the visually impaired students (r = 0.027, n = 150, p = 0.741, two tailed). frustration of the visually impaired students was not significantly correlated with their age (r = - 0.028, n = 150, p = 0.731, two tailed) and similarly, aggression of the visually impaired students was also not significantly correlated with their age (r = 0.093, n = 150, p = 0.258, two tailed). there was a significant positive correlation between adjustment scores and anxiety level of the visually impaired students (r = 0.919, n = 150, p = 0 at p < 0.01, two tailed). visually impaired students frustration and aggression level were also positively correlated with social and personal adjustment scores (r = 0.887, n = 150, p = 0 at p < 0.01, two tailed) and (r = 0.664, n = 150, p = 0 at p < 0.01, two tailed), respectively. the correlation between anxiety and the frustration level of the visually impaired students (r = 0. 961, n = 150, p = 0 at p < 0.01, two tailed) were positive and statistically significant. similarly, anxiety and aggression level of the visually impaired students (r = 0.727, n = 150, p = 0 at p < 0.01, two tailed) were statistically significant and positively correlated with each other. the correlation between frustration and aggression level of the visually impaired adolescents (r = 0. 637, n = 150, p = 0 at p < 0.01, two tailed) were found to be positive and statistically significant (table 6). the respondents were interviewed and their recent case history was obtained from the class teachers, and the presence of disorders was determined by enquiring the subjects directly and having discussions with their teacher. among the 150 respondents, 29 twenty - nine did not suffer from any disorder with a low mean frustration score of 24.03. twenty - six respondents had one psycho - physiological disorder with a mean frustration score of 67.38. fifty - two respondents had three psycho - physiological disorders with a mean frustration score of 84.44. twenty - nine students did not have any disorder with a mean aggression score of 43.58. twenty - nine respondents did not have any disorder with a mean adjustment score of 48.17. twenty - six had one psycho - physiological disorder with a mean adjustment score of 84.42. the respondents were interviewed and their recent case history was obtained from the class teachers, and the presence of disorders was determined by enquiring the subjects directly and having discussions with their teacher. among the 150 respondents, twenty - nine did not suffer from any disorder with a low mean frustration score of 24.03. twenty - six respondents had one psycho - physiological disorder with a mean frustration score of 67.38. fifty - two respondents had three psycho - physiological disorders with a mean frustration score of 84.44. twenty - nine students did not have any disorder with a mean aggression score of 43.58. twenty - nine respondents did not have any disorder with a mean adjustment score of 48.17. twenty - six had one psycho - physiological disorder with a mean adjustment score of 84.42. in this study, an effort was made to investigate the psychological problems of visually impaired adolescent students. in general, when the cause for impairment was analyzed among the students, nearly half of the visually impaired respondents (48.0%) were blind due to inborn deformities. in general, the data revealed the prevalence of medium levels of social and personal adjustment, anxiety, frustration and aggression among the respondents. it was confirmed that when there is an increase in anxiety, there is proportionate increase in maladjustment or poor adjustment. the adolescent stages did not have a significant relationship with the student s adjustment, anxiety, frustration and aggression levels. the above results were in line with the findings of already reported studies (9, 21, 22). it could be deduced that most of the visually impaired adolescents make appropriate social and personal adjustments based on their level of frustration. in general, when there is an increased level of frustration (depression), there is an increase in maladjustment or poor adjustment. the rate of adjustment (personal and social) varies in accordance with the rate of aggression (23). there was a positive correlation between adjustment and anxiety level among visually impaired adolescents and the adjustment parameter did not have the same relationship with frustration and aggression level among the respondents. furthermore, escalation in the level of anxiety tends to increase the aggression level, which depicts that there is a strong relationship between anxiety and aggression level among visually impaired adolescents. there as a relationship between frustration and aggression level among visually impaired adolescents. when they have increased frustration they tend to be aggressive in nature this might be due to their inabilities to overcome the frustration hence they develop a defensive attitude. the findings also indicated that there is a prevalence of learning disability, communication disorders and educational retardation due to hearing loss. gender of the visually impaired students does have a significant relationship with anxiety, frustration, aggression and adjustment level. the tendency of aggression among the male students might be due to the existence of a male dominant society. they have been conditioned knowingly or unknowingly to behave aggressively if their desires or interests or needs are not actualized. this assertion could be compared with the study on the impact of emotional maturity on stress and self - confidence of adolescents, which projects that adolescent boys tend to have significantly higher stress than girls and girls tend to have significantly higher self - confidence (25). it could be deduced that gender and frustration level among visually impaired students are independent. a significant portion of the visually impaired students irrespective of their gender identity had been experiencing frustration. however, most of the visually impaired students with respect to their gender identity had increased or decreased levels of anxiety, aggression and adjustment. in contrary to the above findings, there was no significant gender difference with respect to psychological problems among visually impaired subjects (26). it is evident that there is a strong relationship between the occurrence of psycho - physiological disorders and anxiety, frustration, aggression and adjustment among visually impaired students. increase in the scores of psychological disorders as mentioned above is directly related to an increase in the number of psycho - physiological disorders among visually impaired adolescents. it was found that the irritable bowel syndrome (ibs) was associated with a higher frequency of generalized anxiety disorder (28). it was also reported that there was a strong significant relationship between depression and anxiety with headaches and irritable bowel syndrome (ibs) (29). when the relationship between anger and psycho - physiological disorders was examined, focusing mostly on anger when it is suppressed or held in rather than expressed behaviorally, as predicted, tension headache sufferers were found to have significantly more anger than non - pair controls (30). the psychosocial implications for better understanding of psychological and psycho - physiological problems can be drawn from this study. assessing anxiety, frustration, aggression and adjustment level of adolescents may facilitate teachers and parents to get to know the level of psychological problems and help the affected individuals overcome the problems with the help of counselors. the teachers, parents and counselors can easily understand the demographic variables and how far they are related to the students psychological problems. this study indicated that the students who are affected by psycho - somatic disorders may realize the cause for the disorders might be due to maladjustment, anxiety, frustration and aggression. awareness programs can be put in place for teachers, parents and students to understand the psychological problems of the subjects. collection of data was done individually for the visually impaired students with strain, spending more time was considered as a major limitation of this study. in the future, this work can be extended by including independent variables such as neurotic tendencies and phobias of the subjects. the dependent variables like scholastic performance, creativity, psychosomatic problems like dermatitis, skin eruptions, eating disorders, backache, hiccoughs and menstrual disturbances can also be included. a similar study can also be done for orthopedic students including all the above variables.	background : visual impairment tends to evoke more discomfiture than any other disability. primarily, the biggest issue may be that blindness is visible. furthermore, visual impairment develops serious medical, psychological, social and economic problems.objectives:the focus of the current study was to investigate the psychological and psycho physiological problems of visually impaired adolescent students.patients and methods : purposive sampling was adopted to select 150 visually impaired students (71 males and 72 females) from five schools in coimbatore city of the tamil nadu state, india. anxiety, frustration, aggression and social and personal adjustment levels of the visually impaired students were measured in this study using taylor s manifest anxiety scale, frustration test, aggression scale and the adolescent adjustment inventory, respectively.results:anxiety (2 = 185.66, p = 0 at p < 0.01), frustration (2 = 167.23, p = 0 at p < 0.01) and aggression (2 = 57.66, p = 0 at p < 0.01) were significantly related to adjustment among visually impaired students. the adjustment score had a significant positive correlation with anxiety (r = 0.919, p = 0 at p < 0.01), frustration (r = 0.887, p = 0 at p < 0.01) and aggression levels (r = 0.664, p = 0 at p < 0.01), anxiety was significantly correlated with frustration (r = 0. 961, p = 0 at p < 0.01) and aggression levels (r = 0.727, p < 0.01) and frustration was significantly correlated with aggression level (r = 0. 637, p = 0 at p < 0.01) of visually impaired adolescents. there was a positive relationship between psycho - physiological disorders and anxiety frustration, aggression and adjustment among visually impaired students.conclusions:visually impaired students exhibited significant levels of psychological and psycho - physiological problems.
the nomenclature used to describe the gastric pathology in the mouse stomach has never been standardized. inconsistent terminology hinders progress toward developing and interpreting models that can help elucidate the molecular and cellular progression of metaplastic pathology. establishing terminology that is specific for particular pathologic features is necessary to accurately classify cellular changes at key stages. later, we attempt to define the most commonly observed lesions in a way that we hope will guide interpretation of future experiments. first, however, we must define some key pathology terms we use to describe the lesions : hyperplasia, metaplasia, and dysplasia. hyperplasia refers to a pathologic lesion characterized by expansion of a normal cell lineage that resides in the tissue where it normally is found. metaplasia refers to the presence of an otherwise normal cell lineage (or lineages) in a tissue where such a lineage is not normally found. dysplasia is the presence of cells with abnormal cellular features and implies that the cells, which could resemble either normal or metaplastic lineages, have acquired mutations or epigenetic alterations that provide increased risk for malignant (eg, invasive) progression. parietal cells, whose primary job is to secrete acid, also are known as oxyntic cells. thus, oxyntic atrophy is the pathologic process characterized by loss of parietal cells. in human beings and mouse models, loss of parietal cells usually correlates with the onset of metaplastic lesions, such that oxyntic atrophy has been termed the sine qua non for metaplasia.1, 2 in human beings and mice, chronic helicobacter infection can lead to loss of parietal cells in the corpus of the stomach.3, 4, 5, 6 oxyntic atrophy is diagnosed easily on h&e staining because the absence of highly eosinophilic parietal cells is obvious. during oxyntic atrophy, mature chief cells (digestive enzyme secreting or zymogenic), which are mixed h&e positive, also are absent. work in mouse models and human beings suggests that the loss of mature chief cells may not simply be because they all die similar to parietal cells, but rather that chief cells, in response to loss of parietal cells, change their differentiation state. specifically, they reprogram into metaplastic mucous cells.7, 8, 9, 10, 11 such a reprogramming of cell fate also is known as transdifferentiation. for a more definitive analysis beyond h&e, cell - type and lineage - specific markers can be used with immunofluorescent or immunohistochemical techniques : for example, antibodies against the proton pump, h+/k+adenosine triphosphatase (atpase) (or subunit) will label only mature parietal cells, whereas antibodies against the basic helix - loop - helix transcription factor, mist1 (a15), will label only chief cells.2, 7, 12 foveolar cells are the simple columnar mucous cells lining the surface of the stomach and extending downward toward the gastric gland (figure 1). they face the harshest conditions, being closest to the lumen of the stomach, and turn over the fastest.13, 14 gastric units are shaped roughly like a funnel, with the glandular portion (the part with the parietal and chief cells) below the neck of the funnel, and the foveolar cells in the wide mouth. foveolar hyperplasia (figure 1) usually is associated with an increase in proliferation in the normal progenitor cells in the isthmus of the gastric unit. a common cause of foveolar hyperplasia in mice and human beings is an increase of gastrin. increased signaling through the epidermal growth factor (egf) receptor (eg, by increased abundance of its ligand transforming growth factor) also causes foveolar hyperplasia ; human mntrier disease is caused by such overactive signaling.17, 18 interestingly, oxyntic atrophy and foveolar hyperplasia often are linked. long - term loss of parietal cells causes decreased stomach acid (hypochlorhydria), which causes gastrin - secreting cells in the antrum of the stomach (g cells) to secrete gastrin in an attempt to stimulate parietal cell function. mucous neck cell hyperplasia connotes expansion of normal mucous neck cells (figure 1). a related term often used by pathologists, especially veterinary pathologists, is mucous metaplasia, which often may be the same lesion as mucous neck cell hyperplasia. a reason for the possible confusion is that mucous metaplasia typically is diagnosed by conventional histochemical staining (h&e, periodic acid it describes a lesion characterized by abnormally increased numbers of mucous - expressing neck cells in the glands of the stomach (ie, not in the foveolar / pit region). in our experience, thus, mucous metaplasia is often a misnomer because no new (metaplastic) cell lineages are found in the stomach. mucous neck cells may have a metaplastic look when they expand markedly because in the normal stomach they usually are difficult to see, given that their cytoplasms do not stain with h&e, and those nonstaining cytoplasms are localized predominantly in the lumen of the gland because parietal cells occupy the vast majority of the basement membrane. when mucous neck cells expand significantly, they do so at the expense of parietal cells, giving a morphology that appears as if a new population of cells has appeared. on immunohistochemical or immunofluorescent staining, however, the cells in these lesions label exclusively with mucous neck cell lineage markers (including griffonia simplicifolia lectin (gs - ii), trefoil factor 2 (tff2), mucin 6 (muc6), and gastrokine 3 (gkn3)).21, 22 thus, this lesion is best described as mucous neck cell hyperplasia. mucous neck cell hyperplasia has been reported in a number of settings with alterations of ion channels (eg, loss of kvlqt1) or endocrine cell influences. in our experience the expansion of mucous neck cells usually is not associated with markedly increased proliferation within the mucous neck cell population, but rather appears to reflect either an increased production of mucous neck cells from multipotent progenitors or a slowing of differentiation of mucous neck cells into chief cells.7, 23 it is important to note that combinations of mucous neck cell hyperplasia can exist in association with other truly metaplastic lesions. for example, spasmolytic polypeptide (tff2)-expressing metaplasia (spem), which will be discussed later, often is characterized by abundant mucous - containing cells (figure 1). spem may look like mucous neck cell hyperplasia on histochemical stain, and it can combine with mucous neck cell hyperplasia. loss of parietal cells in mice or human beings correlates with the induction of metaplasia in the corpus glands.2, 7, 10, 12, 24, 25, 26 in particular, as mentioned previously, this oxyntic atrophy is characterized by loss of mature chief cells. as mentioned earlier, mouse models and examination of human tissue suggest that many of these chief cells do not die, but actually transdifferentiate or reprogram into a metaplastic mucous - secreting lineage : spem. spem shares many features with the cells that populate the gland in embryonic and juvenile stomach.7, 11, 12, 27 namely, spem is characterized by expression of lower levels of chief cell proteins (such as the digestive enzyme pepsinogen c and, in mice, gastric intrinsic factor), with markers of mucous neck cells (tff2, gkn3, and muc6) (figure 2). the pattern of corpus units that have no parietal cells and abundant basal cells co - expressing chief and neck cell markers is similar to the organization, at least at the superficial level, of units in the more distal stomach (the antrum or pylorus) (figure 1). spem also may represent a reparative lineage equivalent to the ulcer - associated lineages identified in association with healing mucosa in inflammatory bowel disease. indeed, recent studies have noted that spem develops at the edges of healing gastric ulcers and contributes to ulcer healing.30, 31 as mentioned, it is thought that spem cells arise in part via reprogramming / transdifferentiation of chief cells. the key evidence is that lineage mapping studies in mice using the mist1 reporter of chief cell differentiation have shown that spem cells emerging during loss of parietal cells were once mist1-positive (ie, they were chief cells). the chief cells that reprogram after loss of parietal cells down - regulate expression of chief cell differentiation markers (eg, the endogenous mist1 gene) and begin to express high levels of proteins that were expressed in mucous neck cell lineages, including tff2 and muc6.25, 26, 32 thus, the metaplastic cells can be identified in the base of gastric glands (the normal niche for chief cells) by strong immunolabeling for tff2, which is the origin for the moniker spem.33, 34, 35 this lineage often can be identified by pink staining in diastase - resistant periodic acid most importantly, spem glands usually show, especially at their bases, hybrid cells, which express both chief cell markers (eg, intrinsic factor in mice) and mucous cell markers (eg, tff2, muc6, or gs - ii lectin).2, 25, 26, 36 spem is a true metaplasia because spem - type cells are not normal corpus lineages, but resemble deep antral gland cells. spem can not be determined definitively in the antrum, where deep mucous gland cells show many of the same characteristics. it is a lesion specifically of glands from the corpus / body (also known as fundic - type glands) that develop metaplasia. spem likely occurs with similar characteristics in all mammals, but has been documented definitively in mice, rats, gerbils, and human beings.10, 33, 34, 35, 37 the organization of spem glands, as mentioned, superficially resembles the antrum, with tff2-positive cells also labeling with a lower abundance of chief cell markers (gastricintrinsic factor in mice, or pepsinogen c), and the center of proliferation moves from near the gastric lumen (at the isthmus, where the funnel of pit / foveolar cells meet the deeper glandular cells) in the normal oxyntic gland, toward the base in spem glands. spem cells often show proliferation with tff2/gif dual - positive cells also labeling with ki-67 or nucleotide analogs such as bromodeoxyuridine, especially in the presence of inflammation. investigations have identified some biomarkers of spem that do not appear in normal corpus lineages including he-4 (wfdc2) and cd44 variant 9. it also is clear in human patients and in animal models that morphologically apparent spem lineages often can show varying levels of intestinalizing transcripts including tff3, mal2, cftr, and muc4.36, 39 this intestinalized spem is morphologically identical to other spem lineages and can be recognized only by immunostaining with specific intestinal markers. although both spem and intestinal metaplasia are present in the stomachs of human beings with atrophic gastritis secondary to h pylori infection,2, 12 few instances of documented intestinal metaplasia exist in mouse models. in human stomach, alcian blue staining of intestinal - type goblet cells often is used to define intestinal metaplasia. however, murine deep antral mucous cells and spem often both are strongly alcian blue positive. muc2 and tff3 labeling of cells with intestinal goblet cell like morphology provides the most specific indication of intestinal metaplasia (figure 2c). notably, expression of tff3 and muc2 has been reported in lineages with immunocytochemical evidence of spem, so there may be intermediates of intestinalized spem that reflect evolution of metaplastic phenotypes.36, 40 expression of nuclear cdx1 and cdx2 has been noted in human intestinal metaplasia, but has been less apparent in mouse models. more recently, cdx1-positive cells and muc2-positive goblet cells have been observed in mist1-kras (g12d) mice, but cdx2 expression was not observed. the expression of cdx1 rather than cdx2 may reflect the more prominent expression of cdx1 in the duodenum, consistent with the concept that intestinal metaplasia in the stomach may represent more specifically a indeed, previous studies have suggested that intestinal metaplasia in the human corpus may reflect a duodenal lineage paradigm based on pdx1 expression. it is notable that forced expression of the intestinal master regulatory transcription factor cdx2 in the stomach caused complete intestinalization of the stomach mucosa.45, 46 however, it is difficult to interpret these results within the context of adult - onset metaplasia because the gastric epithelium in these mice was specified during development with forced expression of an intestine - promoting transcription factor in cells that otherwise would have become stomach. thus, the epithelium in this case simply may be (developmentally) intestine, rather than metaplastic stomach. only specific marker staining can differentiate between spem with intestinalizing characteristics and frank intestinal metaplasia with the presence of goblet cells and villin - expressing absorptive cells. the presence of glands penetrating into the submucosa (sometimes referred to as gastritis cystica profunda) usually is considered a dysplastic lesion in human beings. for example, the presence of gastritis cystica profunda in human beings has been noted decades after gastrojejunostomy for ulcer disease and is considered a preneoplastic lesion. although invasive submucosal glands have been reported in h pylori infected mongolian gerbils, some investigations have indicated that invasive glands are reversible after bacterial eradication. recent detailed studies in gerbils have noted that invasive submucosal glands usually are connected to spem glands within the mucosa. numerous mouse models have assumed that invasive glands are synonymous with dysplasia or even invasive neoplasia (malignancy).3, 4, 50 however, we are unaware of any models in which investigators have shown truly neoplastic qualities in these lesions. as noted in mouse models of colon cancer, in our experience, the mouse stomach has a propensity for extension of metaplastic lesions. in some occasions, tremendous proliferation in the epithelium can result in cross - sectional cuts of the tissue that appear invasive but are not. in other cases, the epithelial cells in the mucosa actually may breach the relatively thin muscularis mucosa of the mouse stomach. although these glands thus technically might appear to be invasive, the examples we have observed in the literature and in our own studies do not show any additional features of neoplasia. in other words, such invasive glands remain symmetrical and are indistinguishable from surrounding metaplastic glands that are not apparently in the submucosa. furthermore, the glands in question typically do not show other signs of dysplasia / neoplasia, such as : loss of nuclear polarity, cells piling up with nuclear crowding, irregular gland forms with an invasive - seeming leading edge, nuclear atypia, or surrounding stromal hyperplasia (desmoplasia). in addition, these invasive glands usually are populated by tff2-positive lineages (figure 2d) and do not show proliferative rates any greater than associated intramucosal spem. furthermore, we are unaware of models showing metastatic behavior typical of invasive adenocarcinomas of the stomach in human beings. thus, there is no evidence that such glands actually invade the way human tumors do. therefore, the neoplastic implications of seemingly invasive submucosal glands, which might be assumed in human beings to be premalignant lesions, are unclear in mouse models. the interpretation of dysplasia therefore is fraught with controversy even among human pathologists diagnosing lesions in patients. the importance of a dysplasia diagnosis is essentially that it is prognostic for increased progression to cancer. given that no actual metastatic or truly invasive adenocarcinoma models in the mouse stomach seem to exist, it is difficult to define the term dysplasia in a standard way in mouse models. if used, it must be strictly defined based on which atypical, potentially neoplastic features are present in the lesion in question. in human beings, the aberrant morphologies generally are defined by patterns in h&e staining, including the following : multilayered cells lacking polarity, complex glandular patterns with irregular intervening lumens (back - to - back cribriform glands), altered and inconsistent nuclear morphology and positioning, and irregular chromatin patterns (eg, prominent nucleoli). proliferative rates are expected to be high, but metaplastic lineages also can show high proliferative rates. no present studies have defined specific mutations that might define a transition to cancer in mice. to our knowledge, there is only 1 purely genetically driven mouse model of epithelial carcinogenesis that results in invasive metastatic tumors that invade to the serosal surface (and do not just form the seemingly invasive submucosal glands discussed earlier) and then metastasize to regional lymph nodes.52, 53 in this model, large t antigen was expressed as a transgene in parietal cell progenitors, which eventually led to an invasive carcinoma that was always neuroendocrine in morphology and molecular phenotype. no specific model of intestinal type cancer in the gastric body that is driven by a controlled genotype of mouse (as opposed to tumors induced by mutagens) has been reported. h pylori is a gram - negative bacteria that inhabits more than 50% of the global population. chronic h pylori infection can trigger changes in the fundic mucosa that correlate with increased risk for a subset of individuals to gastric adenocarcinoma. most pathologists recognize chronic atrophic gastritis as the first lesion indicating an increased risk for progression of an h pylori infection to gastric cancer. this lesion pairs a chronic immune cell infiltrate (including resident plasma cells, establishment of lymphoid follicles) with oxyntic atrophy (loss of parietal cells). work over the past several years by our groups and others in both human beings47, 56, 57 and rodents26, 34, 35 has established that spem is a common metaplastic phenotype observed in the atrophic human and rodent stomachs. in the setting of a chronic inflammatory infiltrate that does not resolve in human beings, spem may progress to intestinal metaplasia.2, 12 correa originally described the association of intestinal metaplasia with the development of intestinal - type gastric cancer. when intestinal - type gastric adenocarcinoma develops, it usually occurs in the setting of both spem and intestinal metaplasia lesions. thus, the presence of metaplastic cell lineages in the stomach carries increased risk for progression to neoplasia. the adenocarcinomas caused by h pylori in this setting are exceedingly heterogeneous from patient to patient, but they vary on a spectrum from neoplastic glands with more obviously gastric differentiation to ones with obvious intestinal differentiation.54, 56 in general, these types of gastric cancers originally were referred to as intestinal - type because they arise in the background of intestinal metaplasia. it is easy to assume that the cancers, which have varying degrees of intestinal morphology, thus arise from the intestinal metaplasia, but that may be too simplistic an interpretation.12, 32 an alternative view, for example, is that inducing postmitotic, differentiated cells to re - enter the cell cycle is an inherently risky process that can induce, unmask, or generate mutations owing to massive changes in the chromatin landscape. thus, abundant metaplasia simply may reflect a sign of abundant, risky, reprogramming events.11, 60 a review of studies over the past 20 years that show spem phenotypes in association with varying degrees of gastric neoplasia is detailed later. in particular, we review the pathologic phenotypes of metaplasia in these models (summarized in table 1) and how they inform our knowledge of the evolution of preneoplastic lesions. mice infected with helicobacter felis recapitulate much of the pathology associated with h pylori infection observed in human beings, including parietal cell loss and metaplasia, and these bacteria have been used for years specifically in the c57bl/6 mouse strain to study metaplasia.4, 5, 6, 61 mouse - adapted clinical isolates of h pylori from infected human beings also can be used to recapitulate the chronic infection and metaplasia seen in patients. the host inflammatory response, however, is thought to be critical both in human beings and in mouse models.3, 36, 63, 64, 65 in general, the immune response can be categorized along 2 distinct paradigms : innate and adaptive. innate immunity includes the epithelial cells themselves (eg, parietal cells produce tremendous microbial killing via acid) along with phagocytic cells such as monocytes and granulocytes, in particular neutrophils, that are recruited rapidly to areas of injury or infection. the slower, adaptive immune response is associated with antibody - producing b cells, antigen - recognizing cd4 t - cells, and cytotoxic cd8 t cells. studies in the early 1990s focused on understanding how the immune system contributed to the development of gastric pathology associated with helicobacter infection. the inflammatory infiltrate observed in h felis infected mice is mixed, containing populations of lymphocytes, neutrophils, and macrophages within the metaplastic mucosa. roth identified that t cells were necessary for helicobacter - associated parietal cell loss because t - cell deficient mice did not develop oxyntic atrophy or metaplasia. murine models also have aided in the further understanding of metaplastic events occurring after parietal cell loss. six months after helicobacter infection, mice developed oxyntic atrophy and tff2-expressing metaplasia.5, 6 in further studies, the mucous cell lineage expansion was identified as spem, derived from the transdifferentiation of mature chief cells. spem is highly proliferative throughout the corpus in h felis infected mice.5, 6, 37 although the metaplasia is extensive, intestinal metaplasia as seen in human beings is notably absent from helicobacter - infected mice. however, there is evidence for intestinalization of spem during chronic h felis and h pylori infection.35, 37, 40 a caveat of using helicobacter infection to study oxyntic atrophy and metaplasia is that alterations, especially to the immune system, may affect the competency of helicobacter to induce parietal cell loss, a prerequisite for metaplasia development. infection models also induce large - scale metaplasia slowly (after approximately 6 months) and asynchronously. acute drug treatments that induce parietal cell death directly have some advantages in that they rapidly and synchronously induce metaplasia, bypassing chronic immune mechanisms usually required for parietal cell loss. this rapid and direct induction of spem allows study of specific stages of metaplasia using both molecular and histologic techniques. treatment with the parietal cell specific protonophore drug dmp-777 leads to rapid loss of parietal cells within 3 days, followed by induction of spem after 1014 days of treatment.10, 34 loss of parietal cells is abrogated when animals are pretreated with omeprazole before dmp-777 administration. although no proliferative chief cells were identified in the normal mucosa, scattered proliferative spem cells were identified after dmp-777 treatment. interestingly, in both mice and rats, administration of dmp-777 for up to 2 years led to prominent continuous metaplasia, but never resulted in any dysplastic or neoplastic lesions. the absence of dysplastic lesions in the face of chronic metaplasia appears to be caused by a lack of inflammation, likely because of the other action of dmp-777 as a cell - permeant elastase inhibitor that could serve to block neutrophil function and innate immunity.10, 34, 37 mice given 3 consecutive daily doses of 5 mg of fully emulsified bolus doses of tamoxifen had a 90% reduction in parietal cell mass in the stomach by 3 days after the first injection.26, 69 high - dose tamoxifen - treated mice also developed spem within 3 days of treatment with scattered proliferative spem cells identified. the pattern of spem induction by high - dose tamoxifen resembles that induced by dmp-777 and also is ameliorated by omeprazole. l635 is a chiral molecular cousin of dmp-777, which retains parietal cell protonophore activity, but lacks the neutrophil elastase inhibitory capacity. l635 treatment causes rapid induction of parietal cell loss and spem in the presence of a prominent inflammatory milieu. the rapid induction of oxyntic atrophy combined with a strong inflammatory infiltrate in the gastric mucosa leads to accelerated development of a highly proliferative and intestinalized spem lineage within 3 days of treatment that is remarkably similar to spem observed after infection with h felis. therefore, l635 treatment provides a rapid model for the development of highly proliferative intestinalizing spem. located in the antrum, g cells produce gastrin to both regulate gland homeostasis and promote acid secretion from parietal cells, primarily through the stimulation of histamine release from enterochromaffin - like cells.71, 72 gastrin also functions as a growth factor for pit cells and exerts anti - apoptotic activity at the transcriptional level.73, 74, 75 in the stomach, endogenous gastrin levels increase in response to parietal cell loss and increase epithelial cell proliferation and foveolar hyperplasia. the insulin - gastrin mouse model uses the mouse insulin promoter to drive expression of a human gastrin transgene, producing moderate increases in circulating gastrin levels. the insulin - gastrin mice on the fvb background develop spem and then submucosal lesions in the gastric corpus that express tff2 by 20 months of age. although the cause of parietal cell loss is unclear, a chronic state of acid hypersecretion may contribute to the oxyntic atrophy. gastrin null mice develop antral tumors by 12 months of age.77, 78 in the corpus, the mucosa generally is thinner, with fewer parietal cells present. dmp-777 treatment in gastrin null mice causes an accelerated induction of spem in 13 days, as compared with 10 days in wild - type mice.10, 25 however, gastrin null mice do not develop foveolar hyperplasia, which typically occurs as a result of oxyntic atrophy, consistent with the concept that gastrin is a major driver of foveolar hyperplasia. several mouse models induce changes spontaneously, consistent with the development of spem in the corpus of the stomach. these models may initiate with a normal mucosa and then develop increasing levels of atrophy and metaplasia. claudin 18 levels are high in the normal stomach, but prominently are reduced in the metaplastic stomach. hayashi examined the impact of claudin-18 loss in the stomachs of claudin 18 null mice. these mice showed few parietal cells at birth and never developed a normal complement of parietal cells during the postnatal period. instead, claudin 18 null mice showed proliferative spem that was associated with a significant neutrophil infiltrate and increases in il1 expression. the claudin 18 null stomachs also showed high levels of the spem marker he-4 (wfdc2). although the metaplastic spem lineages in the claudin 18 null mice were highly proliferative, these mice were studied only up to 16 weeks of age, at which time no invasive or dysplastic lesions were reported. kruppel - like factor 4 is down - regulated in gastric cancers, and loss of kruppel - like factor 4 is associated with poor prognosis.80, 81 in the kruppel - like factor 4 null mouse, the stomach shows marked oxyntic atrophy from birth and both foveolar hyperplasia and mucous cell metaplasia. some intrinsic factor expressing cells remain at the bases of corpus glands that are dominated by a tff2-expressing mucous cell lineage. although no dual labeling for intrinsic factor and tff2 was performed, the staining for tff2 appeared at the base of the glands, consistent with the suggestion that these glands represented spem. interestingly, however, the investigators noted that there was little proliferation among the spem lineages and even at up to a year of age, no intestinal metaplasia, dysplasia, or invasive lesions developed. thus, this model resembles the stable benign metaplasia after long - term administration of dmp-777. runx3 is a transcription factor that is highly expressed in chief cells with lower levels of expression in surface cells and mucous neck cells. interestingly, runx3 null mice do maintain parietal cells, although they appear small and acid secretion is variable. intestinalized spem can be observed with expression of muc2 and cdx2 in deep gland regions, but no goblet cells appear to be present. these findings are consistent with an association of runx3 loss with intestinalization.85, 86 although runx3 null mice do not develop any evidence of dysplasia or penetrating glands, they do show increased susceptibility to n - methyl - n - nitrosourea (mnu)-induced dysplasia in the corpus. thus, this model suggests that regulation of intestinalization focused in chief cell lineages may promote metaplastic progression without complete parietal cell loss. the phenotype of this model also may show aspects of mucous neck cell hyperplasia accentuated by the small size of remaining parietal cells. increased cyclic adenosine monophosphate is the principal driver of parietal cell acid secretion mediated through activation of the h2-histamine receptor. lopez - diaz examined a model of chronic acid overproduction in the h / k - cholera toxin mouse. this mouse showed increased acid secretion from an early age, as would be expected for parietal cells with increased expression of cholera toxin and increased cyclic adenosine monophosphate production. although the mice showed a normal compendium of cell lineages in the gastric corpus at 2 months of age, by 7 months of age the mice developed increasing levels of parietal cell loss until loss was severe by 1516 months of age. the parietal cell loss at the later time points was associated with the presence of spem. the loss of parietal cells appears to relate to the development of autoantibodies against parietal cell antigens, especially h / k - atpase. no invasive glands were noted in these mice at the later stages, although measures of proliferation were not determined. several studies conducted on characterizing gene and protein expression of metaplasia in the murine and human stomachs have yet to identify a dominant signaling pathway driving neoplasia.25, 37, 89 however, different signaling pathway aberrations in gastric cancer have been characterized, identifying kras, fibroblast growth factor receptor 2, epidermal growth factor receptor (egfr), erbb2, and met using single - nucleotide polymorphism arrays. these and other signaling pathways have been investigated for their role in the development of metaplasia. up - regulation of phospho - erk in metaplasia has been noted in both human beings and mice.43, 91 however, ras mutations have been observed in only approximately 9% of gastric cancers. nevertheless, recent gene - profiling studies have noted a signature for up - regulation of kras activity in more than 40% of intestinal - type human gastric cancers.92, 93 choi recently examined the induction of metaplasia in mist1;lsl - kras(g12d) mice (mist1-kras mice). after tamoxifen induction, these mice expressed activated kras in gastric chief cells, and after 4 weeks developed proliferative metaplasia. three months after induction, intestinal goblet cells expressing muc2 were evident, progressing to invasive glands 4 months after induction. importantly, treatment of the mice with a mek inhibitor, selumetinib, caused arrest of the metaplasia and allowed recrudescence of normal gastric lineages. the normal gastric lineages appeared to cause extrusion of the metaplastic glands from the gastric mucosa into the lumen. these findings indicate that activation of ras is involved at all of the key steps in the development of metaplasia, from transdifferentiation of chief cells into spem, to further differentiation of intestinal metaplasia and the promotion of invasive changes. hayakawa have suggested that mist1;lsl - kras(g12d) mice develop metaplasia from scattered isthmal mist1-expressing cells. this interpretation is at odds with previous studies in k19-kras(g12d)-expressing mice that showed a phenotype dominated by foveolar hyperplasia. matsuo recently confirmed this phenotype of induced foveolar hyperplasia after targeting of kras expression to the isthmal progenitor cells using the runx1 er1 promoter. in that investigation, occasional chief cells also were targeted by er1, and active kras in those cells appeared to give rise to spem. thus, the majority of evidence suggests that ras activation in chief cells leads to the development of spem. smad3 null mice were first characterized extensively for the spontaneous development of colonic adenocarcinoma. although development of colon cancers required colonization of animals with helicobacter hepaticus, observations at the jackson laboratories (bar harbor, me) indicated that gastric tumors evolved in the absence of any infection. detailed analysis of these mice subsequently showed that smad3 null mice developed spem by 6 months of age and invasive lesions in the proximal corpus by 10 months of age. invasive lesions, thought to be derived from the first gland of the corpus, generally showed tff2-positive cells along with large numbers of dclk1-positive tuft cells. these results suggested that loss of smad3 can lead to the development of proximal gastric neoplasia. shinohara sought to evaluate the role of bmp signaling in the differentiation of gastric lineages in the h / k - noggin mouse. inhibition of bmp signaling through overexpression of noggin resulted in a reduction of parietal cell numbers and expansion of spem lineages expressing tff2. although there was increased proliferation in the mucosa, most of this proliferation was abolished when the transgenic mice were crossed onto a gastrin null background, a finding consistent with a role for proliferation in foveolar hyperplasia in these mice rather than spem. importantly, the overexpression of noggin in the stomach caused an augmentation of metaplastic changes after infection with h felis or h pylori. these findings suggest that bmp signaling generally is anti - inflammatory and that loss of this signaling may promote metaplasia development. the role of egfr and its ligands in alterations in gastric mucosal lineage differentiation has been well established. overexpression of tgf in the gastric corpus is associated with severe foveolar hyperplasia in mntrier disease in human beings.17, 101 overexpression of tgf in the stomachs of metalothionein (mt)-tgf mice leads to a similar phenotype.102, 103 these studies all suggested that overexpression of tgf altered the differentiation of gastric progenitors to the production of surface mucous cells over gland cells (ie, parietal, mucous neck, and chief cells). nevertheless, other studies have suggested that alterations in egfr activation can influence the induction of metaplasia. waved-2 mice, which have an attenuating mutation of the egfr, showed enhanced development of spem after loss of parietal cells induced by dmp-777. these effects seemed to be specific to particular egfr ligands because although tgf null mice showed no alteration in spem induction, amphiregulin null mice showed an enhanced induction of spem after dmp-777 treatment, similar to that observed in waved-2 mice. just as interestingly, amphiregulin - deficient mice older than 1 year of age developed spem spontaneously. by 18 months of age, more than 40% of amphiregulin null mice the goblet cell intestinal metaplasia evolved in glands also containing spem, and cells with intermediate morphology with dual expression of tff2 and muc2 were observed at the interface between spem and intestinal metaplasia lineages. these findings showed that egfr - mediated signaling was involved in the evolution of metaplastic lineages. insights into different aspects of the inflammatory response show the impact of immune cells in the progression of metaplasia. the expression of the proinflammatory cytokine interferon (ifn), typically expressed by t - helper (th)1-polarized t cells, is increased in helicobacter - infected mice. transgenic overexpression of ifn using elements of the parietal cell specific driver h / k - atpase (atp4b) promoter caused a prominent inflammatory infiltrate, oxyntic atrophy, epithelial cell proliferation, and spem after 3.5 months. spem and immune infiltration still were present in transgenic (tg)-ifn mice crossed to rag1 mice that lacked mature t and b cells. therefore, ifn alone can drive metaplastic changes in the corpus without the assistance of t or b cells. parietal cells undergo apoptosis in hip1-related (hip1r) mice and develop a robust inflammatory response and spem by 12 months of age. double - knockout mice, null for both hip1r and ifng, have delayed metaplasia development, but mice at 12 months of age appear to have similar phenotypic spem as control hip1r null mice. therefore, ifn is not required for spem induction after parietal cell loss. human autoimmune gastritis occurs when t cells target parietal cells for apoptosis in the stomach, causing robust metaplasia that predisposes affected individuals to endocrine carcinoids. transgenic balb / c mice engineered with cd4 + t cells that recognize the parietal - specific antigen h / k - atpase develop spem by 24 months of age. by 12 months of age, txa23 mice have extensive inflammation with areas of aberrant gland morphology and penetrating submucosal glands. the phenotype observed in this model may differ from most other models of metaplasia because these mice are on a balb / c background.110, 111 specifically, t cells in balb / c mice express higher levels of il4 than ifn, thereby driving a th2-predominant response. most investigations in the stomach use c57bl/6 mice, which tend toward a th1 predominant response wherein t cells express higher levels of ifn. therefore, the type of metaplasia in the txa23 atrophic gastritis model perhaps is indicative of a th2-driven environment paired with parietal cell loss. polymorphisms in the il1b gene are observed in a variety of human, including gastric, adenocarcinomas. il1 is part of the il1 family of cytokines that function as alarmins by promoting the inflammatory response. transgenic mice expressing human il1 using the h / k - atpase promoter develop spontaneous inflammation, oxyntic atrophy, and spem with invasive lesions at late time points. helicobacter increases the susceptibility and speed at which gastric pathology develops in h / k - il1b mice. conversely, il1-receptor antagonist treatment can inhibit the progression of metaplasia and inflammatory cell infiltration. together, these findings support il1 as a major driver of inflammatory cell recruitment that promotes the progression of metaplasia. stromal - derived factor 1 (sdf1) typically is expressed by cancer - associated fibroblasts and is found to be up - regulated in helicobacter - infected gastric mucosa. transgenic mice expressing sdf1 using the h / k - atpase promoter do not show recruitment of inflammatory cells, but show hyperproliferative gastric epithelial cells and focal areas of spem with dilated glands at late time points (18 months). similarly, when tg - sdf1 mice are crossed to proinflammatory tg - il1 mice there is increased epithelial cell proliferation and accelerated spem development in the corpus. these data indicate that although sdf1 does not drive recruitment of inflammatory cells, it does appear to promote epithelial changes and promote spem, especially in the setting of atrophic gastritis. oshima developed transgenic mice expressing ptgs2 and mpges-1 in progenitor cells, driven by elements from the cytokeratin 19 promoter. the resulting mice showed recruitment of m2 polarized macrophages into the mucosa, subsequently causing the development of spem. treatment using the nonsteroidal anti - inflammatory drug meloxicam ameliorated the inflammation and reversed the metaplastic gland morphology. in an effort to determine the role of specific cytokines in this process mice null for tnf had decreased inflammation, reducing metaplasia overall. however, no overt change in inflammation or metaplasia was observed in l1r1 mice. therefore, cyclooxygenase 2 and prostaglandin e2 can drive metaplastic changes in the stomach through the recruitment of m2-polarized macrophages in a tnf-dependent pathway. activated -catenin activity, but not mutations in apc, is observed in 51% of human intestinal - type gastric cancers. k19-promoter expressing cells driving wnt1 expression caused suppression of normal gastric epithelial cell differentiation that was accompanied with increased proliferation and tff2-positive cells. by 7 to 18 weeks the gan mice were developed by crossing k19-wnt1 mice to k19-c2me mice (described earlier), thereby activating wnt signaling in progenitor cells with a coordinated immune cell infiltration. this results in highly proliferative spem that drives the rapid development of tumors in the corpus. furthermore, amelioration of the inflammatory response using a ccl2 chemokine inhibitor or clodronate treatment to deplete macrophages causes tumor regression in gan mice. thus, activation of the wnt signaling pathway can drive metaplasia formation, however, inflammation is necessary for metaplasia progression. il11 belongs to the il6 family of cytokines that induces signal transduction through the il11ra. il11 is not expressed during the early stages of an inflammatory response associated with helicobacter infection, but gradually increases during chronic inflammation and intestinal metaplasia. il11 expression normally is localized to parietal cells, therefore its expression in the stomach is lost when parietal cells die (eg, in the setting of oxyntic atrophy in response to helicobacter infection). nevertheless, exogenous treatment of il11 in wild - type mice leads to parietal cell loss within 24 hours, an infiltration of polymorphonuclear cells, and the emergence of spem. thus, it is hypothesized that il11 is released by dying parietal cells to recruit inflammatory cells into the stomach and promote the development of spem. a member of the il1 family of cytokines, il33 is expressed normally in the gastric mucosa, localizing to cells in the surface cell zone. il33 synergizes to promote a th2 inflammatory response that activates innate and adaptive immune cells. il33 generally is classified as an alarmin because of its release into the extracellular environment by necrotic or apoptotic cells. il33-treated mice develop spem and a robust inflammatory response in the lungs, intestine, and stomach, indicating that il33 is sufficient to recruit inflammatory cells and drive mucous production within respiratory and gut epithelial tissue.64, 119 however, il33 is expressed only in the normal stomach by cells in the surface cell (upper pit / foveolar) zone in the corpus, and those cells are not thought to undergo cell death during helicobacter infection. therefore, although administration of il33 is capable of recruiting inflammatory cells and inducing spem, it remains to be seen if endogenous il33 functions in this capacity. data from mouse models indicate that the induction of metaplasia involves at least 2 phases. parietal cell loss induced by helicobacter infection requires the action of specific lymphocytic populations and specific immune modulatory molecules. in a second phase, after, or concomitant to, the loss of parietal cells, spem develops, at least in part, from transdifferentiation of chief cells. the utilization of a number of drug - induced methods to ablate parietal cells acutely can bypass the initial inflammatory phase required for parietal cell loss. similarly, genetic models, such as directed activation of ras in chief cells, can bypass parietal cell loss to induce metaplasia via transdifferentiation. constitutive activated ras in these metaplastic cells seems to be sufficient to maintain and expand metaplasia, although ras activation also may recruit macrophages to support the progression of metaplasia. mouse models of immune modulator overexpression similarly can induce both parietal cell loss and spem induction. it seems evident that inflammatory mediators are required for induction of more aggressive and proliferative metaplasia. what remains unclear from mouse models is the explicit connection between the metaplastic / atrophic lesions and progression of carcinogenesis. thus far, none of the models of metaplasia progress to true metastatic, or even regionally invasive, adenocarcinoma. in our experience, even the mouse models termed dysplastic seem predominantly to be exuberantly proliferative metaplastic or reactive lesions. with the exception of the mist1-kras mouse and some aged mice on the amphiregulin null background, mouse models of metaplasia also have not faithfully recapitulated the induction of goblet cell containing intestinal metaplasia. this could suggest that extensive intestinal metaplasia as seen in human beings is central to the development of adenocarcinoma. alternatively, intestinal metaplasia in human beings may represent an adaptive response, occurring over years of chronic injury rather than a true preneoplastic lesion. further studies are needed to discern the molecular mediators of key transition points along the metaplasia - to - carcinoma sequence : at the point of transdifferentiation of chief cells into spem, at the transition of spem into intestinal metaplasia, and at the point of true dysplastic transition from within the metaplastic milieu (spem or intestinal metaplasia). moreover, the search will continue for further mouse models that can extend investigations to true models of gastric adenocarcinoma development.	intestinal - type gastric adenocarcinoma evolves in a field of pre - existing metaplasia. over the past 20 years, a number of murine models have been developed to address aspects of the physiology and pathophysiology of metaplasia induction. although none of these models has achieved true recapitulation of the induction of adenocarcinoma, they have led to important insights into the factors that influence the induction and progression of metaplasia. here, we review the pathologic definitions relevant to alterations in gastric corpus lineages and classification of metaplasia by specific lineage markers. in addition, we review present murine models of the induction and progression of spasmolytic polypeptide (tff2)expressing metaplasia, the predominant metaplastic lineage observed in murine models. these models provide a basis for the development of a broader understanding of the physiological and pathophysiological roles of metaplasia in the stomach.
the term hypospadias is derived from greek and refers to a rent (spadon) on the ventrum of the penis. a number of surgical procedures and technical modifications for distal hypospadias has been developed and refined through the years to allow for a simple single stage repair that has lessened patients discomfort and decreased complications and cost effectiveness without sacrificing functional and cosmetic results. tubularised incised plate (tip) repair described by snodgrass in 1994 provides a technique with a possible low complication rate for correcting distal hypospadias, creating a neourethra of normal diameter irrespective to the plate, glans configuration and location of the meatus. the aim of the present study was to evaluate the results of a new technique (mirs technique) which is a modified version of thiersch - duplay urethroplasty, in comparison to the snodgross urethroplasty. this prospective comparative study was carried out in a tertiary care hospital of northern india over a period of 3-year from march 2010 to march 2013, included 120 patients of anterior (distal penile, subcoronal, coronal and glanular) hypospadias without chordee. we assigned patients in two groups, group = 1 and group = 2, each group having 60 patients. they underwent either mirs technique (group 1 n = 60) or snodgrass technique (group 2 n = 60). mirs technique (named after first and second author who gave this technique) this technique also called mush and shab 's technique is used for anterior (distal) types of hypospadias that is glanular, coronal and subcoronal. proposed urethral plate is outlined at a width of 9 - 12 mm depending on the age of patient and size of the phallus, by two iongitudinal lines which are joined by a transverse line proximal to meatus. cathterisation is done with a 6 - 8 fr silastic cathter (haiyan kangyuan medical instruments co. ltd, china). incision is made along the proposed markings and the urethal plate is tubularised over the cathter by continuous through and through polyglactin inverting suture, with care not to close the distal extent (meatus) too tightly. second layer coverage of neourthra is derived from adjacent facial tissues (dartos + bucks fascia) in the region of shaft, and deep glanular tissues in the glanular part, using interrupted 6/0 polyglactin sutures. the third layer is derived from approximation of skin over the second layer [figures 1 - 4 ]. (a - k) demonstrate the various steps of snodgrass technique (a - d) demonstrate the various steps of mirs technique (a - c) demonstrate the postoperative photographs of mirs technique (a) demonstrate the postoperative photograph of snodgrass technique patients are discharged the next morning. the advantages with this technique are minimal tissue trauma, no degloving of shaft skin, no undermining of the urethral plate or lateral skin flaps, no midline incision of the urethral plate, no sealing flap from prepuce and no disturbance of prepuce. minimal the tissue trauma, lesser the vascularity is compromised and better will be a healing process. this minimal disturbance of anatomy in the first instance does not hamper the results of further interventions if needed. the mean age of the patients was 4 years (range : 2 - 6 years). the maximum number of cases were distal penile type (n = 25 in each group). the mean operative time was 55 min (range : 43 - 70 min) in group 1 and 71.9 min (range : 60 - 81 min) in group 2 (p < 0.001) [tables 1 - 3 ]. urethrocutaneous fistula developed in 2 and 4 patients in group 1 and 2, respectively. fistula closure was done at least 3 months postoperatively and there was no significant difference in success rate between the two groups. three cases of glanular dehiscence were detected (one in group 1 and 2 in group 2) ; the patient in group 1 had a successful repair using the already preserved prepuce. there was transit prepucial oedema in 13 patients in group 1 due to tight dressing, as compared to 4 patients in group 2, the other complications like haematoma, infection, dehiscence and gangrene of skin flaps was less in our group compared to snodgrass class but not reaching the statistical significance. meatal stenosis occurred in 3 patients in group 1 as compared to only 1 patient in group 2. type of hypospadias and number of cases studied the patients from both the groups were mostly discharged in the next morning following the operative procedure, only few patients from both groups required discharge with some delay. one patient from group 1 and eight patients from group 2 had some analgesic requirement in the immediate postoperative period. data were analysed with help of means, standard deviations and percentage statistics. for parametric data, student 's t - test was applied for nonparametric data, chi - square or fischer exact test, whichever appropriate, was used. the choice of operation for repair of distal hypospadias is determined by a number of factors including the configuration of the glans and meatus and associated degree of penile curvature. the ultimate goal is a penis that is not only functionally normal but cosmetically as well. complications are common after hypospadias repair, ranging from fistula to complete loss of the neo - urethra requiring total reconstruction. in 1994, snotgrass described tip urethroplasty for distal penile hypospadias repair. an important step in the snodgrass repair is the interposition of a barrier layer (flap) between the neourethra and the overlying skin in order to decrease the rate of urethrocutaneous fistula formation. the most popular flap used is the preputial flap ; however, mobilisation and ventral transposition of the flap around one side of the penile shaft may cause penile torsion, especially if the flap is of inadequate length and laid on with tension. moreover, dissection of the flap may jeopardise the blood supply to the dorsal skin, which is often used for resurfacing closure, and may thus predispose to skin loss and failure of the repair. to avoid penile torsion, a modification of the way in which the preputial flap is immobilised has been described. a window is created in the flap at the midline, and the penile shaft is pulled through it in order to transfer the dartos flap ventrally over the neourethra. the size of the flap may, however, be inadequate to cover the repair when the ventral skin is deficient, and another modification in flap creation was described, which is to raise the ventral dartos flap to cover the neourethra. this technique was claimed to be associated with a low fistula rate and easier harvesting and mobilisation of the flap to cover the neourethra. difference between mirs technique and snodgrass technique : no midline incision is made in mirs technique as compared with snodgrass technique.prepuce is not disturbed at all in mirs technique whereas in snodgrass technique sealing flap is dissected from the prepuce.no degloving of the penis and creation of byars flap is done in mirs technique whereas in snodgrass technique there is complete degloving of the penile shaft and creation of byars flaps. prepuce is not disturbed at all in mirs technique whereas in snodgrass technique sealing flap is dissected from the prepuce. no degloving of the penis and creation of byars flap is done in mirs technique whereas in snodgrass technique there is complete degloving of the penile shaft and creation of byars flaps. the central focus of our technique is to make a minimally invasive procedure which does not disturb the natural anatomical planes of the organ beyond what is required to correct the defect. the defect in the distal penile hypospadias without chordee for which we have recommended the procedure is failure of tublisation of the distal urethral plate, otherwise the tissues are adequate. hence, we are assisting the nature in tubularsing the deficient portion of distal urethral plate without disturbing the rest of the organ. another factor that may affect penile alignment is the degree of penile skin degloving during hypospadias repair : complete degloving to the penoscrotal junction or limited to the area around the urethral meatus. turial. recommended limited degloving of the penile skin in order to limit the need for a large covering layer of the neourethra, whereas sozubir and snodgrass performed complete degloving of the penile skin to provide full erection and prevent postoperative torsion or chordee. we achieved results comparable to snodgrass technique without degloving the penile skin, thereby not only save the operative time but also give less trauma to phallus. we stented all patients in both groups for about 1-week, which allowed drainage of the urinary bladder and prevented voiding due to the surgery. it also helps haemostasis, reduces postoperative bleeding and in the same time this short period, avoids the problem of catheter blockage and bladder irritation. proponents of stenting argue that it keeps the dorsal midline incision stretched open and limits premature healing, which would obviate the benefit of the dorsal incision. mean operative time in group 2 (snodgrass group) in our study was 71.9 min which is comparable to mean operative time in another study. complications like haemotoma, infection, dehiscence and gangrene of skin, urethrocutaneous fistula were less frequent in mirs technique compared to snodgrass technique. among these analgesic requirement (p = 0.32) however meatal stenosis occurred in three patients in case of mirs technique, whereas only one patient developed meatal stenosis in snodgrass technique. transient prepucal oedema was significantly (p = 0.036) more common in mirs technique (group 1) as compared to snodgrass technique (group 2). this was less in group 2 because prepuce is not usually preserved in snodgrass technique. unsatisfactory cosmetic appearance was present in 3.33% of patients in group 2 in our study which is comparable to another study by roy and saha. glanular dehiscence occurred in 3.33% patients in group 2 in our study which is also comparable to other studies. postoperative penile rotation and cathether blockage occurred in 3.33% patients each in group 2 in our study which is comparable to the study by alsharbaini and almaramhy. our technique (mirs technique also called mush and shab 's technique) is providing results comparable to snodgrass urethroplasty with few added advantages. one of the main advantages being that the technique is simple and easy to learn and thus short learning curve. it is time saving, requires less analgesic in the postoperative period and preserves the precious prepuce. the other advantages with mirs technique are there is minimal tissue traumano degloving of shaft skin, no undermining of urethal plate or lateral skin flaps, no midline incision of urethal plate, no sealing flap from prepuce, no disturbance with prepuce. there is minimal tissue trauma no degloving of shaft skin, no undermining of urethal plate or lateral skin flaps, no midline incision of urethal plate, no sealing flap from prepuce, no disturbance with prepuce. hence, cases of distal hypospadias should also be corrected by our technique so that it will benefit the patients and reduce the operative time.	background : there is no single, universally applicable technique for hypospadias repair and numerous techniques have been practised from time to time. we compare the results of our new technique (mirs technique also called mush & shab 's technique) to snodgross urethroplasty. mirs technique is a modified version of thiersch - duplay urethroplasty.material and methods : this prospective comparative study was carried out in a tertiary care hospital of northern india over a period of 3 years from march 2010 to march 2013 and included 120 patients of anterior (distal penile, subcoronal, coronal and glanular) hypospadias without chordee. they underwent either mirs technique (group 1 n = 60) or snodgrass technique (group 2 n = 60). follow - up was at 1-week, 1-month, 3 months and 6 months.results:the mean operative time was 55 min (range : 43 - 70 min) in group 1 and 71.9 min (range : 60 - 81 min) in group 2 (p < 0.001). urethrocutaneous fistula developed in two and four patients in group 1 and 2, respectively. fistula closure was done at least 3 months postoperatively, and there was no significant difference in success rate between the two groups. three cases of glanular dehiscence were detected (one in group 1 and two in group 2) ; the patient from group 1 had a successful repair using the already preserved prepuce.conclusion : mirs modification of thiersch - duplay technique for distal hypospadias is a time saving procedure with a lower overall complication rate. valuable local tissue is preserved to deal with any complication that may occur. analgesic requirement was significantly lower in this minimally traumatic technique. as it is less time consuming, simple and easy to learn with a short learning curve, this technique deserves application in cases of distal hypospadias.
ninety - nine purpose - bred male and female rm (macaca mulatta) of indian genetic background were used with the approval of the oregon national primate research center animal care and use committee, under the standards of the us national institutes of health guide for the care and use of laboratory animals. these animals were specific - pathogen free (spf) as defined by being free of cercopithicine herpesvirus 1, d - type simian retrovirus, simian t - lymphotrophic virus type 1, rhesus rhadinovirus, and mycobacterium tuberculosis. mhc-1 genotyping for common mamu alleles such as mamu - a01/-a02 and -b08/-b17 was performed by sequence - specific priming pcr, as described. the 99 rm include 15 rhcmv / siv vector - vaccinated rm (710 years of age) with aviremic control of siv infection (14 with sivmac239, 1 with sivmac251) after ir challenge (5 with short - term follow up ; 10 with medium- to long - term follow up), 52 rm (419 years of age) vaccinated with rhcmv / siv or control rhcmv vectors or left unvaccinated prior to sivmac239 challenge (42 ivag, 10 iv), 12 siv+ rm (512 years of age ; 8 with progressive sivmac239 infection, 1 with spontaneously controlled sivmac239 infection, 3 with art - suppressed sivmac251 infection), and 20 siv - nave rm (414 years of age) used as negative controls (these include 4 rm vaccinated with rhcmv / siv vectors) or as siv and rhcmv vector - nave recipients in the adoptive transfer experiments. early (3 years. rhcmv / gag, rev / tat / nef, env and pol-1 and pol-2 vectors were administered subcutaneously at a dose of 5 10 plaque forming units per vector. the control ag - expressing rhcmv vector was used at a total dose of 2.5 10 plaque forming units to match the total dose of the rhcmv / siv vectors. all siv challenges (ir, ivag, iv) used a repeated limiting dose protocol using dosing designed to require > 1 challenge for infection of > 60% of challenged rm, and to infect all or nearly all challenged rm with 10 (weekly) challenges for ir or ivag inoculation (300 focus forming units) and 3 (every 3 week) challenges for iv inoculation (0.2 focus - forming units). rm were considered siv - infected (and challenge discontinued) with the onset of plasma viral load 30 c. eq./ml and the de novo development of cd4 + and cd8 + t cell responses to sivvif, an siv ag not included in the rhcmv / siv vectors. rm were considered controllers if plasma viral load became undetectable (95% cd4 + t cells and the siv - infected preparations were > 50% siv+ following enrichment. siv+ vs. siv t cells were then incubated with microbead - purified cd8 + t cells at an effector : target ratio of 40:1 under the same conditions used for peptide - specific flow cytometric intracellular cytokine analysis. following incubation, stimulated cells were stored at 4c until staining with combinations of fluorochrome - conjugated mabs including : sp34 - 2 (cd3 ; pacific blue, percp - cy5.5), l200 (cd4 ; amcyan), sk-1 (cd8 ; apc, percp - cy5.5), cd28.2 (cd28 ; pe, pe - texasred), dx2 (cd95 ; apc, pe), 15053 (ccr7 ; pacific blue), b56 (ki-67 ; fitc), mab11 (tnf- ; apc, fitc, pe), b27 (ifn- ; apc, fitc), and fn50 (cd69 ; pe, pe - texasred). analysis was performed using flowjo software (tree star). in all analyses, gating on the lymphocyte population was followed by the separation of the cd3 t cell subset and progressive gating on cd4 + and cd8 + t cell subsets. antigen - responding cells in both cd4 + and cd8 + t cell populations were determined by their intracellular expression of cd69 and either or both of the ifn- and tnf cytokines. after subtracting background, the raw response frequencies were memory corrected, as previously described. in selected experiments, cells responding to siv peptides by production of either or both of ifn- and tnf titres of sivenv - specific abs were determined by neutralization of tissue culture - adapted sivmac251 using a luciferase reporter gene assay. immunohistochemistry was performed using a biotin - free polymer approach (golden bridge international, inc.) on 5m tissue sections mounted on glass slides, which were dewaxed and rehydrated with double - distilled h2o. heat induced epitope retrieval (hier) was performed by heating sections in 0.01% citraconic anhydride containing 0.05% tween-20 in a pressure cooker set at 122125c for 30 sec. slides were incubated with blocking buffer (tbs with 0.05% tween-20 and 0.5% casein) for 10 min. for apobec3 g, slides were incubated with rabbit anti - apobec3 g slides were washed in 1x tbs with 0.05% tween-20, endogenous peroxidases blocked using 1.5% (v / v) h2o2 in tbs (ph 7.4) for 10 min., incubated with rabbit polink-2 hrp and developed with impact dab (3,3-diaminobenzidine ; vector laboratories). for isg15 staining, after the hier step, the slides were loaded on an intellipath autostainer (biocare medical) and stained with optimal conditions determined empirically that consisted of a blocking step using blocking buffer (tbs with 0.05% tween-20 and 0.5% casein) for 10 min. and an endogenous peroxidase block using 1.5% (v / v) h2o2 in tbs (ph 7.4) for 10 min. rabbit anti - isg (1:250 ; sigma hpa004627) was diluted in blocking buffer and incubated for 1h at room temperature. tissue sections were washed and developed using the rabbit polink-1 hrp staining system (golden bridge international, inc.) according to manufacturer s recommendations. all slides were washed in h2o, counterstained with hematoxylin, mounted in permount (fisher scientific), and scanned at high magnification (x200) using the scanscope cs system (aperio technologies) yielding high - resolution data from the entire tissue section. representative regions of interest (500 mm) were identified and high - resolution images extracted from these whole - tissue scans. the percent area of the t cell zone that stained for apobec3 g and isg15 were quantified using photoshop cs5 and fovea tools. the rm used in the vaccine efficacy analysis were randomly assigned to vaccine groups with randomization stratified to balance groups for expression of protective mhc alleles. the criteria for categorizing post - challenge rm into protected vs. unprotected groups were established previously. experimenters were not explicitly blinded to the treatment assignments of the rm, nor were the analyses conducted by blinded investigators. all statistical analyses were conducted using nonparametric and model - independent analysis procedures either for the main analysis or as a sensitivity analysis, and in every sensitivity analysis the result was qualitatively consistent with the main analysis. the only exceptions were the time series analyses, which were conducted with two model - based (regression) approaches ; the residuals of these analyses were evaluated and found to be consistent with homoschedasticity and normality requirements, and the results were consistent across approaches. for comparisons of continuous - valued data from independent samples, we applied bivariate mann - whitney u tests, also known as wilcoxon rank sum tests. for comparisons of dichotomous values across groups, we applied fisher s exact tests. we estimated confidence bounds for binomial proportions using the wilson score method, as described in agresti and coull. we compared group means of positivity frequencies for which we have repeated binary measures on individual rm using mixed effects logistic regression (with individual rm mean deviations from group means modeled as a normally - distributed random effect). we compared confidence intervals for rm groups to confidence intervals for individual rm (from other groups) by directly determining overlap of the intervals (and we used the estimated random effect variance and estimated group means to conduct z - tests in sensitivity analyses, which yielded consistent results). we conducted time series analyses using standard linear regression with time as the primary predictor, and we used gaussian first - order autoregressive process models in sensitivity analyses, which yielded consistent results. all tests were conducted as two - tailed tests with a type - i error rate of 5%. ninety - nine purpose - bred male and female rm (macaca mulatta) of indian genetic background were used with the approval of the oregon national primate research center animal care and use committee, under the standards of the us national institutes of health guide for the care and use of laboratory animals. these animals were specific - pathogen free (spf) as defined by being free of cercopithicine herpesvirus 1, d - type simian retrovirus, simian t - lymphotrophic virus type 1, rhesus rhadinovirus, and mycobacterium tuberculosis. mhc-1 genotyping for common mamu alleles such as mamu - a01/-a02 and -b08/-b17 was performed by sequence - specific priming pcr, as described. the 99 rm include 15 rhcmv / siv vector - vaccinated rm (710 years of age) with aviremic control of siv infection (14 with sivmac239, 1 with sivmac251) after ir challenge (5 with short - term follow up ; 10 with medium- to long - term follow up), 52 rm (419 years of age) vaccinated with rhcmv / siv or control rhcmv vectors or left unvaccinated prior to sivmac239 challenge (42 ivag, 10 iv), 12 siv+ rm (512 years of age ; 8 with progressive sivmac239 infection, 1 with spontaneously controlled sivmac239 infection, 3 with art - suppressed sivmac251 infection), and 20 siv - nave rm (414 years of age) used as negative controls (these include 4 rm vaccinated with rhcmv / siv vectors) or as siv and rhcmv vector - nave recipients in the adoptive transfer experiments. early (3 years. rhcmv / gag, rev / tat / nef, env and pol-1 and pol-2 vectors were administered subcutaneously at a dose of 5 10 plaque forming units per vector. the control ag - expressing rhcmv vector was used at a total dose of 2.5 10 plaque forming units to match the total dose of the rhcmv / siv vectors. all siv challenges (ir, ivag, iv) used a repeated limiting dose protocol using dosing designed to require > 1 challenge for infection of > 60% of challenged rm, and to infect all or nearly all challenged rm with 10 (weekly) challenges for ir or ivag inoculation (300 focus forming units) and 3 (every 3 week) challenges for iv inoculation (0.2 focus - forming units). rm were considered siv - infected (and challenge discontinued) with the onset of plasma viral load 30 c. eq./ml and the de novo development of cd4 + and cd8 + t cell responses to sivvif, an siv ag not included in the rhcmv / siv vectors. rm were considered controllers if plasma viral load became undetectable (95% cd4 + t cells and the siv - infected preparations were > 50% siv+ following enrichment. siv+ vs. siv t cells were then incubated with microbead - purified cd8 + t cells at an effector : target ratio of 40:1 under the same conditions used for peptide - specific flow cytometric intracellular cytokine analysis. following incubation, stimulated cells were stored at 4c until staining with combinations of fluorochrome - conjugated mabs including : sp34 - 2 (cd3 ; pacific blue, percp - cy5.5), l200 (cd4 ; amcyan), sk-1 (cd8 ; apc, percp - cy5.5), cd28.2 (cd28 ; pe, pe - texasred), dx2 (cd95 ; apc, pe), 15053 (ccr7 ; pacific blue), b56 (ki-67 ; fitc), mab11 (tnf- ; apc, fitc, pe), b27 (ifn- ; apc, fitc), and fn50 (cd69 ; pe, pe - texasred). analysis was performed using flowjo software (tree star). in all analyses, gating on the lymphocyte population was followed by the separation of the cd3 t cell subset and progressive gating on cd4 + and cd8 + t cell subsets. antigen - responding cells in both cd4 + and cd8 + t cell populations were determined by their intracellular expression of cd69 and either or both of the ifn- and tnf cytokines. after subtracting background, the raw response frequencies were memory corrected, as previously described. in selected experiments, cells responding to siv peptides by production of either or both of ifn- and tnf titres of sivenv - specific abs were determined by neutralization of tissue culture - adapted sivmac251 using a luciferase reporter gene assay. immunohistochemistry was performed using a biotin - free polymer approach (golden bridge international, inc.) on 5m tissue sections mounted on glass slides, which were dewaxed and rehydrated with double - distilled h2o. heat induced epitope retrieval (hier) was performed by heating sections in 0.01% citraconic anhydride containing 0.05% tween-20 in a pressure cooker set at 122125c for 30 sec. slides were incubated with blocking buffer (tbs with 0.05% tween-20 and 0.5% casein) for 10 min. for apobec3 g, slides were incubated with rabbit anti - apobec3 g (1:100 ; sigma hpa001812) diluted in blocking buffer overnight at 4c. slides were washed in 1x tbs with 0.05% tween-20, endogenous peroxidases blocked using 1.5% (v / v) h2o2 in tbs (ph 7.4) for 10 min., incubated with rabbit polink-2 hrp and developed with impact dab (3,3-diaminobenzidine ; vector laboratories). for isg15 staining, after the hier step, the slides were loaded on an intellipath autostainer (biocare medical) and stained with optimal conditions determined empirically that consisted of a blocking step using blocking buffer (tbs with 0.05% tween-20 and 0.5% casein) for 10 min. and an endogenous peroxidase block using 1.5% (v / v) h2o2 in tbs (ph 7.4) for 10 min. rabbit anti - isg (1:250 ; sigma hpa004627) was diluted in blocking buffer and incubated for 1h at room temperature. tissue sections were washed and developed using the rabbit polink-1 hrp staining system (golden bridge international, inc.) according to manufacturer s recommendations. all slides were washed in h2o, counterstained with hematoxylin, mounted in permount (fisher scientific), and scanned at high magnification (x200) using the scanscope cs system (aperio technologies) yielding high - resolution data from the entire tissue section. representative regions of interest (500 mm) were identified and high - resolution images extracted from these whole - tissue scans. the percent area of the t cell zone that stained for apobec3 g and isg15 were quantified using photoshop cs5 and fovea tools. the rm used in the vaccine efficacy analysis were randomly assigned to vaccine groups with randomization stratified to balance groups for expression of protective mhc alleles. the criteria for categorizing post - challenge rm into protected vs. unprotected groups were established previously. experimenters were not explicitly blinded to the treatment assignments of the rm, nor were the analyses conducted by blinded investigators. all statistical analyses were conducted using nonparametric and model - independent analysis procedures either for the main analysis or as a sensitivity analysis, and in every sensitivity analysis the result was qualitatively consistent with the main analysis. the only exceptions were the time series analyses, which were conducted with two model - based (regression) approaches ; the residuals of these analyses were evaluated and found to be consistent with homoschedasticity and normality requirements, and the results were consistent across approaches. for comparisons of continuous - valued data from independent samples, we applied bivariate mann - whitney u tests, also known as wilcoxon rank sum tests. for comparisons of dichotomous values across groups, we applied fisher s exact tests. we estimated confidence bounds for binomial proportions using the wilson score method, as described in agresti and coull. we compared group means of positivity frequencies for which we have repeated binary measures on individual rm using mixed effects logistic regression (with individual rm mean deviations from group means modeled as a normally - distributed random effect). we compared confidence intervals for rm groups to confidence intervals for individual rm (from other groups) by directly determining overlap of the intervals (and we used the estimated random effect variance and estimated group means to conduct z - tests in sensitivity analyses, which yielded consistent results). we conducted time series analyses using standard linear regression with time as the primary predictor, and we used gaussian first - order autoregressive process models in sensitivity analyses, which yielded consistent results. all tests were conducted as two - tailed tests with a type - i error rate of 5%.	established infections with the human and simian immunodeficiency viruses (hiv, siv) are thought to be permanent with even the most effective immune responses and anti - retroviral therapies (art) only able to control, but not clear, these infections14. whether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of hiv - infected individuals. we recently reported that ~50% of rhesus macaques (rm) vaccinated with siv protein - expressing rhesus cytomegalovirus (rhcmv / siv) vectors manifest durable, aviremic control of infection with highly pathogenic sivmac2395. here, we demonstrate that regardless of route of challenge, rhcmv / siv vector - elicited immune responses control sivmac239 after demonstrable lymphatic and hematogenous viral dissemination, and that replication - competent siv persists in multiple sites for weeks to months. however, over time, protected rm lost signs of siv infection, showing a consistent lack of measurable plasma or tissue - associated virus using ultrasensitive assays, and loss of t cell reactivity to siv determinants not in the vaccine. extensive ultrasensitive rt - pcr and pcr analysis of tissues from rhcmv / siv vector - protected rm necropsied 69172 weeks after challenge did not detect siv rna or dna over background, and replication - competent siv was not detected in these rm by extensive co - culture analysis of tissues or by adoptive transfer of 60 million hematolymphoid cells to nave rm. these data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory t cell - mediated immune surveillance elicited and maintained by cmv vectors.
	multistage models of carcinogenesis proposed to account for the observed patterns of tumor development in the skin, liver, lung, bladder and other organs involve initiation of neoplastic change in a few cells by a threshold dose of carcinogen followed by conversion of these latent tumor cells into an autonomous cancer by further doses of the same and/or other carcinogens, and/or noncarcinogenic promoting agents. in the rat urinary bladder, neoplastic change can be initiated by a few weeks treatment with low doses of n - butyl - n-(4-hydroxybutyl)nitrosamine (bbn) or n-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (fanft) or by a single, low dose, intravesicular instillation of n - methyl - n - nitrosourea (mnu). very few animals treated thus will develop bladder cancer unless exposed subsequently to some further regime which will promote tumor growth from the initiated cells. many different factors will stimulate tumor growth in the initiated rat bladder, including further low doses of complete bladder carcinogens, dietary factors such as metabolites of tryptophan or deficiency of vitamin a, the food additives saccharin and cyclamate and some alkylating agents such as cyclophosphamide and methylmethane sulfonate. new and published evidence is reviewed which supports the belief that these and other factors are promoters or later stage carcinogens in the bladder. the difficulties of defining a promoter and of identifying markers of promotion, i.e., of distinguishing the second from the later stages of carcinogenesis in the urinary bladder, are discussed with reference to the action of promoters in the mouse skin initiation / promotion model. however, in terms of their effect on an initiated population on the risk of developing cancer, it is suggested that such a distinction is largely irrelevant. since both second and later stage carcinogens accelerate tumor development in an initiated urothelium, they both have the potential to lower the age at which bladder cancer becomes symptomatic. they are thus as important as are initiating carcinogens in determining the patterns of age - related neoplastic disease in any population.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.
maternal and child health issues take a central stage in global health agenda with the concerted efforts of many players and a number of initiatives. this has been followed by a corresponding commitment on targets, interventions, and guidelines to avert health risks and life threating dangers to women in pregnancy and childbirth. despite the efforts of many global health players, data from the world health organization (who), the global health observatory (gho) 2015 data, and the united nations millennium development goals 2015 report show that pregnancy - related complications are still the leading causes of death amongst women in the reproductive age in developing countries. the risk of a woman in developing countries dying from a maternal - related cause during a lifetime is about 33 times higher compared to a woman living in a developed country even though most of these complications are preventable with improved access to quality health care service. globally, 11% of all births occur to adolescents aged 15 to 19 years, and 95% of these births occur in developing countries. international studies have shown that early child bearing is the biggest challenge to young people, as it is associated with increased vulnerability to poor health as well as a long lasting impact on social, livelihood, education, physical, mental health, including risks of maternal death. adolescent pregnancy is often not the result of a deliberate choice, but rather the absence of choices which include little or no access to schooling, lack of information that positively influence behavior or quality health services, and lack of empowerment, among others. studies indicate in some parts of the world, notably in sub - saharan africa, 35% of pregnancies among adolescents are unplanned or unwanted during pregnancy that resulted due to unsafe sexual practices and lack of awareness. to prevent, minimize or treat unfavourable outcomes of pregnancy, who recommends that all pregnant women should attend at least four anc visits as a minimum with skilled attendant throughout pregnancy, commencing as early as possible in the first trimester. linked to this, in south africa, the national department of health has adopted a minimum of four anc follow - up visits as recommended by the who for women without any risk factors. this is guided by following the early booking visit (preferably 75 %) being blacks. the district has the most challenging road infrastructure for the effective delivery of health services compared to the other four districts in the province. the jtg district economy is based on mining (11 manganese and 3 iron ore mines) and mixed farming, but there is a high level (37.7% youth (25 - 34) official unemployment rate and low level of income opportunities. close to forty five percent (43.1%) of the households are female - headed. there are 44 health facilities of which five are community health center, 37 clinics and 2 district hospitals - the two district hospitals are both located in gasegoyana local municipality. the district has poor and substantial fluctuation in maternal and child health outcomes records between 2009 and 2013. according to the national committee on confidential enquires into maternal deaths (nccemd) report, in 2009/10, the maternal mortality rate (mmr) was 129.9 per 100,000 live births. in 2010/11 and 2011/12 the confidential enquiry report by the committee for 2013/14 reported that the high mmr significantly slowed down to 93.2 per 100,000 live births in 2013/14. these substantial year - to - year fluctuations in mmr outcomes records could partially linked to uptake of anc services by pregnant mothers. it was against this background that this study was conducted to assess factors that influence the poor uptake of anc in jtg district, south africa. a cross - sectional study was conducted at all public health facilities in jtg district between september and november 2014 using mixed method approach. quantitative data was obtained using a standardized anonymous questionnaire adapted from validated tools, which included information on maternal reproductive knowledge, attitude and practice, socio - economic status, utilization of anc services, delivery and postnatal visits (six - week), facility performances and supply side constraints in provision of maternal and child health services. health facilities sampled include 2 district hospitals, 5 community health center (chcs) and 37 clinics. questionnaires were translated from english into two local languages afrikaans and setswana languages and back translated into english to ensure accuracy qualitative data was collected through semi - structured interviews and focus group discussion (fgd) from purposely - selected participants that included high reproductive health risk groups such as teenage mothers, mother who do not utilise anc services at health services and traditional births attendants. the fgds were captured using a digital recorder. the information then was transcribed for analysis where similar emergent teams were differently categorised. lay counsellors who were trained for two days on data collection tools and research ethics collected the data. lay counsellors signed a confidentiality agreement prior to data collection. use of anc services was determined by the participants self - report and was confirmed from clinical records. mothers attending six - week immunization for their infants in all public health facilities in jtg district were the study population from which the sample was drawn. by means of the 2011 census, the six - week immunization and reproductive health services utilization rate data from the 2012/13 district health information system (dhis) was used to quantify the number of mothers expected to attend six - week immunization visit for their infants per facility over the study period. for the facility - based survey, a precision based sample size was calculated taking into account the expected annual utilization of anc services, a precision level of 2 - 3% ; and 95% confidence level. this provided a sample size of 383 respondent mothers who will be attending six - week immunization services for their infants in all public health facilities within the district. sample size was allocated for each facility proportionate to their size as determined by the number of six - week immunizations coverage for 2013/14. univariate, and multivariate logistic regression models were fitted to identify factors influencing the total number of anc visits and late anc booking. results were expressed using descriptive statistics and where necessary, data was disaggregated by different sociodemographic and economic status of respondents. we used a dichotomous dependent or outcome variable of the study on pregnant women s use of anc visit, categorized into : - those who attend at least 4 anc visits, and those who do not during the time of pregnancy. the literature shows that the relationship between a binary outcome variable of study such as attending at least 4 anc visits during the time of pregnancy or failure and predictor variables that affect the outcome variable can successfully be explained using the binary logistic regression model. binary logistic regression analysis was performed to identify predictor variables that influence the number of anc service utilization. odds ratios arising from binary logistic regression analysis are estimated based on the maximum likelihood estimation technique. the logit model is used quite extensively for the estimation of odds ratios in economic studies involving a dichotomous outcome. consider a dependent variable y with 2 possible outcomes (1, 0). the expression y=1 represents the event that a mother has failed to attend 4 anc visits. the expression y=0 represents the event that a pregnant mother has attended at least 4 anc visits successfully. previous studies revealed that utilization of anc services is highly influenced by different sociodemographic, economic factors as well as specific individual - level characteristics both at the client and service providers side. continuous predictable variables had to be dichotomised in order to perform pearson s chi - square tests of association. for instance, the pearson chi - square test of association is used to test the null hypothesis that two factors such the dependent variable and independent variable are independent of each other, against the alternative hypothesis that the two factors are significantly associated with each other. the pearson chi - square test of association is used only for the screening of variables. accordingly, we included several theoretically pertinent sociodemographic and economic independent variables (indicators) that influence uptake of pregnant women s anc services. respondent women s age was classified into age group of respondents less than 20 years (adolescent group), and age group older than or equal to 20 years of age (middle and older women). women s education level was defined in terms of the formal education system of south africa : school not attended, elementary (grade 1 - 7), high school (8 - 12), college or above. marital status of mothers categorized as married, living together, widowed / separated, and single. distance to health facility for anc services categorized as more than or equal to one hour or less. other independent variables include income, level of a mother, individual health seeking behavior and service provider s attitude. household assets, including ownership of durable goods (such as car, fridge, televisions, stove and washing machine). dwelling characteristics included were source of drinking water, sanitation facilities, and construction materials and others used as predictor variables for binary logistic regression analysis. the identification of influential predictor variables was done based on odds ratios. in binary logistic regression analysis, influential predictor variables are characterized by odds ratios that are significantly different from 1, 95% confidence intervals of odds ratios that do not contain 1, and p - values that are smaller than 0.05, at the 5% level of significance. the study was approved by the northern cape provincial health research and ethics committee (phrec), reference number nc - phrec-2014/021. a cross - sectional study was conducted at all public health facilities in jtg district between september and november 2014 using mixed method approach. quantitative data was obtained using a standardized anonymous questionnaire adapted from validated tools, which included information on maternal reproductive knowledge, attitude and practice, socio - economic status, utilization of anc services, delivery and postnatal visits (six - week), facility performances and supply side constraints in provision of maternal and child health services. health facilities sampled include 2 district hospitals, 5 community health center (chcs) and 37 clinics. questionnaires were translated from english into two local languages afrikaans and setswana languages and back translated into english to ensure accuracy qualitative data was collected through semi - structured interviews and focus group discussion (fgd) from purposely - selected participants that included high reproductive health risk groups such as teenage mothers, mother who do not utilise anc services at health services and traditional births attendants. the fgds were captured using a digital recorder. the information then was transcribed for analysis where similar emergent teams were differently categorised. lay counsellors who were trained for two days on data collection tools and research ethics collected the data. lay counsellors signed a confidentiality agreement prior to data collection. use of anc services was determined by the participants self - report and was confirmed from clinical records. mothers attending six - week immunization for their infants in all public health facilities in jtg district were the study population from which the sample was drawn. by means of the 2011 census, the six - week immunization and reproductive health services utilization rate data from the 2012/13 district health information system (dhis) was used to quantify the number of mothers expected to attend six - week immunization visit for their infants per facility over the study period. for the facility - based survey, a precision based sample size was calculated taking into account the expected annual utilization of anc services, a precision level of 2 - 3% ; and 95% confidence level. this provided a sample size of 383 respondent mothers who will be attending six - week immunization services for their infants in all public health facilities within the district. sample size was allocated for each facility proportionate to their size as determined by the number of six - week immunizations coverage for 2013/14. univariate, and multivariate logistic regression models were fitted to identify factors influencing the total number of anc visits and late anc booking. results were expressed using descriptive statistics and where necessary, data was disaggregated by different sociodemographic and economic status of respondents. we used a dichotomous dependent or outcome variable of the study on pregnant women s use of anc visit, categorized into : - those who attend at least 4 anc visits, and those who do not during the time of pregnancy. the literature shows that the relationship between a binary outcome variable of study such as attending at least 4 anc visits during the time of pregnancy or failure and predictor variables that affect the outcome variable can successfully be explained using the binary logistic regression model. binary logistic regression analysis was performed to identify predictor variables that influence the number of anc service utilization. odds ratios arising from binary logistic regression analysis are estimated based on the maximum likelihood estimation technique. the logit model is used quite extensively for the estimation of odds ratios in economic studies involving a dichotomous outcome. consider a dependent variable y with 2 possible outcomes (1, 0). the expression y=1 represents the event that a mother has failed to attend 4 anc visits. the expression y=0 represents the event that a pregnant mother has attended at least 4 anc visits successfully. previous studies revealed that utilization of anc services is highly influenced by different sociodemographic, economic factors as well as specific individual - level characteristics both at the client and service providers side. continuous predictable variables had to be dichotomised in order to perform pearson s chi - square tests of association. for instance, the pearson chi - square test of association is used to test the null hypothesis that two factors such the dependent variable and independent variable are independent of each other, against the alternative hypothesis that the two factors are significantly associated with each other. the pearson chi - square test of association is used only for the screening of variables. accordingly, we included several theoretically pertinent sociodemographic and economic independent variables (indicators) that influence uptake of pregnant women s anc services. respondent women s age was classified into age group of respondents less than 20 years (adolescent group), and age group older than or equal to 20 years of age (middle and older women). women s education level was defined in terms of the formal education system of south africa : school not attended, elementary (grade 1 - 7), high school (8 - 12), college or above. marital status of mothers categorized as married, living together, widowed / separated, and single. distance to health facility for anc services categorized as more than or equal to one hour or less. other independent variables include income, level of a mother, individual health seeking behavior and service provider s attitude. household assets, including ownership of durable goods (such as car, fridge, televisions, stove and washing machine). dwelling characteristics included were source of drinking water, sanitation facilities, and construction materials and others used as predictor variables for binary logistic regression analysis. the identification of influential predictor variables was done based on odds ratios. in binary logistic regression analysis, influential predictor variables are characterized by odds ratios that are significantly different from 1, 95% confidence intervals of odds ratios that do not contain 1, and p - values that are smaller than 0.05, at the 5% level of significance. the study was approved by the northern cape provincial health research and ethics committee (phrec), reference number nc - phrec-2014/021. of 383 sample mothers approached for study participation, 272 agreed to participate in the study and completed the survey questionnaire which resulted in a 72% of response rate. out of 272 mother - infant pairs interviewed at 6 weeks immunization visits, 69.37% had secondary education. regarding the type of employment sector, government and company employment jointly account for only 15.27%. most of the unemployed mothers depend for their source of income on child support grant, disability grant, and donations. however, for 54.76% respondent mothers, the total monthly income was less than or equal to one thousand rand (100 us dollars). about one - fifth (18.68%) of the respondents were teenagers younger than 20 years of age, 71.96% were single mothers, close to half (44.69 %) of mothers reported they had their first baby before the age of 20 years. out of the 18.68% respondent mothers who were teens in this study, 94%, and 82.35% of them regarding ages of the respondents, 15 years was the youngest age and 45 years was the oldest age. about 16.91% of the respondents were married ; 9.56% were living together ; 1.84% were separated / divorced ; and 1.11% were widowed. about two - third (71.96%) respondents were single mothers. in this study, only 28.62% respondents own houses, while 6.32% live in rented houses ; 57.52% live with their relatives, and 9.67% lived with a spouse / partner. with regards to the main source of water used for drinking, 93.38 % respondents were using tap water either inside the house (16.54%) ; or yards (15.44%) or (61.76%) public taps. close to ninety percent (88.97%) of the houses were connected to electricity, and for more than 80.44% of the respondents, electricity was the main type of fuel for cooking in the house. 21.72% households were using flush toilets, 43.82% pit latrines private, and 31.46% ventilated pit latrines. table 1 presents a summary of the socioeconomic and demographic characteristics of the study respondents (table 1). sociodemographic characteristics and descriptive analysis of the study respondents close to half (49 %) of the teenage pregnant women attended at least four anc visit during their pregnancy. however, the majority (64.71%) of teenage mothers booked for the first anc visit after 12 weeks into pregnancy. only (35.29%) of teenage pregnant women booked for an early anc first visit before 12 weeks of pregnancy as recommended by the south african department of health and who guidelines. more than ninety percent (92%) teenage pregnant mothers attended their first anc visits at public health facilities, and a few mothers attended private health facilities (6%) or both public and private facilities (2%). regarding how pregnant women felt about their pregnancy, 25.49% of respondent teenage mother responded that the pregnancy was wanted, while the majority (74.51%) reported that their pregnancies were unwanted at that time. about 2 percent (1.96%) of teenage pregnant mothers completed college or above whilst the majority (86.27%) were in secondary education. 82.35% of them were single mothers, while 5.88% were married, 9.80% were living together, and 1.96% were separated / divorced. the bulk of them (94%) were unemployed, and for the majority of (58.7 %) respondent teenage mothers their total monthly income was less than or equal to one thousand rand (or about $ 100). a number of predictor variables influencing the use of anc services were studied for two outcome variables using the multiple binary logistic regression models : (1) number of anc visits (4 vs. < 4) ; and (2) time of attendance of anc (early vs. late). we estimated the odds ratios (ors) to assess the strength of the associations for the 95% of confidence intervals (cis). regarding the number of anc visits, in multivariable regression after adjusting for employment status, marital status, relationship with the father of the child, waiting time, fear of hiv test, partner support, and education level, we found that mother s age (or=2.11 ; 95%ci = 1.04 - 4.27) ; distance to the nearest health facility (or=3.38 ; 95%ci = 1.45 - 7.87) ; and client service satisfaction (or=8.58 ; 95%ci = 2.10 - 34.95) are significantly associated with poor uptake of anc services (table 2). this suggests that the probability greater number of anc visits is 2.1 times more likely higher with age group older than or equal to 20 years compared to adolescents. increased distance (more than or equal to one hour) to anc services, is 3.3 times more likely to negatively impact uptake of anc visits. none of the predictor variable were strongly associated with the outcome variable time of first anc (early or late) attendance (table 3). a comparison of table 2 with table 3 shows that the independent variables did not affect both number of anc visits and anc booking the same way. estimates obtained from binary logistic regression analysis on number of anc visits estimates obtained from binary logistic regression analysis on time of first anc visits in addition to the surveys questionnaires, we conducted a focus group discussion to reveal a wealth of detailed information and deep insights on the utilization of anc services and provide recommendations on strategies to tackle the challenges and achieve better health outcomes. for each group 10 - 15 (i) women who fully or partially missed reproductive health services ; (ii) high risk reproductive health groups which include adolescents, mothers who plan to give birth at home, late presenters to reproductive health, and those women who have unplanned pregnancies ; (iii) traditional birth attendants, and we found that teenage pregnancy was more common in the jtg district and most of it was unplanned pregnancy. according to adolescents focus group informants, the main reason for unwanted pregnancy among teens was peer pressure from friends and economic dependency. some of the teens had not completed school, were not married and the support they were getting from the child s father were not consistent. according to one of the teenage pregnant women : at the earlier months of my pregnancy, he was there. most of the time his cell phone is off and he is not responding to my calls. because the child s father is no longer able to support us, i will apply for the government child social grant. i will also request my parents to look after my child so that i will be able to look for work. another teenage participant responded, for me everything is still fine with this child s father and he is still supporting me, although i had one more child from different father. regarding knowledge about benefits of early and adequate number of anc visits, the majority of respondents were aware of the benefits. they mentioned the role played by community healthcare workers. according to one of the respondents : the community healthcare workers are helping us to use health care services, remind patients about the date to go to the clinic visits. sometimes they assist patients to the clinic, they encourage patients to take their pills as well as educate them on government s policy with regard to their health. i think they are supporting the healthcare system in reaching the communities. however, some other participants suggested, there is a need to have adequate nurses, and adequate space at the facility as well as priority should be given for pregnant women. the problem is that the clinics use a supermarket approach in providing health services, which means first come first served. this leads to a long waiting time for pregnant women and can be seen by other patients from the community which teen pregnant mother do not want. in our district there are many facilities operating with only a nurse who has to perform all the services which include antenatal, family planning, chronic and also emergencies. on average one nurse has to see 35 patients a day but they often see more than that number daily. of 383 sample mothers approached for study participation, 272 agreed to participate in the study and completed the survey questionnaire which resulted in a 72% of response rate. out of 272 mother - infant pairs interviewed at 6 weeks immunization visits, 69.37% had secondary education. regarding the type of employment sector, government and company employment jointly account for only 15.27%. most of the unemployed mothers depend for their source of income on child support grant, disability grant, and donations. however, for 54.76% respondent mothers, the total monthly income was less than or equal to one thousand rand (100 us dollars). about one - fifth (18.68%) of the respondents were teenagers younger than 20 years of age, 71.96% were single mothers, close to half (44.69 %) of mothers reported they had their first baby before the age of 20 years. out of the 18.68% respondent mothers who were teens in this study, 94%, and 82.35% of them regarding ages of the respondents, 15 years was the youngest age and 45 years was the oldest age. about 16.91% of the respondents were married ; 9.56% were living together ; 1.84% were separated / divorced ; and 1.11% were widowed. about two - third (71.96%) respondents were single mothers. in this study, only 28.62% respondents own houses, while 6.32% live in rented houses ; 57.52% live with their relatives, and 9.67% lived with a spouse / partner. with regards to the main source of water used for drinking, 93.38 % respondents were using tap water either inside the house (16.54%) ; or yards (15.44%) or (61.76%) public taps. close to ninety percent (88.97%) of the houses were connected to electricity, and for more than 80.44% of the respondents, electricity was the main type of fuel for cooking in the house. 21.72% households were using flush toilets, 43.82% pit latrines private, and 31.46% ventilated pit latrines. table 1 presents a summary of the socioeconomic and demographic characteristics of the study respondents (table 1). sociodemographic characteristics and descriptive analysis of the study respondents close to half (49 %) of the teenage pregnant women attended at least four anc visit during their pregnancy. however, the majority (64.71%) of teenage mothers booked for the first anc visit after 12 weeks into pregnancy. only (35.29%) of teenage pregnant women booked for an early anc first visit before 12 weeks of pregnancy as recommended by the south african department of health and who guidelines. more than ninety percent (92%) teenage pregnant mothers attended their first anc visits at public health facilities, and a few mothers attended private health facilities (6%) or both public and private facilities (2%). regarding how pregnant women felt about their pregnancy, 25.49% of respondent teenage mother responded that the pregnancy was wanted, while the majority (74.51%) reported that their pregnancies were unwanted at that time. about 2 percent (1.96%) of teenage pregnant mothers completed college or above whilst the majority (86.27%) were in secondary education. 82.35% of them were single mothers, while 5.88% were married, 9.80% were living together, and 1.96% were separated / divorced. the bulk of them (94%) were unemployed, and for the majority of (58.7 %) respondent teenage mothers their total monthly income was less than or equal to one thousand rand (or about $ 100). a number of predictor variables influencing the use of anc services were studied for two outcome variables using the multiple binary logistic regression models : (1) number of anc visits (4 vs. < 4) ; and (2) time of attendance of anc (early vs. late). we estimated the odds ratios (ors) to assess the strength of the associations for the 95% of confidence intervals (cis). regarding the number of anc visits, in multivariable regression after adjusting for employment status, marital status, relationship with the father of the child, waiting time, fear of hiv test, partner support, and education level, we found that mother s age (or=2.11 ; 95%ci = 1.04 - 4.27) ; distance to the nearest health facility (or=3.38 ; 95%ci = 1.45 - 7.87) ; and client service satisfaction (or=8.58 ; 95%ci = 2.10 - 34.95) are significantly associated with poor uptake of anc services (table 2). this suggests that the probability greater number of anc visits is 2.1 times more likely higher with age group older than or equal to 20 years compared to adolescents. increased distance (more than or equal to one hour) to anc services, is 3.3 times more likely to negatively impact uptake of anc visits. none of the predictor variable were strongly associated with the outcome variable time of first anc (early or late) attendance (table 3). a comparison of table 2 with table 3 shows that the independent variables did not affect both number of anc visits and anc booking the same way. estimates obtained from binary logistic regression analysis on number of anc visits estimates obtained from binary logistic regression analysis on time of first anc visits in addition to the surveys questionnaires, we conducted a focus group discussion to reveal a wealth of detailed information and deep insights on the utilization of anc services and provide recommendations on strategies to tackle the challenges and achieve better health outcomes. for each group 10 - 15 (i) women who fully or partially missed reproductive health services ; (ii) high risk reproductive health groups which include adolescents, mothers who plan to give birth at home, late presenters to reproductive health, and those women who have unplanned pregnancies ; (iii) traditional birth attendants, and (iv) healthcare service providers. we found that teenage pregnancy was more common in the jtg district and most of it was unplanned pregnancy. according to adolescents focus group informants, the main reason for unwanted pregnancy among teens was peer pressure from friends and economic dependency. some of the teens had not completed school, were not married and the support they were getting from the child s father were not consistent. according to one of the teenage pregnant women : at the earlier months of my pregnancy, he was there. most of the time his cell phone is off and he is not responding to my calls. because the child s father is no longer able to support us, i will apply for the government child social grant. i will also request my parents to look after my child so that i will be able to look for work. another teenage participant responded, for me everything is still fine with this child s father and he is still supporting me, although i had one more child from different father. regarding knowledge about benefits of early and adequate number of anc visits, the majority of respondents were aware of the benefits. they mentioned the role played by community healthcare workers. according to one of the respondents : the community healthcare workers are helping us to use health care services, remind patients about the date to go to the clinic visits. sometimes they assist patients to the clinic, they encourage patients to take their pills as well as educate them on government s policy with regard to their health. i think they are supporting the healthcare system in reaching the communities. however, some other participants suggested, there is a need to have adequate nurses, and adequate space at the facility as well as priority should be given for pregnant women. the problem is that the clinics use a supermarket approach in providing health services, which means first come first served. this leads to a long waiting time for pregnant women and can be seen by other patients from the community which teen pregnant mother do not want. in our district there are many facilities operating with only a nurse who has to perform all the services which include antenatal, family planning, chronic and also emergencies. on average one nurse has to see 35 patients a day but they often see more than that number daily. findings from this study indicate that many of the opportunities for anc benefits continue to be missed in the jtg district, particularly among teenagers. there is a need to design innovative strategy and encourage a positive move towards using anc services among teenagers as recommended by the south africa department of health and who guidelines. our study results showed that out of the 18.68% respondent mothers who were teens in this study, less than half (49%) of them had attended four or more anc visits during their pregnancy. only (35.29%) of teenage pregnant women booked for an early anc first visit before 12 weeks of pregnancy. the study observed that 94%, and 82.35% of them were unemployed and single mothers respectively. furthermore, results from multiple logistic regression analysis elucidate that mother s age, distance to health facility and client s service satisfaction found to be highly influential over the use of anc services. our results support findings from several other studies that show teenage pregnancy (mother s age) linked to increased risks of maternal, and infant mortality and morbidity partially due to lower utilization of anc services.[27,29 - 31 ] according to who and national department of health (ndoh) guidelines, all pregnant women should attend at least four anc visits during pregnancy and the first visit should happen before 12 weeks of gestation for healthy pregnancy outcomes. anc provides the opportunity to better clinical management of risk causes through early detection, treatment of anomalies of pregnancy as well as preventive health services. however, to fully benefit from these interventions, it is vital that pregnant women should start the visit as early in their pregnancy and attend at least four anc visits. in this study, both the focus group discussions and the logistic regression analysis results showed that the importance of the quality of services at health facility for increased uptake of anc services. during the focus group discussion, research respondents indicated that teenagers do not want to be seen by other community members while attending anc services. generally, this is due to the expected fear of religious belief, low level of education, cultural factors, ethical and social norms of the society, which do not appreciate teenage pregnancy out of marriage. if such perceptions are informed by fear of being judged then this could be a strong predictor for both late anc attendance and lower utilization of anc services by teenagers. a recent publication from world bank research output confirms that there is a widely accepted truth that socio - cultural factors shape perceptions, cognition, and preferences in durable ways. previous studies point to a strong causal links between client - friendly quality of services and utilization of health care services.[33 - 37 ] adolescent pregnancy is often not the result of a deliberate choice, but rather the absence of choices which include little or no access to school, lack of information that positively influence behavior or good quality health services and lack of empowerment among others. hence pregnancy and anc use in developing countries could be associated with various inter - related markers of the social determinants of health[27 - 29,38 ] present study showed that the fact that 74.52% of teenage pregnant mothers did not want to be pregnant during that time, and 86.27 % teenage mothers only had secondary education, and the bulk of them (94%) are unemployed, and for the majority (58.7%) respondent teenage mothers the total monthly income is less than or equal to one thousand rand (or about $ 100) shows the influences of the social determinants of health. teenagers engage in risky sexual behaviors, not only due to lack of knowledge but also due to unfavorable decisions linked to their socio - demographic and economic conditions. lower socio - economic status is a risk factor for both unplanned and unwanted pregnancies and healthy decisions.. thus, there is a need to address the social determinant of health factors along with improvement in health systems to provide adolescent friendly services. following the millennium development goals (mdgs), which was adopted by the international community in 2000, many developing countries have been able to make significant progress in the reduction of maternal and child mortalities. although undeniably, there have been remarkable gains witnessed, still there is a long way to go with focused approach particularly in low - resource geographic settings and socio - economically disadvantaged population groups. as many of the root causes of poor health and health seeking behaviors are linked to the social determinants of health, and empirical evidence shows that there is a strong bi - causal links between poverty and poor health status people at a lower socio - economic conditions are often disproportionately vulnerable to ill - health. effective implementation of sustainable development goals (sdgs) agenda tailored to country s holistic development need can address many of the health related challenges. bu this needs everyone to contribute their part : governments, the private sector, civil society and individuals. the findings of the study are limited to this district and can not be generalized to the northern cape province or the country. the study findings may not be representative of all pregnant mothers who do not attend six - week immunization due to child illness or death. the findings of the study are limited to this district and can not be generalized to the northern cape province or the country. the study findings may not be representative of all pregnant mothers who do not attend six - week immunization due to child illness or death. this study concludes that several factors influence uptake of anc services by pregnant women in john taolo gaetsewe district. use of anc was significantly influenced by mother s age, longer distance to health facility, and poor quality of anc services provided at health facilities among others. furthermore, tackling the social determinants of health are critical to empower women and avoid unwanted pregnancies and the associated risks. moreover, there is a need for community awareness, particularly in rural areas on the who s and south african national department of health guidelines regarding the number of anc visits and booking time to improve maternal and child health outcomes in jtg district. there is an urgent need to improve the quality of adolescent reproductive health services tailored to their health and developmental needs.collaborative efforts across countries, governments, partners and the un agencies, are required to tackle the social determinants of health and advance an era of more equitable and healthier people, irrespective of where one lives as aspired by sdg.preventing early pregnancy, through creating opportunities for empowerment, along with individual s healthy life style and health seeking behavior is critical. there is an urgent need to improve the quality of adolescent reproductive health services tailored to their health and developmental needs. collaborative efforts across countries, governments, partners and the un agencies, are required to tackle the social determinants of health and advance an era of more equitable and healthier people, irrespective of where one lives as aspired by sdg. preventing early pregnancy, through creating opportunities for empowerment, along with individual s healthy life style and health seeking	background and objectives : in resource - limited settings, the uptake of antenatal care visits among women, especially teenage pregnant women, is disturbingly low. factors that influence the uptake of anc services among teenage women is largely understudied and poorly understood in john taolo gaetsewe (jtg), a predominantly rural and poor district of south africa. the aim of this study was to determine the factors that influence uptake of anc services among teenage mothers in jtg district.methods:a cross - sectional health facility - based study utilising mixed method was conducted in all public health facilities (n=44) at jtg district. mother - infant pairs (n=383) who brought their infants for six - week first dpt immunisation during the study period were enrolled in the study. structured questionnaires were used to collect data on demographic, socio - economic and uptake of anc indicators.results:out of 272 respondent mothers, 18.68% were adolescent mothers (13 - 19 years). the logistic regression analysis shows that mother s age (or=2.11 ; 95%ci = 1.04 - 4.27) ; distance to the nearest health facility (or=3.38 ; 95%ci = 1.45 - 7.87) ; and client service satisfaction (or=8.58 ; 95%ci = 2.10 - 34.95 are significantly associated with poor uptake of anc services.conclusion and global health implications : there is a need to improve the quality of adolescent reproductive health services tailored to their health and developmental needs. moreover, addressing the social determinants of health that affect individual s healthy life style and health seeking behavior is critical.
cognitive neuroscientists are intensively studying the cognitive processes and neural systems underlying theory of mind (tom ; saxe and kanwisher, 2003), the ability to understand that other people have separate mental representations (i.e., beliefs or desires) that guide their behavior. most recent studies (e.g., saxe and kanwisher, 2003 ; mitchell, 2008) have used a false belief (fb) task (wimmer and perner, 1983) to measure people s ability to reason about other people s beliefs and a false photograph (fp) task (zaitchik, 1990) as a control story that putatively relies on the same reasoning processes as the fb task without involving references to another person s mental representations. another set of studies has compared fb stories to stories in which the physical cause of an event has to be deciphered (fletcher., 1995 ; all of these tasks have been inherited from the field of developmental psychology, where they have been used to study normal and abnormal (i.e., autism) development. neuroimaging studies that have used the fb and fp material published by saxe and kanwisher (2003) have consistently reported that the right temporo - parietal - junction (rtpj), an area between the posterior superior temporal sulcus and the inferior parietal lobule, is activated by fb reasoning (aichhorn., 2009 ; saxe., 2009) the rtpj, however, is also activated by changes in working memory load (todd., 2005), multisensory conflict (balslev., 2005), attentional functions (corbetta., 2008), sense of agency (farrer and frith, 2002), and out - of - body experiences (blanke and arzy, 2005 ; see decety and lamm, 2007 for a meta - analysis). this heterogeneous list emphasizes the importance of designing tom paradigms that control for auxiliary, non - tom processes that activate the rtpj. tom paradigms involve linguistic analysis, the maintenance, retrieval, and manipulation of information within working memory, reasoning, inhibition of competing interpretations, and attention. comprehension of fb and fp stories may differentially involve processes such as working memory that are separate from tom but modulate rtpj activity. therefore, a deeper understanding of the cognitive processes recruited by the fb and fp tasks is necessary before conclusions regarding brain mechanisms can be drawn. theory of mind studies have reported that reaction times are faster to comprehension questions concerning fb than fp stories (saxe and kanwisher, 2003 ; saxe and powell, 2006), suggesting that fb stories are easier to comprehend. this result has been used to rule out arguments that brain activations for fb stories could be due to greater difficulty or time - on - task. here, we confirmed differences in difficulty using a broader set of measures and evaluated several factors that might underlie this result. we checked the cognitive equivalence of fb and fp texts by measuring the time taken to read the story as well as to read and respond to the comprehension question. we also obtained working memory span (wms) scores to check for possible correlations between wms and text comprehension. finally, we conducted a linguistic analysis of fb and fp texts to test their linguistic equivalence. fifteen participants (nine males ; mean age = 25) performed the experiment in exchange for payment. participants were native english speakers between the ages of 18 and 32, were nave concerning the purpose of the experiment, and gave written consent following the guidelines set by the human studies committee of washington university. twenty - four fb and 24 fp stories that have been widely used in tom studies (saxe and kanwisher, 2003 ; saxe and powell, 2006 ; saxe., 2006 ; four more blocks were conducted with stories that have been used in other tom studies. however, because these stories have not been used in many studies or did not have comprehension questions associated with them, they were not included in the present analysis. each fb and fp story had two associated comprehension questions, a representation question (rp) and a reality question (rl). the rp question probed the character s mental state in the fb text or the state of the world portrayed by the photograph in the fp text, while the rl question probed the actual outcome of the story in the fb text or the current state of the world (as opposed to when the photograph was taken) in the fp text. an example of each type of story and comprehension question is given in footnote 1.. therefore the experimental design was 2 (text type : fb vs. fp) 2 (comprehension question : rp vs. rl). a moving window paradigm (just., 1982) was used to present the text material. each button - press revealed a new word and covered the previous word with place - holders. the reaction time for a button - press was the estimate of processing time for a word. this method of presentation allowed participants to use peripheral vision to obtain information about the spatial distribution of the text, as in normal reading, but prevented participants from rereading previous sections of the text. as a consequence, after the text was read, a question appeared on a blank screen with two answers located on the bottom left and right. participants selected an answer by pressing a key with the index finger located on the matching side. once a response had been given, a second question was presented on the screen. response time to the comprehension questions was measured from question onset. a working memory span (wms) test (daneman and carpenter, 1980) was run prior to the task in order to estimate wm capacity for each participant. in this test, sentences were presented on the screen, one at a time, and participants had to read each sentence out loud. following the presentation of the last sentence, participants recalled the last word of each sentence. initially, a five trial set was presented in which each trial consisted of two sentences. if a correct response was given in three of the five trials, task difficulty was increased by presenting three sentences per trial. again, if a correct response was given in three of five trials, task difficulty was increased. for the highest difficulty, words could be recalled in any order with the restriction that the word belonging to the last sentence could not be recalled first. fifteen participants (nine males ; mean age = 25) performed the experiment in exchange for payment. participants were native english speakers between the ages of 18 and 32, were nave concerning the purpose of the experiment, and gave written consent following the guidelines set by the human studies committee of washington university. twenty - four fb and 24 fp stories that have been widely used in tom studies (saxe and kanwisher, 2003 ; saxe and powell, 2006 ; saxe., 2006 ; four more blocks were conducted with stories that have been used in other tom studies. however, because these stories have not been used in many studies or did not have comprehension questions associated with them, they were not included in the present analysis. each fb and fp story had two associated comprehension questions, a representation question (rp) and a reality question (rl). the rp question probed the character s mental state in the fb text or the state of the world portrayed by the photograph in the fp text, while the rl question probed the actual outcome of the story in the fb text or the current state of the world (as opposed to when the photograph was taken) in the fp text. an example of each type of story and comprehension question is given in footnote 1. therefore the experimental design was 2 (text type : fb vs. fp) 2 (comprehension question : rp vs. rl). a moving window paradigm (just., 1982) each button - press revealed a new word and covered the previous word with place - holders. the reaction time for a button - press was the estimate of processing time for a word. this method of presentation allowed participants to use peripheral vision to obtain information about the spatial distribution of the text, as in normal reading, but prevented participants from rereading previous sections of the text. as a consequence, after the text was read, a question appeared on a blank screen with two answers located on the bottom left and right. participants selected an answer by pressing a key with the index finger located on the matching side. once a response had been given, a second question was presented on the screen. response time to the comprehension questions was measured from question onset. a working memory span (wms) test (daneman and carpenter, 1980) was run prior to the task in order to estimate wm capacity for each participant. in this test, sentences were presented on the screen, one at a time, and participants had to read each sentence out loud. following the presentation of the last sentence, participants recalled the last word of each sentence. initially, a five trial set was presented in which each trial consisted of two sentences. if a correct response was given in three of the five trials, task difficulty was increased by presenting three sentences per trial. again, if a correct response was given in three of five trials, task difficulty was increased. for the highest difficulty, words could be recalled in any order with the restriction that the word belonging to the last sentence could not be recalled first. fb and fp texts contained a similar number of grammatical sentences per story (fb = 2.6 and fp = 2.4, t(14) = 1.17, p = 0.247, d = 0.34), but a different number of clauses per story (that is, the number of grammatical units consisting of a subject and a predicate) (fb = 4.3 and fp = 3.5, t(14) = 3.22, p = 0.002, d = 0.93). syntactic structure is known to influence comprehension processes (friederici and weissenborn, 2007), as manifested in end - of - sentence wrap - up effects (balogh., 1998) that are thought to reflect mechanisms for integrating information or for checking the completeness of the sentence and its arguments. as a result, the time taken to read each word of a sentence in a story is influenced by the specific syntactic structure of the sentence in which it is embedded. sentences in a text are individual entities when it comes to comprehension mechanisms. given the lack of equivalence between the grammatical structure of the sentences in fb and fp stories and the fact that fb and fp texts had, on average, the same number of sentences per story, we chose sentence reading time rather than story reading time as the index of linguistic processing / comprehension times. an analysis of sentence reading time showed that fb stories were read faster than fp stories (t(14) = 6.77, p = 0.001, d = 0.46). to analyze the comprehension questions, separate anovas [2 (story type : fb vs. fp) 2 (comprehension question : rl vs. rp) ] on response times and accuracy were performed. response times to correctly answered questions were faster in the fb than fp condition (f(1,14) = 8.20, p = 0.013, p2=0.37). no main effect of comprehension question or interaction was found (both fs 0.400, figure 2a) but it did correlate with response time to the comprehension question for all four conditions : fb rp r = 0.82, p = 0.001 ; fb rl r = 0.67, p = 0.006 ; fp rp r = 0.65, p = 0.009 ; fp rl r = 0.73, p = 0.002 (figure 2b). in all cases, most importantly, wms correlated with accuracy in one of the four experimental conditions (figure 2c). accuracy for representation questions about false photos (fp rp), the most difficult question type, was higher for those individuals with higher wms (r = 0.53, p = 0.040). scatter plots for wms correlations with reading time (a), response times (b), and response accuracy (c). the results presented here show that processing of fb and fp stories is not equivalent, raising concerns that these two conditions are not suitable for comparison in tom paradigms. we found that fb stories were read faster than fp stories and replicated previous findings of faster response times to comprehension questions following fb than fp stories. these response times were correlated across subjects with wms scores, indicating the importance of working memory during the comprehension phase of the task. the most interesting result, however, was that comprehension questions following fp stories were particularly difficult when they required subjects to manipulate the information provided by the story in order to reconstruct the original state of the world, as in the case of representation questions (i.e., in the fp story presented in footnote 1, the apple hanging on the tree as shown in the photo as opposed to the apple lying on the ground after being blown off the tree by the wind). only in this condition was comprehension accuracy correlated with wms scores, consistent with a particularly heavy involvement of working memory. finally, linguistic analyses showed that the increased difficulty and greater working memory requirements of representation questions following fp than fb stories were not due to the linguistic features of the questions, consistent with the hypothesis that they instead arose from the need to reorganize how information from fp stories was structured in memory in order to arrive at a correct response. it is possible that the moving window paradigm, in which words were presented one at a time, placed increased demands on working memory. since these increased demands applied to both fb and fp stories, however, the use of this paradigm does not explain the observed difference between story types. at most since the information targeted by representation questions was presented at the beginning of the text in 87.5% of fp stories, the greater difficulty and larger working memory loads associated with representation questions following fp stories might have reflected a recency effect (deese and kaufman, 1957). however, 100% of fb stories also presented representation information prior to belief information without any cost in accuracy, reinforcing the view that the representation of fb stories was more flexible and less structured for a particular type of question than the representation of fp stories. a more flexible representation of fb information would also explain the faster reading times for this condition. developmental studies also suggest that fb and fp stories are not equivalent. while fb and fp tasks are comparable in difficulty in 3- to 5-year - olds (zaitchik, 1990 ; leekam and perner, 1991 ; leslie and thaiss, 1992) no correlation between performance in the two tasks is found when children s age is partialed out. therefore, the fact that they tend to be similar in difficulty in one age bracket seems more a coincidence than an index of a common underlying reasoning process (perner and leekam, 2008). perner and leekam (2008) also point out the lack of equivalence between fb and fp tasks since both beliefs and photos are representations of a reality, but the fb misrepresents its target (in the chocolate story, the place where the chocolates are currently stored) while the fp shows its target correctly (in the apple story, the apple hanging on the tree at the time the photo was taken). the results reported here suggest two factors separate from tom reasoning that might be responsible for reported activation differences between fb and fp stories. the first is related to language processing, since we found that fb stories were read faster than fp stories and the two types of stories were not matched in their syntactic structure. it is widely agreed that the right hemisphere plays an important role in language processing (jung - beeman, 2005), specifically during high level tasks involving comprehension of metaphors (mashal., 2007) and jokes (coulson and wu, 2005), drawing inferences (mason and just, 2004), generating sentence endings (kircher., 2001), and detecting inconsistencies in stories (meyer., 2000). interestingly, manipulations of the syntactic structure of sentences produce activity differences in right posterior sts (friederici., 2009). therefore, the rtpj activations reported in tom studies could be related to uncontrolled differences in linguistic features of the stories. this possibility is supported by the fact that results originally attributed to differences in mental processes have been later shown to be due to linguistic features of the experimental material. (1999) reported that right hemisphere damage (rhd) patients showed difficulty with tom stories but not with control stories. (2008) retained happ.s original tom set but created a new set of control stories that were better matched in linguistic difficulty. they found that rhd patients were not selectively impaired in tom, although they were significantly worse overall than age - matched controls. similarly, neuroimaging studies that have used material different than the stories of saxe and colleagues (e.g., saxe and kanwisher, 2003) have tended to report less clear results (fletcher., 1995 ; happ., 1999 a second factor that is separate from tom processing and distinguishes fp and fb conditions is working memory load. response times to comprehension questions were slower following fp than fb stories and comprehension accuracy was significantly worse for fp stories followed by representation questions than for the other conditions. wms scores correlated with the time to answer comprehension questions in all four conditions and with the accuracy in answering representation questions following fp stories, indicating that working memory load is greater under fp conditions. importantly, the magnitude of rtpj deactivation co - varies with working memory load (todd., 2005), consistent with other results showing that deactivations become larger as task difficulty increases (mckiernan., 2003). the dependence of rtpj responses on working memory load was observed in a non - tom paradigm, consistent with the view that rtpj activity is modulated by a more basic process than tom that is nonetheless likely operative during tom paradigms. therefore, rtpj activity may be more positive in fb than fp conditions because it involves a lower working memory load. the absolute polarity of responses in rtpj is somewhat inconsistent across studies. while most papers find a relative activation for fb and a relative deactivation for fp (saxe and kanwisher, 2003 ; saxe., 2006, 2009 ; aichhorn., 2009 ; scholz., 2009), 2006 ; saxe and powell, 2006 ; kobayashi., 2007) or two deactivations (mitchell, 2008). the reasons for this inconsistency are unclear, but may reflect the fact that studies typically use a block - like design that does not differentiate task phases or components such as text processing, memory maintenance, or the manipulation of information in order to respond to the comprehension question. different processes within a single trial have been shown to activate and deactivate the rtpj, with the polarity of the observed response at any time point as the sum of the responses to the component processes (shulman., 2003). therefore, it may not be surprising that the overall polarity of the rtpj response in tom paradigms differs across studies depending on the duration and timing of the complex mixture of processes involved in fb and fp conditions. irrespective of the observed polarity of the overall response, however, conditions involving higher working memory loads, such as the fp condition, will produce less positive rtpj responses. theory of mind studies suggest that rtpj is a key component of the network for belief reasoning. according to this proposal, fb reasoning is carried out by a specialized module (i.e., rtpj) that is very effective in reasoning about mental states while the brain module specialized for other types of reasoning is not as effective. we suggest instead that the demands on basic processes needed to successfully perform both conditions are not equivalent and as a result, the areas modulated by these processes are differentially activated. mind reading is a highly valued ability in any social environment and the amount of practice that we carry out from early childhood likely far exceeds that for other types of causal reasoning. we suggest that this extensive practice allows for a more flexible mental representation of fb than fp stories in terms of the frame of reference and for a more straightforward manipulation of information when a reality check is necessary, and results in the use of fewer resources during processing. therefore, rtpj activity may not be related to mental state reasoning or any type of reasoning per se, but to a more basic process that is shared to different extents by fb and fp stories, such as the use of working memory. if extensive practice makes us more proficient when reasoning about mental states, we would expect children to show no differences in rtpj activation, since they are not yet experts and reasoning about fb requires as much resources as reasoning about fp. as they become more proficient with the former type of reasoning saxe. (2009) observed this pattern of results in children aged 611. while rtpj was recruited equally for fb and control stories in younger children, it was only recruited (i.e., rtpj activation) for mental reasoning in older children. although the results indicated activations for each child in both conditions, the baseline for this comparison was another condition that had shown a large deactivation in the group analysis. therefore, it is quite possible that if the task data were referenced to a resting baseline, most of the younger children would actually show a deactivation for both tasks (indexing a greater difficulty in both of them), while older kids would only show a deactivation for the control stories, which continued to be difficult, but not for the fb stories. taken together, the current results show that comparisons of fb and fp tasks may not cleanly isolate tom processes. the behavioral differences between the two types of stories indicate that the inferential processes necessary to comprehend the text and the difficulty level of these processes are not comparable. while we do not claim that wm differences are the sole determinant of the lack of equivalence between fb and fp conditions, our results do show that these conditions differ in their cognitive processing requirements. in conclusion, more attention should be given to the lack of equivalence between contrasting conditions in tom paradigms before drawing general conclusions about the functional role of brain areas such as the rtpj. the results reported here suggest two factors separate from tom reasoning that might be responsible for reported activation differences between fb and fp stories. the first is related to language processing, since we found that fb stories were read faster than fp stories and the two types of stories were not matched in their syntactic structure. it is widely agreed that the right hemisphere plays an important role in language processing (jung - beeman, 2005), specifically during high level tasks involving comprehension of metaphors (mashal., 2007) and jokes (coulson and wu, 2005), drawing inferences (mason and just, 2004), generating sentence endings (kircher., 2001), and detecting inconsistencies in stories (meyer., 2000). interestingly, manipulations of the syntactic structure of sentences produce activity differences in right posterior sts (friederici., 2009). therefore, the rtpj activations reported in tom studies could be related to uncontrolled differences in linguistic features of the stories. this possibility is supported by the fact that results originally attributed to differences in mental processes have been later shown to be due to linguistic features of the experimental material. (1999) reported that right hemisphere damage (rhd) patients showed difficulty with tom stories but not with control stories. (2008) retained happ.s original tom set but created a new set of control stories that were better matched in linguistic difficulty. they found that rhd patients were not selectively impaired in tom, although they were significantly worse overall than age - matched controls. similarly, neuroimaging studies that have used material different than the stories of saxe and colleagues (e.g., saxe and kanwisher, 2003) have tended to report less clear results (fletcher., 1995 ; happ., 1999 ; a second factor that is separate from tom processing and distinguishes fp and fb conditions is working memory load. response times to comprehension questions were slower following fp than fb stories and comprehension accuracy was significantly worse for fp stories followed by representation questions than for the other conditions. wms scores correlated with the time to answer comprehension questions in all four conditions and with the accuracy in answering representation questions following fp stories, indicating that working memory load is greater under fp conditions. importantly, the magnitude of rtpj deactivation co - varies with working memory load (todd., 2005), consistent with other results showing that deactivations become larger as task difficulty increases (mckiernan., 2003). the dependence of rtpj responses on working memory load was observed in a non - tom paradigm, consistent with the view that rtpj activity is modulated by a more basic process than tom that is nonetheless likely operative during tom paradigms. therefore, rtpj activity may be more positive in fb than fp conditions because it involves a lower working memory load. the absolute polarity of responses in rtpj is somewhat inconsistent across studies. while most papers find a relative activation for fb and a relative deactivation for fp (saxe and kanwisher, 2003 ; saxe., 2006, 2009 ; aichhorn., 2009 ; scholz., 2009), some studies find two activations (perner., 2006 ; saxe and powell, 2006 ; kobayashi., 2007) or two deactivations (mitchell, 2008). the reasons for this inconsistency are unclear, but may reflect the fact that studies typically use a block - like design that does not differentiate task phases or components such as text processing, memory maintenance, or the manipulation of information in order to respond to the comprehension question. different processes within a single trial have been shown to activate and deactivate the rtpj, with the polarity of the observed response at any time point as the sum of the responses to the component processes (shulman., 2003). therefore, it may not be surprising that the overall polarity of the rtpj response in tom paradigms differs across studies depending on the duration and timing of the complex mixture of processes involved in fb and fp conditions. irrespective of the observed polarity of the overall response, however, conditions involving higher working memory loads, such as the fp condition, will produce less positive rtpj responses. theory of mind studies suggest that rtpj is a key component of the network for belief reasoning. according to this proposal, fb reasoning is carried out by a specialized module (i.e., rtpj) that is very effective in reasoning about mental states while the brain module specialized for other types of reasoning is not as effective. we suggest instead that the demands on basic processes needed to successfully perform both conditions are not equivalent and as a result, the areas modulated by these processes are differentially activated. mind reading is a highly valued ability in any social environment and the amount of practice that we carry out from early childhood likely far exceeds that for other types of causal reasoning. we suggest that this extensive practice allows for a more flexible mental representation of fb than fp stories in terms of the frame of reference and for a more straightforward manipulation of information when a reality check is necessary, and results in the use of fewer resources during processing. therefore, rtpj activity may not be related to mental state reasoning or any type of reasoning per se, but to a more basic process that is shared to different extents by fb and fp stories, such as the use of working memory. if extensive practice makes us more proficient when reasoning about mental states, we would expect children to show no differences in rtpj activation, since they are not yet experts and reasoning about fb requires as much resources as reasoning about fp. as they become more proficient with the former type of reasoning, differences in rtpj activity saxe. (2009) observed this pattern of results in children aged 611. while rtpj was recruited equally for fb and control stories in younger children, it was only recruited (i.e., rtpj activation) for mental reasoning in older children. although the results indicated activations for each child in both conditions, the baseline for this comparison was another condition that had shown a large deactivation in the group analysis. therefore, it is quite possible that if the task data were referenced to a resting baseline, most of the younger children would actually show a deactivation for both tasks (indexing a greater difficulty in both of them), while older kids would only show a deactivation for the control stories, which continued to be difficult, but not for the fb stories. taken together, the current results show that comparisons of fb and fp tasks may not cleanly isolate tom processes. the behavioral differences between the two types of stories indicate that the inferential processes necessary to comprehend the text and the difficulty level of these processes are not comparable. while we do not claim that wm differences are the sole determinant of the lack of equivalence between fb and fp conditions, our results do show that these conditions differ in their cognitive processing requirements. in conclusion, more attention should be given to the lack of equivalence between contrasting conditions in tom paradigms before drawing general conclusions about the functional role of brain areas such as the rtpj. 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.	theory of mind (tom), the ability to reason about other people s thoughts and beliefs, has been traditionally studied in behavioral and neuroimaging experiments by comparing performance in false belief and false photograph (control) stories. however, some evidence suggests that these stories are not matched in difficulty, complicating the interpretation of results. here, we more fully evaluated the relative difficulty of comprehending these stories and drawing inferences from them. subjects read false belief and false photograph stories followed by comprehension questions that probed true (reality questions) or false beliefs (representation questions) appropriate to the stories. stories and comprehension questions were read and answered, respectively, more slowly in the false photograph than false belief conditions, indicating their greater difficulty. interestingly, accuracy on representation questions for false photograph stories was significantly lower than for all other conditions and correlated positively with participants working memory span scores. these results suggest that drawing representational inferences from false photo stories is particularly difficult and places heavy demands on working memory. extensive naturalistic practice with tom reasoning may enable a more flexible and efficient mental representation of false belief stories, resulting in lower memory load requirements. an important implication of these results is that the differential modulation of right temporal parietal junction (rtpj) during tom and false photo control conditions may reflect the documented negative correlation of rtpj activity with working memory load rather than a specialized involvement in tom processes.
in total hip arthroplasty, anterior or posterior tilt of the pelvis can affect the position of acetabular component in the sagittal plane of the body as compared with its anatomic position in the pelvis. the rotation of pelvis leads to anterior tilt (where the upper portion of the pelvis tips forward) and posterior tilt (upper portion of the pelvis tips backward). the aging process and spinal deformities may lead to an altered sagittal balance. the sacrum and pelvis therefore forms a rigid structure (sacropelvis), which translates and rotates around the bicoxofemoralaxis for the necessary compensatory balance. of importance is the fact that each individual possesses a pelvic equilibrium which is specific and is different in standing and lying positions ; however there are variations in the position of pelvis from the lying to the standing position. consequently the orientation of the acetabular cup version changes dynamically from supine to standing position. in the presence of a normal range of motion of the spine, the pelvis rotates around the transverse axis giving some room for adjustment during movements of the prosthetic hip. we now report a patient who had recurrent dislocations after total hip arthroplasty because of excessive pelvic tilting. this was a 69-year old female with painless limp without any significant history of trauma, with associated severe degenerative kypho - scoliosis. total hip arthroplasty was performed by a posterolateral approach in lateral decubitus position, using a ceramic on metal articulation. a 48 mm acetabular cup (pinnacle) 28 mm metal head and a standard offset stem (summit) [depuy, usa ] postoperative radiographs showed inclination of the cup 45 on anterior posterior view and anteversion 24 on the lateral view [fig. 2 ]. the hip dislocated anteriorly, postoperative three months during standing position while walking, and was confirmed by radiography [fig. 3 ]. revision was done with a constrained liner (duraloc shell, 50 mm constrained liner 28 50 mm depuy usa). postoperative radiographs showed inclination of the cup 45 and acetabular component anteversion 24 in supine position. 4 ], the relevant pelvic parameters were studied but only open reduction with exchange of constrained polyethylene liner was done because patient 's guardian refused re - revision of second acetabular cup. during the postoperative period the patient was maintained in the hip brace at 15 abduction and 15 flexion for six months by locking the joint in the brace and allowing only about 75 of flexion of the hip joint and also preventing adduction during walking in standing position. the patient had been amenable to postoperative prophylactic care and the tendency to dislocation eventually had resolved. apart from routine radiographs, sacral tilt (st) and measured acetabular tilt (mat) were determined in lateral radiographs for evaluation of any pelvic tilt, as described by lazennec. measurements were made using a digitized computer system pacs (picture archiving and communication system piview star version 5, infinitt, seoul korea). st measured in supine and standing position was 49 and 16 and mat in supine and standing position was 25 and 60 respectively [fig. the sagittal position of pelvis is a key factor in impingement as well as dislocation ; it is not vertical nor is it static. in supine position, the pelvis tilts anteriorly, which decreases anteversion of the acetabular component, and in the standing and sitting position, the reverse happens and anteversion is increased. optimal adjustment of the acetabular cup is of utmost importance for ensuring stability and maximum range of motion of the prosthetic joint, without causing impingement on the femoral neck. after total hip arthroplasty increased anterior tilt of acetabular component can cause posterior impingement and anterior dislocation, similarly, posterior tilt or retroversion can cause anterior impingement and posterior dislocation. impingement was prevalent even when components were implanted in the anatomic safe zone in a study of 162 retrieved acetabular components. with increased trend of using hard on hard bearing surfaces, minimizing the risk of impingement which leads to catastrophic events like chip fracture, metallosis, and loosening in these bearing surfaces, it is worth reckoning that component orientation is made in static lying position, whereas prosthesis dislocates out of the result of a dynamic phenomenon which occurs during change in position during the movements of daily life. this is a reasonable explanation, as to why an anatomically oriented cup, placed in supine position may functionally prove to be wrongly oriented, so favoring subsequent dislocations. the patient described here had a fixed kyphoscoliotic deformity, which was compensated by pelvis being tilted posteriorly with a more vertical position of the sacrum. this verticalized the cup and placed the hip in hyperextension during change of position from supine to standing position. a retroverted pelvis led to functional anteversion that was more than the normal range, causing uncovering of the femoral head anteriorly. even though the cup was placed appropriately in accordance with the pelvic bony landmarks it led to posterior impingement and anterior dislocation. this patient had constant low values of st in supine and standing position, signifying posterior tilt of the pelvis. st showed variation of 33 from supine to standing position, which was much more than normal variation. reported normal range of pelvic extension from supine to standing position is less than 1015. the patient was clearly an outlier among the 57% of people who demonstrate excessive hyperextension of hip from supine to standing position. there is significant inter - subject and intra - subject variation in st and mat values from supine to standing position which is directly influenced by the mechanics of lumbosacral junction and resulted in a dynamic functional anteversion that was high outside the safe zone. these require unique adjustment in acetabular cup alignment to maximize stability and avoid impingement and, or dislocation after total hip arthroplasty. st and mat are functional angles and the orientation of acetabulum in supine and standing positions and can be accurately extrapolated from the correlation between pelvic parameters. we would conclude that there are outliers in the pelvis tilt among the patient who undergo total hip arthroplasty. a detailed analysis of individual sagittal balance allows a precise evaluation of the amount of sagittal pelvic hyper - rotation which influences acetabular cup orientation and allows preventive or prospective adjustment of the cup. therefore we intend in future to request a preoperative lateral x - ray view of the spine - pelvis in the upright, supine position and lateral decubitus to evaluate the global sagittal balance, and to plan corrective adaptation of pelvic balance.	introductionthe orientation of acetabular component is influenced by pelvic tilt, body position and individual variation in pelvic parameters. most post - operative adverse events may be attributed to malposition of the component in the functional position. there is evidence that orientation of the pelvis changes from the supine to standing position. authors report a case of recurrent dislocation after total hip arthroplasty due to excessive pelvic tilting.presentation of case a 69-year old female with coxarthrosis had undergone total hip replacement with recurrent dislocation of the hip on bearing weight in spite of using constrained acetabular component.discussionour case report substantiates the influence of pelvic tilt, incurred by a sagittal deformity of spine, on dynamic orientation of the acetabular cup which was positioned in accordance with the anatomic landmarks alone. if the reference is only bony architecture and dynamic positions of the pelvis are not taken into account, improper functional orientation of the acetabular cup can result in sitting and standing positions. these can induce instability even in anatomically appropriately oriented acetabular component.conclusionthe sagittal position of pelvis is a key factor in impingement and dislocation after total hip arthroplasty. pelvic tilting affects the position of acetabular component in the sagittal plane of the body as compared with its anatomic position in the pelvis. we suggest a preoperative lateral view of spine - pelvis, in upright and supine position for evaluation of a corrective adaptation of the acetabular cup accordingly with pelvic balance.
recovery following spinal cord injury (sci) is limited because of axonal damage, demyelination, and scar formation. in addition to the formation of a central hemorrhagic lesion devoid of normal neurons and glia, oligodendrocytes and astrocytes in the white matter near the impact site are reduced by about 50% by 24 h after injury. recently, the use of stem cell for neurodegenerative disease has been widely investigated as a therapeutic strategy [46 ]. neural stem cells have been used for the treatment of neurological diseases such as sci or stroke. numerous studies have reported that the survival and differentiation of grafted cells into neural cells correlate with behavior improvement. however, these cells are limited for clinical application because of insufficient cell supply, risk of immune rejection, and ethical problems. since mesenchymal stem cells (mscs) can be readily isolated and their numbers increased in vitro and differentiated into several types of mature cells including neurons, adipocytes, cartilage, and skeletal hepatocytes under appropriate conditions, a new therapeutic strategy has been a valuable source for central nervous stem (cns) disease [10, 11 ]. human umbilical cord blood - derived mscs (hucb - mscs) have therapeutic potential and are attractive because these cells are readily available and are less immunogenic as compared to other sources of stem cells, such as bone marrow or adipose. an alternative strategy of stem cell therapy is protection of injured cells and promotion of endogenous cell regeneration. several studies have reported that stem cells might provide a better environment for damaged tissue and save remaining neurons by neurotrophic factors or cytokines [13, 14 ]. however, the specific mechanism of the mscs for these assertions remains controversial and ill - explored. nevertheless, msc treatment of sci has been reported as a candidate that supplies angiogenic, antiapoptotic, and mitogenic factors as well as migration toward damaged tissue. recently, mscs have been used in clinical treatment and were shown to be effective in the treatment of various pathologies although evidence for distinct therapeutic mechanism was lacking. the normal spinal cord contains endogenous neural progenitor cells (npc) and oligodendrocyte precursor cells (opcs). nevertheless, production of new neurons and oligodendrocytes by endogenous cells into the spinal cord may be very restricted after injury. furthermore, cell transplantation studies have demonstrated that exogenous stem cells differentiate only very poorly when grafted into the spinal cord. thus, the environment of the spinal cord appears to be highly restrictive for the differentiation of opcs. if this environmental restriction can be changed by hucb - msc in sci, opcs may be able to supply new neurons and oligodendrocytes. however, it is not known whether survival and differentiation generated from endogenous cells are influenced by transplanted hucb - mscs. in the present study, we show that the transplantation of hucb - mscs confers therapeutic effects in a rat experimental sci model. we investigated whether transplantation of hucb - mscs improved the functional recovery and improved the proliferation and genesis of resident endogenous cells within the spinal cord by hucb - mscs. human ucbs were obtained from normal full - term pregnant woman. the protocol for human subjects adhered to the guidelines outlined by the institutional review irb board of the catholic university of korea (seoul, republic of korea). all animal protocols were approved by the institutional animal care and use committee of catholic university medical school. forty - five adult male sprague - dawley rats weighting between 270 and 300 g were employed in our experiments. briefly, rats were deeply anesthetized with ketamine - xylazine cocktail (80 mg / kg of ketamine, 10 mg / kg of xylazine). under a dissecting microscope, the skin and muscles overlying the thoracic cord were separated and retracted, the t9 vertebral level was removed by laminectomy, and the underlying spinal cord segment was exposed by slitting the dural sheath. the impact rod of the nyu impactor was centered above t9 and dropped from a height of 25 mm to induce an incomplete partial sci. following lesion, the dorsal back musculature was sutured and the skin closed with surgical clips. after surgery, the animals were kept on a thermostatically regulated heating pad until completely awake. the urinary bladder of all rats was emptied manually two times per day until recovery of urinary function. rats were assigned randomly to one of the following two major groups : one group of rats were treated with 5 l phosphate - buffered saline (pbs) as the control group. the second group of rats received transplanted with hucb - mscs (3 10 cells/5 l). in experiment 1, the hucb - mscs was designed to test the therapeutic effectiveness (n = 26), and, in experiment 2, these cells were designed to evaluate the proliferation of endogenous cells after transplantation (n = 12). the weight - drop injury level was based on our experience with the model to produce spontaneous recovery at a basso - beatti - bresnahan (bbb) score of 4. once the 46 rats were subjected to contusion sci, they were divided randomly into the two groups. using a 25-gauge syringe (hamilton, reno, nv) fixed in a stoelting stereotaxic frame (dae jong) at 7 days after injury, hucb - mscs were transplanted into the spinal cord (0.5 mm from the midline, 1.5 mm down from the dura, and 5 mm rostral from the contusion epicenter). each rat received a 5 l injection in the contusion site over a 10 min period each time. the cannula of the hamilton syringe was left in place after injection for an additional 5 min. all animal received antibiotics (gentamicin sulfate, 30 mg / kg / day) during the first week after transplantation. sprague - dawley rats (n = 12) were injected with 50 mg / kg brdu (sigma - aldrich, st. louis, mo, usa) intraperitoneally each day for 7 days to label the newly generating cells after transplantation. the motor function restoration after spinal cord contusion was observed by open - field bbb locomotor ratio scale. the scale used for measuring hind - limb function with these procedures ranges from a score of 0, indicating no spontaneous movement, to a maximum score of 21, with an increasing score indicating the use of individual joints, coordinated joint movement, coordinated limb movement, weight - bearing, and other functions. behavioral testing was performed weekly on each hindlimb from the postoperative day to 7 weeks after sci. spinal cord contusion and cell transplantation were separately performed in double - blinded experiments by different investigators. to study functional recovery and differentiation of transplanted hucb - mscs, rats from each group were sacrificed at 1 and 2 weeks (pbs, n = 3 ; transplantation, n = 3) after transplantation and the others were examined by the bbb locomotor test 6 weeks after transplantation (pbs, n = 7 ; transplantation, n = 7). also, to study endogenous cell proliferation after transplantation, rats from each group were sacrificed at 2 h and 1 week after the last brdu injection (n = 5). all the rats were deeply anesthetized with a ketamine - xylazine cocktail (80 mg / kg of ketamine, 10 mg / kg of xylazine) and then perfused transcardially with 0.01 m pbs (ph 7.4), followed by 4% paraformaldehyde (pfa) in 0.01 m pbs. the spinal cord was removed from each rat and postfixed in 4% pfa for 4 hours. postfixed tissue was cryoprotected in 0.1 m phosphate buffer (ph 7.4) containing 15% and 30% sucrose solution at 4c. the spinal cords were embedded in oct compound and stored at 70c. to examine the cavity volume, 14 m thick serial transverse sections were prepared from 20 mm long spinal cord stumps (1 mm each for rostral and caudal to the lesion epicenter). also, to compare the coexpression of various cell - type - specific markers and brdu cells, 10 m thick serial coronal sections were prepared as described above. coronal sections were collected from cell transplantation site to the injury epicenter sites and mounted on gelatin - coated slides. single staining was used to identify newly generated cells after transplantation. for brdu immunohistochemistry, the sections were warmed for 20 min and washed with 0.01 m pbs for 10 min. sections were incubated in 50% formamide-2x standard saline citrate at 60c for 2 h, subsequently treated with 2 n hcl at 37c for 30 min to denature deoxyribonucleic acid, and then incubated in 0.1 mol / l boric acid at room temperature for 10 min to neutralize residual acid. the sections were incubated with rat anti - brdu (1 : 100 ; abcam, cambridge, uk) or mouse anti - brdu (1 : 100 ; dakocytomation, glostrup, denmark). subsequently, sections were incubated for 1 h at room temperature with fluorescence - conjugated secondary antibody or biotinylated antibody ; the latter was reacted with avidin peroxidase for 30 min (abc - kit ; vectastain elite ; vector laboratories, burlingame, ca) followed by detection solution (0.25 mg / ml diaminobenzidine, 0.03% h2o2, 0.04% nicl). to determine the fate of newly generated cells after transplantation, double - fluorescent immunolabeling was performed, combining brdu labeling with one of cell - specific phenotypic markers listed below. we used mouse anti - nestin (1 : 100 ; millipore, billerica, ma) to identify neural stem progenitor ; mouse anti - ng-2 chondroitin sulfate proteoglycan (anti - ng-2 ; 1 : 100 ; millipore) to identify oligodendrocyte progenitor ; mouse anti-2,3-cyclic nucleotide 3-phosphodiesterase (anti - cnpase ; 1 : 100 ; millipore), mouse antimyelin basic protein (anti - mbp, 1 : 100 ; millipore), rabbit anti - glial fibrillary acidic protein (anti - gfap ; 1 : 500 ; millipore) to identify astrocytes. after washing, samples were incubated in alexa 488-conjugated goat anti - rat igg (1 : 200 ; vector laboratories), alexa 546-conjugated goat anti - mouse igg (1 : 200 ; vector laboratories), or alexa 546-conjugated goat anti - rabbit igg (1 : 200 ; vector laboratories) for 1 h. fluorescently stained slides were stored at 20c and observed using a fluorescence microscope equipped with a spot digital camera or a model lsm 510 confocal scanning laser microscope (zeiss, jena, gemany). apoptosis was detected by the terminal deoxynucleotidyl - transferase - mediated d - utp - biotin nick end (tunel) assay using the in situ cell death detection kit (roche, indianapolis, in) developed using the cy2-conjugated streptavidin (jackson laboratories, west grove, pa). brdu cells were counted within a reticule of a specified area (0.0682 mm) positioned in the ependymal and parenchymal region (dorsal (above the corticospinal tract), lateral, and ventromedial region of the residual white matter) in sections. white matter regions were counted in six randomly chosen sections per 1 mm length of spinal cord, and the numbers were averaged. for measurement of the cavity volume, rats at 6 weeks after transplantation were used. the area of the cavity in the damaged spinal cord was measured in images of the sections using imagej version 1.38 image analyzer software (national institutes of health, bethesda, md) on consecutive sections at an interval of 70 m. the volume of the cavity was then calculated by multiplying the average area by the depth of the spinal cord. the bbb score and cell counts were subjected to the paired t - test or one - way anova for transplantation and pbs - treated groups of rats. we assessed the recovery of hindlimb function with the bbb locomotor scale from 1 day to 6 weeks after sci. in the case of sci rats, the motor function scores of mscs - injected rats (11.07 0.3) were significantly higher than the pbs - injected rats (9.25 0.3) at 7 weeks after sci. the behavioral data from the bbb locomotor scores demonstrated that mscs - treated rats were dramatically improved in neurological function (p < 0.005, figure 1). in addition, the spinal cords of mscs - injected rats had cavities much smaller than those of the pbs - injected rats. the cavity volume of mscs - treated rat was 0.82 0.14 mm on average, whereas the pbs - treated rats showed a volume of 2.12 0.28 mm. these results for cavity volume were significantly different between the mscs - treated and pbs - treated rats. thus, msc transplantation led to a significant improvement of behavior as well as reduction of cavity volume after sci. mscs promoted the functional recovery and reduced the cavity volume following transplantation in sci (figure 1). since an effect of hucb - mscs was evident, we investigated whether newly generated cells were enhanced by the transplanted cells. it has been suggested that oligogenesis by endogenous opcs and survival of these cells can contribute to self - repair after myelin loss. with the thought that these processes might be stimulated recovery to cns injury, an experiment was done to investigate the proliferation endogenous generated cells by daily injection of brdu during the 7 days after transplantation. brdu - positive cells were counted in the ependymal and parenchymal regions (figures 2(a) and 2(b)) as previously described [23, 25 ]. proliferation of the newly generated cells increased greatly in hucb - mscs - treated rats as compared with pbs - treated rats (figure 2(c)). this data demonstrated that hucb - mscs could enhance proliferation of endogenous cells within the spinal cord. functional recovery in response to therapeutic grafting of stem cells after sci is related to the differentiation of grafted cells into glial cells, including astrocytes or oligodendrocytes. appropriately, an experiment was done to examine if the transplantation of mscs could enhance the differentiation of endogenous opcs into astrocytes or oligodendrocytes by performing immunostaining for brdu and several phenotype markers including differentiating oligodencrocyte markers ng2, cnpase, the mature oligodendrocyte marker mbp, gfap typical of astrocytes, and the neural stem cell marker nestin. cells in the ependymal and parenchymal region were counted in sections from the injury epicenter to cell transplantation site. one and 2 weeks after cell transplantation, the numbers of brdu positive cells were significantly increased compared with the pbs group (figure 3). in the ependymal region, brdu - labeled nestin and gfap cells were increased compared with the pbs group at 1 and 2 weeks (figure 3). the numbers of brdu - labeled ng2 positive cells were also significantly increased compared with the pbs group in the parenchymal region (figure 3). also, brdu - labeled cells displaying strong immunoreactivities for cnpase, mbp, or gfap in the cell transplantation group were evident. but these immunoreactivities were weak for those rats treated with pbs (figure 4). these data suggest that hucb - mscs are an influential microenvironment within the spinal cord. to investigate whether transplantation of mscs have a protected injured spinal cord cells from apoptosis, a tunel assay was performed on sections obtained from the injury site on 2 weeks after transplantation. numerous tunel - positive (green) cells were observed at the injury site in pbs - treated rats. the number of tunel positive cells was significantly lower in msc - treated rats than in pbs - treated rats (figure 5(b)). taken together, these results indicate that hucb - mscs not only promote oligogenesis in the spinal cord but also have a neuroprotective effect relative with cavity volume (figure 1(c)). in this study, hucb - mscs that were transplanted after sci survived in and around the injured site and were able to ameliorate some of the behavior effects of sci, as measured by spontaneous limb movement in an open - field test, hind limb extension, and toe spread. in addition, the cavities of msc - treated rats were much smaller than pbs - injected rats. cavity formation is a characteristic of progressive tissue necrosis, which follows the initial primary cell destruction in sci. therefore, reduction of the cavity volume means that transplanted mscs after sci have a neuroprotective effect. the presently indicated therapeutic effect of hucb - mscs in sci agrees with previous data, but the exact mechanisms to improve the functional deficits remain to be elucidated. a prior study showed that transplanted cells ameliorated the functional recovery through the integration into spinal cord tissue and establishment of some connections within the injured area of the spinal cord. however, the transplantation of hucb - mscs could not solely account for functional recovery after sci. other possibility may be various beneficial actions of endogenous neurogenesis or oligogenesis within the adult spinal cord which is largely mediated via trophic influences. previous studies have indicated that mscs could produce trophic factors, cytokines, and other neuroprotective factors in stroke or traumatic brain injury [28, 29 ]. bms cells secrete more than 20 cytokines in vitro, and hucb - mscs can secrete a number of cytokines and chemokines. therefore, these factors and some of the other cytokines secreted by hucb - mscs may function as survival and differentiation factors for neural progenitor cells and then play an important role in the proliferation and differentiation of neural tissue and in the increase of central nerve system plasticity [31, 32 ]. to understand whether the transplanted hucb - mscs are capable of restoring the production of endogenous cells, we studied the mechanisms that contributed to functional recovery by determining the endogenous cell proliferation and differentiation into glial cells following transplantation. compared to the control group, transplanted cells increased endogenous cell division within the sci area and a subpopulation of newly dividing cells. also, in the received, the transplanted cells, immature and mature oligodendrocytes, and astrocytes were stimulated. these observations support the possibility that factors produced by hucb - mscs activate nearby oligogenesis, and that activation of the astrocytes increases in oligogenesis, since astrocytes are located in close proximity to neural stem cells and express several factors that independently increase oligogenesis. in addition, some of transplanted cells were brdu - positive cell. but, these cells are not differentiated neural lineage markers. in agreement with the present findings, a previous study reported not only extensive oligogenesis of newly born cells after sci but also that mscs promote oligogenesis in neural stem cells in vitro [24, 33 ]. however, msc - treated rats displayed markedly reduced apoptotic cell death in the injured site. these results suggest that functional recovery might result in endogenous oligogenesis and neuroprotection stimulated by trophic factors secreted into transplanted cells. the collective results support the view that hucb - mscs transplantation is beneficial in sci by virtue of their growth factor secretion and ability to provide physical support to growing axons. further studies are needed to confirm that the benefit obtained from hucb - mscs persists at later time points and/or to improve the efficacy of the transplanted hucb - mcss. also, the mechanisms underlying functional recovery after transplantation of hucb - mscs remain to be further investigated. we have shown that stem cell therapy of hucb - mscs may provide more of functional recovery in spinal cord injury such as reduction of cavity volume, increasing of cell proliferation and endogenous oligogenesis, and decreasing of apoptosis. therefore, the author suggests that promotion of oligogenesis by hucb - mscs may provide a scientific basis for the potential use of these cells as a therapeutic tool for the treatment of other disease.	numerous studies have shown the benefits of mesenchymal stem cells (mscs) on the repair of spinal cord injury (sci) model and on behavioral improvement, but the underlying mechanisms remain unclear. in this study, to investigate possible mechanisms by which mscs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood - derived mscs (hucb - mscs) on the endogenous cell proliferation and oligogenesis after sci. sci was injured by contusion using a weight - drop impactor and hucb - mscs were transplanted into the boundary zone of the injured site. animals received a daily injection of bromodeoxyuridine (brdu) for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem / progenitor cells was evident. behavior analysis revealed that locomotor functions of hucb - mscs group were restored significantly and the cavity volume was smaller in the mscs - transplanted rats compared to the control group. in mscs - transplanted group, tunel - positive cells were decreased and brdu - positive cells were significantly increased rats compared with control group. in addition, more of brdu - positive cells expressed neural stem / progenitor cell nestin and oligo - lineage cell such as ng2, cnpase, mbp and glial fibrillary acidic protein typical of astrocytes in the msc - transplanted rats. thus, endogenous cell proliferation and oligogenesis contribute to msc - promoted functional recovery following sci.
the study included a total of 67 norwegian, 3 swedish, and 2 danish vd patients. the patients were recruited at the department of vascular surgery, oslo university hospital, aker, and were seeking surgical treatment for abdominal aorta aneurism repair, carotid or femoral endarterectomy, or percutaneous transluminal angioplasty of the femoral arteries. clinical oral examinations were performed at the hospital ward to determine the periodontal status of the patient. the first group (vd with cp) was made up of patients undergoing treatment for vd and diagnosed with cp (n=35, mean age=67.9, sd=6.9, range : 51.385.0). the second group (vd without cp) was made up of patients undergoing treatment for vd who were assessed without cp (n=37, mean age=71.1, sd=7.8, range : 55.484.8). demographic data of the patients with atherosclerotic and aortic aneurysmal vascular diseases (vd) with (w/) and without (wo/) chronic periodontitis (cp) the patients medical records were obtained for general health information. ethnicity and smoking behavior written informed consent was obtained from all participants ; the study was approved by the regional ethical committee (rek sr, no. 08/322b) and was in accordance with the helsinki declaration of 1975, as revised in 2008. nct01358630). the patients in both groups were diagnosed and treated according to standard procedures at the department of vascular surgery, oslo university hospital. diagnosis of cp was based on the classification system of the american academy of periodontology, established in 1999 at the international workshop for classification of periodontal diseases and conditions (31). clinical periodontal status was assessed by a trained dentist (armingohar) as previously described (16). the vascular biopsies were collected under surgical treatment from the walls of aneurisms and during excision of intravascular plaques in carotid or common femoral arteries. saliva samples were collected with the oragene dna self - collection kit (genotek, ottawa, ontario, canada) according to the manufacturer 's instructions. extraction of genomic dna was performed using the masterpure complete dna purification kit (epicentre biotechnologies, madison, wi, usa) as previously described (16). snp allele discrimination assays were performed by the real - time polymerase chain reaction (pcr) amplification method. the alleles of il10 at 592 (rs 1800872) and 1,082 (rs1800896), were detected by 5'nuclease allelic discrimination taqman method using assays by design / demand (applied biosystems, foster city, ca, usa) with locus - specific forward and reverse primers and two allele - specific oligonucleotide probes for each snp of interest (table 2), according to manufacturer 's instructions. the alleles of il10 819 (rs1800871) were detected by 5 nuclease allelic discrimination method using locus - specific forward and reverse primers and two allele - specific oligonucleotide probes for each snp of interest (table 2 ; biochemistry department, university of oslo, oslo research park) as described previously (32). the probes for each allele (listed in table 2) were labeled with fam or vic at the 5 end, and non - fluorescent quencher or usa) using the taqman universal pcr master mix (applied biosystems), along with approximately 20 ng dna template. primer and probe sequences used to detect nucleotide polymorphisms in this study snp, single nucleotide polymorphism ; fw, forward primer ; rv, reverse primer. taqman probes are listed with the polymorphic bases in bold. to confirm the results of the taqman genotyping, part of the il-10 promoter region covering the snp loci of interest was pcr amplified and sequenced. pcr reactions were performed with forward primer 5-acaaatc caagacaacactactaag-3 and reverse primer 5-atgaatacccaagacttctccttgcta-3 sigma - aldrich, mo, usa), and onetaq 2x master mix (new england biolabs, beverly, ma, usa) at an annealing temperature of 55c. the pcr products were purified by exosap - it (affymetrix, santa clara, ca, usa) according to the manufacturer 's protocol. purified amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit (applied biosystems) and the forward and revers primers. sequence reactions were run on an abi prism 3730 dna analyzer (applied biosystems). sequence trimming, quality check and alignment were performed with the sequencher 5.0 program (gene codes corp. the statistical analyses were performed using the spss software (ibm spss statistic version19) and javastat (http://statpages.org/). chi - square and fisher 's exact tests were used for univariate comparisons of allelic and genotypic frequencies of the snp loci between the groups. binary logistic regression was used to adjust the genotypic differences for the effect of age, gender, body mass index (bmi), diabetes, and smoking status. the genotype analyses were performed by comparing each genotype frequency to the pooled total of the other groups. the assembly and prediction of haplotypes for the il10 snp cluster were done by the software the study included a total of 67 norwegian, 3 swedish, and 2 danish vd patients. the patients were recruited at the department of vascular surgery, oslo university hospital, aker, and were seeking surgical treatment for abdominal aorta aneurism repair, carotid or femoral endarterectomy, or percutaneous transluminal angioplasty of the femoral arteries. clinical oral examinations were performed at the hospital ward to determine the periodontal status of the patient. the first group (vd with cp) was made up of patients undergoing treatment for vd and diagnosed with cp (n=35, mean age=67.9, sd=6.9, range : 51.385.0). the second group (vd without cp) was made up of patients undergoing treatment for vd who were assessed without cp (n=37, mean age=71.1, sd=7.8, range : 55.484.8). demographic data of the patients with atherosclerotic and aortic aneurysmal vascular diseases (vd) with (w/) and without (wo/) chronic periodontitis (cp) the patients medical records were obtained for general health information. ethnicity and smoking behavior written informed consent was obtained from all participants ; the study was approved by the regional ethical committee (rek sr, no. 08/322b) and was in accordance with the helsinki declaration of 1975, as revised in 2008. the patients in both groups were diagnosed and treated according to standard procedures at the department of vascular surgery, oslo university hospital. diagnosis of cp was based on the classification system of the american academy of periodontology, established in 1999 at the international workshop for classification of periodontal diseases and conditions (31). clinical periodontal status was assessed by a trained dentist (armingohar) as previously described (16). the vascular biopsies were collected under surgical treatment from the walls of aneurisms and during excision of intravascular plaques in carotid or common femoral arteries. saliva samples were collected with the oragene dna self - collection kit (genotek, ottawa, ontario, canada) according to the manufacturer 's instructions. extraction of genomic dna was performed using the masterpure complete dna purification kit (epicentre biotechnologies, madison, wi, usa) as previously described (16). snp allele discrimination assays were performed by the real - time polymerase chain reaction (pcr) amplification method. the alleles of il10 at 592 (rs 1800872) and 1,082 (rs1800896), were detected by 5'nuclease allelic discrimination taqman method using assays by design / demand (applied biosystems, foster city, ca, usa) with locus - specific forward and reverse primers and two allele - specific oligonucleotide probes for each snp of interest (table 2), according to manufacturer 's instructions. the alleles of il10 819 (rs1800871) were detected by 5 nuclease allelic discrimination method using locus - specific forward and reverse primers and two allele - specific oligonucleotide probes for each snp of interest (table 2 ; biochemistry department, university of oslo, oslo research park) as described previously (32). the probes for each allele (listed in table 2) were labeled with fam or vic at the 5 end, and non - fluorescent quencher or usa) using the taqman universal pcr master mix (applied biosystems), along with approximately 20 ng dna template. primer and probe sequences used to detect nucleotide polymorphisms in this study snp, single nucleotide polymorphism ; fw, forward primer ; rv, reverse primer. taqman probes are listed with the polymorphic bases in bold. to confirm the results of the taqman genotyping, part of the il-10 promoter region covering the snp loci of interest was pcr amplified and sequenced. pcr reactions were performed with forward primer 5-acaaatc caagacaacactactaag-3 and reverse primer 5-atgaatacccaagacttctccttgcta-3 sigma - aldrich, mo, usa), and onetaq 2x master mix (new england biolabs, beverly, ma, usa) at an annealing temperature of 55c. the pcr products were purified by exosap - it (affymetrix, santa clara, ca, usa) according to the manufacturer 's protocol. purified amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit (applied biosystems) and the forward and revers primers. sequence reactions were run on an abi prism 3730 dna analyzer (applied biosystems). sequence trimming, quality check and alignment were performed with the sequencher 5.0 program (gene codes corp. the statistical analyses were performed using the spss software (ibm spss statistic version19) and javastat (http://statpages.org/). chi - square and fisher 's exact tests were used for univariate comparisons of allelic and genotypic frequencies of the snp loci between the groups. binary logistic regression was used to adjust the genotypic differences for the effect of age, gender, body mass index (bmi), diabetes, and smoking status. the genotype analyses were performed by comparing each genotype frequency to the pooled total of the other groups. the assembly and prediction of haplotypes for the il10 snp cluster genotyping was successful in all but two patients, who were excluded from further analysis. in table 3, allelic and genotypic frequencies of the snps il10 592, 819 and 1,082 are listed. allele and genotype frequencies within the il10 gene locus in patients with atherosclerotic and aortic aneurysmal vascular disease (vd) with (w/) or without (wo/) chronic periodontitis (cp) snp, single nucleotide polymorphism. allele designation (1= more frequent, major allele ; 2=less frequent, minor allele). genotype designations. the genotypic differences were adjusted for the effect of age, gender, body mass index, diabetes, and smoking status. using chi - square tests, a significant difference in allelic frequencies was found between the two study groups regarding il10 592 (p=0.03, or=2.23, 95% ci=1.074.68) and il10 819 (p=0.047, or=2.1, 95% ci=1.0034.42) with the c allele being more frequent in the vd group with cp. the frequencies of the il10 592 genotype 1/1 (c / c) showed a significant difference (chi - square, p=0.01, or=3.53, 95% ci=1.339.31) comparing the group of vd patients with cp (n=22) with the group of vd patient without cp (n=12). after adjustment for the risk factors age, gender, bmi, diabetes and smoking status by logistic regression, this difference was significant (p=0.02, or=4.0, 95% ci=1.2812.35), indicating a significant association of the il10 592 c / c genotype with risk for cp among vd patients. the heterozygote il10 592 genotype 1/2 (c / a) frequencies displayed a statistical significant difference comparing the two groups of patients (chi - square, p=0.02, or=0.31, 95% ci=0.120.82), being lower in the vd patients with cp (n=11) compared to those without cp (n=22). this difference was significant (p=0.02, or=0.26, 95% ci=0.080.82) in the logistic regression model, indicating a protection against periodontitis among vd patients with the il10 592 1/2 genotype. the il10 592 genotype was significantly associated with cp in an overall analysis of the individual genotypes using chi - square (p=0.03). this association remained borderline significant in a logistic regression model (p=0.05) (results not shown). the il10 819 genotype 1/1 (c / c) showed a significantly different frequency between the two groups (chi - square, p=0.02, or=3.12, 95% ci=1.198.2), being higher in the vd with cp group (n=22) than in the vd without cp group (n=13). when adjusted for the risk by logistic regression, this difference remained significant (p=0.02, or=3.69, 95% ci=1.211.4), indicating a possible association of the il10 819 c / c genotype with risk for cp among vd patients. the frequencies of the heterozygote il10 819 genotype 1/2 (c / t) displayed statistical significant difference comparing the vd patients with cp group (n=11) with those without cp (n=21) (chi - square, p=0.03, or=0.35, 95% ci=0.130.92). this difference was significant in the logistic regression model (p=0.03, or=0.28, 95% ci=0.090.88) suggesting protection against cp among vd patients with the il10 819 1/2 genotype. no significant allelic or genotypic frequency differences in the il10 1,082 snp were detected comparing the vd patients with and without cp (p>0.05). the predicted il10 haplotype ata (1,082, 819, 592) designated as 1 - 2 - 2 showed a borderline significant difference comparing the two patient groups (chi - square, p=0.047, or=0.47, 95% ci=0.211.06), possibly indicating protection against periodontitis compared to the other haplotypes. estimated il10 locus haplotype frequencies in patients with atherosclerotic and aortic aneurysmal vascular disease (vd) with (w/) or without (wo/) il10 (1,082), il10 (819), and il10 (592) haplotype designation. no statistically significant correlations were found between the risk factors age, gender, bmi, diabetes and smoking status. the results from snp genotyping using real - time pcr amplification method and by dna sequencing were in agreement. cp has been associated with vd and is suggested to be an additional risk factor for cardiovascular diseases independent of confounding factors (5, 6). polymorphisms in inflammatory genes have been investigated for association with periodontal and cardiovascular disease susceptibility, and both shared (1012) and distinct genetic associations (4, 10, 11, 13, 14) have been identified. here, we investigated whether polymorphisms in the il-10 gene promoter region can be associated to cp among patients with vd. three functional snps in the promoter region of the il10 at positions 1,082 (a > g), 819 (c > t) and 592 (c > a) are reported to have a regulatory function on promoter activity (26), and are associated with altered il-10 production (28, 34). these snps have been investigated for association with cp susceptibility, but with inconsistent results (9, 29). ethnic differences in il10 allele and genotype frequencies may be a reason for the discrepancy in reported results ; hence only scandinavian patients were included in the present study (35). the il10 snp at position 592 (c > a) is located in a negative regulatory region, the 819 (c > t) polymorphism may affect an estrogen receptor element (34), while the il10 1,082 (a > g) snp lies within a putative e - twenty - six ets like - transcription binding site and may influence transcriptional activity (27). larsson. showed that the transcription factor sp1 only bound to the g allele at the 1,082 position in the il-10 promoter region resulting in higher il-10 and sp1 mrna expression for gg genotypes in b - cells (28). the three snps are in close linkage disequilibrium, with three main preference haplotypes (1,082, 819, 592) : gcc, acc and ata (26, 34). the gcc haplotype is associated with high, the ata haplotype with low and the acc with an intermediate il-10 production (36, 37). have shown in their study that both il10 592 ca and aa genotypes were more frequent in patients with cp, and that these genotypes were associated with disease severity and lower levels of il-10, tissue inhibitor of metalloproteinase-3 (timp-3), and the inhibitor of osteoclastogenesis opg mrna expression in diseased periodontal tissues (22). cullinan. investigated the possible association between il-10 gene polymorphisms and periodontal disease progression in a prospective longitudinal study in an australian population. they reported that individuals having either the ata / acc or the acc / acc genotype experienced less periodontal disease progression after 5 years compared to individuals with other genotypes (38). in our patients, the putative high il-10 producer alleles (819 c and 592 c) and genotypes (819 c / c and 592 c / c) were associated with cp, while the low producer ata haplotype showed a trend to protection against cp. in addition, the g allele and g / g genotype of the il 1,082 were more frequent among cp patients, but this difference did not reach a significant level comparing the two groups. in contrast to our study, the alleles il10 819 t and 592 a, which are connected with low il-10 levels, have been associated with risk for cp in other caucasian populations (29, 39). similar to our study, the high il10 producer g allele and gg genotype of il 1,082 have been found to be associated with cp in a swedish population (40). while in other studies, the low producer a allele of 1,082 has also been associated with risk for cp (41, 42). these previous findings compared to our findings, suggest that studying populations of similar ethnicity show more consistency and reproducibility. this could be due to increase of il10 genetic effect size in the scandinavian (nordic) population(s) from natural selection and/or variable exposures to environmental and other factors affecting immunocompetence in cp and vd. it is plausible that il-10 can reduce clearance of infectious pathogens by suppressing the protective innate and adaptive immune responses, and therefore is involved in the persistence of bacteria and chronic infections (19, 43). murine models of infectious diseases have shown that even normal levels of il-10 tend to limit the efficiency of immune responses against pathogens, and that the resistance to infection can be improved by reducing il-10 levels (44). additionally, il-10 enhances the release of anti - inflammatory mediators such as il-1 receptor antagonist (18). in a previous study, we found a significantly higher frequency of the allele 1 and a higher frequency of allele 2 of the ilirn vntr among cp patients with vd (16). association studies have shown that homozygous carriers of allele 2 of the il-1ra gene have a longer and more severe pro - inflammatory immune response than persons with other il1rn genotypes (15). thus, it is conceivable that the carriage of allele 2 can be beneficial in combating microbial challenges in the gingival sulcus. indeed, allele 2 carriers have shown reduced vaginal colonization by mycoplasma and resistance to human cytomegalovirus and epstein - barr virus (15). together, these findings indicate that excessive down - regulation of the pro - inflammatory mechanisms could result in reduced efficiency of anti - pathogenic immune responses in periodontal disease. the genotype of cp patients in this study and our previous study (16), associated with excessive anti - inflammatory responses and potentially reduced pathogen clearance, can therefore be detrimental in periodontal disease susceptibility. the number of patients in the present study was limited because it is difficult to recruit patients with life - threatening vd, being prepared for major surgery. the consequence is an increase in the risk for type ii error, the possibility that potential differences between groups might not be detected. yet, we have found a statistically significant difference between the study groups regarding the il10 819 and 592 independent of shared risk factors, indicating that in persons with vd, genotypes of inflammatory factors within the il-10 gene cluster are associated with cp. animal studies and in vitro studies on human aortic endothelial cells have shown anti - atherogenic properties of il-10 (45, 46). genotype and haplotype variants within the il10 gene have been associated with atherosclerotic diseases, but with discrepant results (47, 48). our findings, however, suggest that these gene variants still can have a stronger association to cp than vd. the development of cp may constitute additional burden in the pathogenesis of vd by causing sustained low - graded systemic inflammation and bacterial dissemination in the circulation (49). in this study, we have identified genetic markers that are specifically associated with cp within a group of patients with vd. information about these genetic components or predictive biomarkers may help to identify subgroup of vd patients who are at risk to develop cp, and selectively target them for effective preventive cp interventions.	backgroundchronic periodontitis (cp), atherosclerotic and aortic aneurysmal vascular diseases (vd) are chronic inflammatory conditions with multifactorial etiologies, including involvement of predisposing genetic factors. in a previous study, polymorphisms in the gene for the anti - inflammatory interleukin-1 receptor antagonist were associated with cp in patients with vd.objectivethis study investigates whether polymorphisms in the gene for the anti - inflammatory interleukin-10 (il10) could be related to cp in the same manner.methodsseventy-two patients with vd of whom 35 had cp were genotyped for single nucleotide polymorphisms (snps) in the il10 592 (rs1800872), 819 (rs1800871), and 1,082 (rs1800896) gene by taqman rtpcr method and by dna sequencing.resultsthe c alleles and c / c genotypes of il10 592 and il10 819 frequencies were significantly higher, while the frequencies of the il10 592 (c / a) and il10 819 (c / t) heterozygote genotypes were significantly lower in the vd group with cp compared to those without cp. the il10 haplotype ata frequency (1,082, 819, 592) showed a trend to a significant difference between the two groups indicating protection against cp.conclusionstaken together, our findings suggest an independent association of genetic polymorphisms in the il-10 gene locus with cp in patients with vd. development of cp and the implications on vascular disease emphasize the importance of early detection and adequate treatment of periodontitis among these patients.
one uncommon manifestation is amyloid myopathy (am), the symptoms of which are usually non - specific, typically including progressive proximal weakness with an increased creatine kinase (ck) level, macroglossia and muscle pseudohypertrophy. the diagnosis of am is usually overlooked and it is often misdiagnosed as inflammatory myopathy, even when a muscle biopsy is available. when suspecting am, congo red staining and an immunohistochemistry or immunofluoresence assay should be performed. furthermore, the use of mri for evaluating neuromuscular disorders and inflammatory myopathies has become increasingly common, especially in cases of polymyositis. we present here a case of al with severe and rapidly progressive myopathy as the initial symptom. the patient was a 65-year - old man with a history of hypertension and obesity (weight 144 kg) who presented with proximal muscle weakness in the legs and a 35 kg weight loss. no familial occurrence of muscle diseases was reported. at the first clinical examination after two years of experiencing fatigue, the patient showed slight proximal muscle weakness corresponding to 4/5 on the medical research council (mrc) scale and minor weakening of the tendon reflexes but was able to walk without support. muscle weakness progressed within six months, however, forcing the patient to use a walking aid. he then had grade 3 symptoms on the mrc scale in the shoulder girdle and bilaterally in the thigh muscles, with grade 4 in the distal hand and leg muscle groups. in addition, the new findings included mild dysphagia, macroglossia and an absence of tendon reflexes. laboratory findings included elevated serum creatine kinase (ck) and serum aldolase levels (table 1). serum transferrine, protein electrophoresis and immunofixation findings were normal, whilst serum ferritine was elevated. an electromyography (emg) revealed proximal fibrillation and motor unit potentials of short duration, low amplitude and a polyphasic character and insertional activity. the patient had arrhythmic episodes of supraventricular tachycardia (svt), and a chest x - ray film showed cardiac dilatation. the ejection fraction was 55%, and the myocardium was slightly abnormal, indicating muscle disease. table 1abnormal laboratory test findings.laboratory testlaboratory valuesreference valuess - ck (u / l)1900220040280 (for men)s - aldolase (u / l)18<5e - mcv (fl)1001068298p - albumin (g / l)24353645u - protein (mg / l)2100<200s- ferritine (ug / l)79720250p - alat (u / l)901961070p - gt (u / l)242128415115 muscle magnetic resonance imaging (mri) was performed with t1, short tau inversion recovery (stir) and t2 fat sat axial projections (figure 1). axial t2 fat suppression magnetic resonance imaging of the thighs showed a mildly increased signal intensity in the posterior muscles, while coronal t2 fat suppression magnetic imaging showed a coarse reticular pattern in the subcutaneous fat of the lower extremities. a left vastus lateralis muscle biopsy showed mainly non - specific changes, but there was some evidence suggestive of inclusion body myositis. the abdominal ct results were normal. the provisional diagnosis at this stage was considered to be inclusion body myositis. a gastroscopy was performed on account of elevated liver enzymes and a suspicion of celiac disease, and biopsies from the duodenum, antrum and corpus revealed diffuse mucosal, submucosal and vascular amyloid accumulation. the amount of plasma cells in a bone marrow biopsy was normal. in view of the gastric biopsy findings and the diagnosis of primary amyloidosis, the muscle biopsy was re - examined more specifically. congo red staining eventually showed vascular and extracellular amyloid accumulation, which led to a diagnosis of am. despite the gastrointestinal changes, the patient did not have any clinical symptoms of this. figure 1(a) axial t2 fat suppression magnetic resonance imaging of the patient s thighs. mildly increased signal intensity (b) coronal t2 fat suppression magnetic imaging show a coarse reticular pattern in the subcutaneous fat of the lower extremities. (a) axial t2 fat suppression magnetic resonance imaging of the patient s thighs. mildly increased signal intensity is visible in the posterior muscles. (b) coronal t2 fat suppression magnetic imaging show a coarse reticular pattern in the subcutaneous fat of the lower extremities. after the ibm diagnosis, the patient was treated with prednisone 100 mg / day for two months and then at a decreasing dosage for three months. since the muscle weakness had markedly progressed in spite of this prednisone medication, the treatment was stopped after five months. immunoglobulin 0.4 mg / kg i.v. was given for five days, leading to a minor improvement in muscle strength, so that the treatment was repeated three times monthly. oral melphalan and prednisone treatment was also started after the diagnosis of amyloid myopathy, the total target dose of melphalan being 740 mg, while prednisone was given 100 mg p.o. once a day for four days. only a minor response to the medication was seen as far as the progression of muscle weakness and other symptoms during one year of follow - up was concerned. the first examination of the left vastus lateralis muscle biopsy showed changes suggesting neurogenic atrophy, regeneration and some rimmed vacuoles. nadh - tr and trichrome staining results were normal, but sdh - cox staining revealed some cox - negative fibers. no mch - class i upregulation was seen on the muscle membranes nor any fiber necrosis. although the muscle biopsy findings were mainly non - specific, there was some evidence suggestive of inclusion body myositis. a gastroscopy was performed later on account of elevated liver enzymes and a suspicion of celiac disease, and duodenum, antrum and corpus biopsies revealed diffuse mucosal, submucosal and vascular amyloid accumulation which was resistant to potassium permanganate digestion. an immunohistochemical assay was negative for amyloid a, but the accumulation was positive for lambda light chain staining. the number of plasma cells in a bone marrow biopsy was normal. in view of the gastric biopsy findings and the diagnosis of primary amyloidosis congo red staining eventually showed vascular and extracellular amyloid accumulation, which led to a diagnosis of am (figure 2). an immunohistochemical assay for amyloid a was negative, and kappa and lambda light chain stainings were inconclusive. figure 2(a) biopsy of the vastus lateralis muscle shows the vascular wall and an interstitial accumulation of amyloid (congo red), (b) expression of neonatal myosin in atrophic cells (myosin heavy chain neonatal immunoperoxidase staining, original magnification 20), (c) a cox - negative fiber (succinate dehydrogenase cytochrome oxidase double staining) and (d) a duodenal mucosal biopsy containing amyloid (congo red). scale bar 100 m (figure a (a) biopsy of the vastus lateralis muscle shows the vascular wall and an interstitial accumulation of amyloid (congo red), (b) expression of neonatal myosin in atrophic cells (myosin heavy chain neonatal immunoperoxidase staining, original magnification 20), (c) a cox - negative fiber (succinate dehydrogenase cytochrome oxidase double staining) and (d) a duodenal mucosal biopsy containing amyloid (congo red). scale bar 100 m (figure a the first recognised patient presenting with amyloid involvement in muscle, the condition known as am, was reported by lubarsch in 1929, and it has now been reported in three of the largest series that myopathy is rarely caused by amyloidosis. the symptoms of am are usually non - specific, including progressive proximal weakness with an elevated creatine kinase level, macroglossia and muscle pseudohypertrophy as the typical features. we described a case of al with severe, rapidly progressive myopathy as the initial symptom. the patient had suffered from mild muscle weakness for 2.5 years, followed by a rapid progression of the condition and the onset of other clinical symptoms such as dysphagia and macroglossia. visceral manifestations of al, including cardiomyopathy, nephropathy and gichanges, were also observed. weakness of knee extensors with absent knee reflexes was prominent in the early course of the disease suggesting the diagnosis of ibm although distal involvement in the hands was observed later. although amyloid myopathy is a welldescribed disorder, it is often overlooked, for two reasons : first, it is rarely observed clinically, and second, a broad span of differential diagnoses is needed. it is usually misdiagnosed as inflammatory myopathy, even given a muscle biopsy, as seen here and reported by others. three cases of am mimicking polymyositis have been reported lately, all showing similarities in clinical, neurophysiological and muscle biopsy findings when stained only with hematoxylin - eosin. in all three cases, the original muscle biopsy samples were analyzed retrospectively with congo red staining, which led to a proper diagnosis of am. furthermore, spuler. found that the routine use of congo red - stained sections increased the frequency of a diagnosis of am 10-fold. the present case illustrates the difficulty of differentiating am from inflammatory myopathies : the first suspected diagnosis was inclusion body myositis. the presence of neonatal myosin in some fibers reflects the immaturity / regenerative activity of the muscle and is a sign of a pathological state in the muscle. cox - negative fibers are a common finding in ibm, but non - specific, as they are often seen in the muscle of aged people. am was not suspected until amyloidosis was seen in the gastric mucosal biopsy. congo red staining eventually showed vascular and extracellular amyloid accumulations in the original muscle biopsy sections, which led to a diagnosis of am. the use of mri for the evaluation of inflammatory myopathies and neuromuscular disorders has become increasingly common, especially in cases of polymyositis, and its usefulness for diagnosing am has also been described. reported a case of a patient with the occurrence of abnormal subcutaneous fat in mri and increased t2 signal intensity and minimal signal intensity alternation in the muscles, and established that these findings coupled with minimal muscular involvement constituted a unique guide to a proper diagnosis of primary am. this is consistent with the previous mri findings in 2 patients described by metzler. in particular a hypointense reticular appearance for the subcutaneous fat in mri furthermore, in the case of the patient described by hull., mri also revealed decreased t1 signaling in the bone marrow, suggesting hematologic malignancies. in the present case the muscle mri revealed evidence suggestive of atrophy and oedema in the muscles of the hips, thighs and calves nevertheless, as noticed after the patient s death, the mri had been performed without using a muscle - specific protocol, so that the finding remained inconclusive. in summary, we have reported here on a well - described case of the rarely occurring disease am. this case demonstrates that am can masquerade as ibm, which is known to be a more common disorder. the inflammatory myopathies are increasingly being evaluated using mri, which can be of practical significance in differentiating am from other muscle diseases, targeting affected muscles for biopsy and assessing disease activity. in addition, mri is of use when performed with an appropriate muscle - specific protocol. to clarify the etiological diagnosis of myopathy,	amyloid myopathy (am) is a rare manifestation of primary systemic amyloidosis (al). like inflammatory myopathies, it presents with proximal muscle weakness and an increased creatine kinase level. we describe a case of al with severe, rapidly progressive myopathy as the initial symptom. the clinical manifestation and muscle biopsy were suggestive of inclusion body myositis. am was not suspected until amyloidosis was seen in the gastric mucosal biopsy. the muscle biopsy was then re - examined more specifically, and congo red staining eventually showed vascular and interstitial amyloid accumulation, which led to a diagnosis of am. the present case illustrates the fact that the clinical picture of am can mimic that of inclusion body myositis.
recently we demonstrated that time domain nuclear magnetic resonance (td - nmr) is a very efficient and robust technique to quantify paramagnetic ions in a solution during in situ measurements of electrodeposition reactions [1, 2 ]. it is also efficient in determining the solubility constant, k sp, of paramagnetic ions. such efficiency comes from the fact that this is a nondestructive analysis, which facilitates the coupling with other techniques [14 ]. furthermore, operation and maintenance costs of the equipment are low, compared with current techniques for quantification. this technique is performed with the known cpmg (carr - purcell - meiboom - gill) pulse sequence which measures the transverse relaxation time constant (t 2) of the h nuclei. when in an aqueous solution, the unpaired electrons of the paramagnetic ions interact with the h nucleus in the solvent accelerating the relaxation process [58 ]. the variation of t 2 is inversely proportional to the concentration of the paramagnetic ions. the objective of this work was to compare the relaxometry technique, for the quantification of the paramagnetic ions contained in aqueous electrolytic solutions, using the uv - vis spectroscopic technique and data from the atomic absorption spectroscopy, which was obtained from the literature. one possible application of this technique could be to monitor the process of corrosion, specifically corrosions taking place under the effect of a magnetic field, as it is already known that the magnetic field will alter the speed of the reaction [1, 2, 9 ]. the stock electrolyte solution was prepared with milli - q water, 0.1 mol l cuso45h2o (synth), and 0.1 mol l na2so4 (isofar). in order to obtain the calibration curve, this solution was diluted (using a 0.1 mol l na2so4 solution) down to a concentration of 10 mol l cu. the stock electrolyte solution (watts solution) was prepared with milli - q water, 300 g l niso46h2o (synth), 90 g l nicl26h2o (synth), and 45 g l h3bo3 (fluka). this solution (ni 1.52 mol l) was diluted (using a 45 g l h3bo3 solution) down to a concentration of 10 mol l ni. the stock electrolyte solution was prepared with milli - q water, 0.02 mol l crcl36h2o (vetec), 10 g l nh4cl (synth), 14 g l nacl (synth), 20 g l h3bo3 (fluka), 30 g l glycine (j.t.baker), and 63 g l methanol (vetec). this solution was diluted (using the same solution as before, but with the absence of the chrome ions) down to a concentration of 10 mol l cr. the stock electrolyte solution was prepared with milli - q water, 0.1 mol l mnso4h2o (synth) water, and 0.1 mol l na2so4 (isofar). in order to obtain the calibration curve, this solution was diluted (using 0.1 mol l na2so4 solution) down to a concentration of 10 mol l mn. the measurements were performed in two equipment pieces : a 0.23 t, td - nmr spectrometer (spinlock, crdoba argentine).the home - built unilateral td - nmr sensor (unmr) with 0.33 t (14.2 mhz for h) constructed in a a 0.23 t, td - nmr spectrometer (spinlock, crdoba argentine). the home - built unilateral td - nmr sensor (unmr) with 0.33 t (14.2 mhz for h) constructed in a u - shaped geometry [14, 15 ]. the uv / vis spectrometer used for these experiments was from perkinelmer manufacturer, model lambda 25. the measurements were performed in the wavelength range of 1000 nm to 600 nm. the maximum absorption peaks were situated at 810, 422, and 394 nm for the aqueous solutions of cu, cr, and ni, respectively. the cpmg parameters were /2 and pulses with 6.2 and 12.4 s, respectively. the number of echoes differed for each sample but ranged from 400 to 2000 echoes. the cpmg parameters were /2 and pulses with 3 and 6 s, respectively. the time between each refocusing pulse (2) was 120 s, 2000 echoes were used, the recycling delay was 500 ms, and 300 scans were performed. seven curves were made for each ion ; thereby the used value of t 95% was 2,447 (student 's t - test). the confidence level used for the calculation of the validation parameters for the relaxometry was 95% (table 1). the validation parameters for the relaxometry technique, using the spinlock and the unmr, are shown in table 1. to compare the relaxometry with the spectrophotometry (sp) the solutions of cu, ni, and cr were used due to their intense colors. the validation parameters for the sp are also in table 1. to calculate the limit of detection (lod) and limit of quantification (loq) the following equations were used:(1)lod = x+t95%,loq = x+10,where x is the average of the signals obtained for the blank solutions (7 measurements) and is the standard deviation. the relaxometry measurements using the spinlock showed the best results, with a lower lod (10 mol l) and loq (10 mol l) in comparison to the measurements obtained using the unmr (10 and 10 mol l, resp.). the best efficiency of the spinlock can be explained by the homogeneous magnetic field, which eliminates the diffusion effects that are present in the unmr measurements. another advantage of the spinlock measurements is that the sample is fully analyzed while in the unmr only a slice is analyzed (at a height of 3 mm above the sensor surface). despite the fact the unmr has the highest loq, it has some advantages : it is an open system that allows the analysis of big samples ; it is small and light (less than 2.5 kg) which facilitates its transportation and it has a larger superior limit of quantification than the spinlock. the sensitivity of both spectrometers is similar, differing by approximately 15%. these techniques showed the highest sensitivity towards mn, which can be associated with the fact that the mn has more unpaired electrons than the other ions, which enhance the relaxation effect [5, 8 ]. therefore, manganese has the lowest loq (10 mol l for spinlock and 10 mol l for unmr). the spinlock and unmr linear regression curves for ions ni, cu, cr, and mn are shown in figure 1. the figure is a plot of the ion concentration (mol l) versus r 2 (s), which is the inverse of t 2. the parameters of the regression curves are displayed in table 2 for spinlock, unmr, and sp. it is possible to note a large difference in the intercept value between both techniques (0.4 s for spinlock and 10 s for unmr). this difference can be attributed to the diffusion effects that take place during the unmr measurements. since nothing is truly stationary in a solution and the unmr spectrometer possesses such a large magnetic field gradient, the movement of the particles towards a different location and different magnetic field strength attenuates the measured signal and the apparent relaxation time (which is different from the true relaxation time of the sample) and, thus, increases the value of the intercept of the curves. furthermore, by comparing the intercept value throughout the same technique but for different ions, a slight difference is noted for the cr ions. this difference can be explained in light of the different additives used in the chromium solution which affect the relaxation time of the sample by reducing it. this shows that the composition of the matrix must be taken into account when performing relaxometric measurements, as they can influence the relaxation time of the sample. as expected, for (cu)aq the loq (10 mol l) for sp is higher than the relaxometry technique using spinlock (10 mol l). furthermore, the sp can be used only for quantification of compounds which have absorption bands in the uv - visible region ; because of this the (mn)aq concentration can not be determined by this technique without a pretreatment of the sample. another technique commonly used for quantification of ions is the atomic absorption spectrometry (aas), which has the advantage of being selective, but the disadvantage is its high cost of operation and maintenance of the equipment in addition to having a limitation on the viscosity of the solvent. the loq for the measurements performed in the spinlock spectrometer is approximately of one order of magnitude larger than aas, as shown in table 3. it is worth noting that the relaxometry technique is only sensitive to paramagnetic species and metal ions, whereas aas is not as selective and is able to detect both metal atoms and metal ions. it can be used in solution when the solvents have high - viscosity and it does not require chemical indicators or support electrolytes. a disadvantage of relaxometry is that other species in the solution, such as complexants and other paramagnetic species, can interfere with the analysis. when constructing a calibration curve the matrix composition however, this technique has low cost of operation and maintenance, it does not require specialists for its operation, and it is a good option for use in rapid analysis and for coupling with other techniques. a possible application of this technique is to quantify ions in residual effluents of electroplating industry and other chemical residues in a simple and fast way. with this work we have demonstrated that the relaxometry technique performed with the spinlock spectrometer has a lower loq than the one performed with unmr. this can be explained by the homogeneous magnetic field of the spinlock, which eliminates diffusion effects. nevertheless, unmr has the advantage of being an open system, which does not limit sample size. unmr has proven to be better than sp, as it is able to detect paramagnetic ions without sample pretreatment, which is the case with sp when analyzing, for example, mn ions. the loq obtained through the spinlock spectrometer is of one order of magnitude larger than the one found with aas (10 mol l for spinlock against 10 mol l for aas) but has the advantage of being a much cheaper and more robust technique than aas. in conclusion td - nmr can be used as an alternative method to some quantification techniques, such as aas and sp, due to its fast analyses, easy handling, and no need for sample pretreatment.	we have demonstrated that the relaxometry technique is very efficient to quantify paramagnetic ions during in situ electrolysis measurements. therefore, the goal of this work was to validate the relaxometry technique in the determination of the concentration of the ions contained in electrolytic solutions, cu2 +, ni2 +, cr3 +, and mn2 +, and compare it with other analytical methods. two different nmr spectrometers were used : a commercial spectrometer with a homogeneous magnetic field and a home - built unilateral sensor with an inhomogeneous magnetic field. without pretreatment, manganese ions do not have absorption bands in the uv - visible region, but it is possible to quantify them using relaxometry (the limit of quantification is close to 105 mol l1). therefore, since the technique does not require chemical indicators and is a cheap and robust method, it can be used as a replacement for some conventional quantification techniques. the relaxometry technique could be applied to evaluate the corrosion of metallic surfaces.
decreased serum iron concentration and accelerated loss of nutrients in psoriasis through the hyperproliferation and desquamation has been reported. iron is a central component of heme groups, enzymes that are involved in respiration and deoxyribonucleic acid synthesis. the largest iron pool in our body is heme, the metal is required for oxygen transport in hemoglobin. the second largest pool of iron is the non - heme form stored in ferritin. however, the essential metal can be toxic to cells when present at high concentrations because of its ability to promote the formation of damaging oxidative radicals therefore, humans and animals have evolved highly efficient mechanisms for iron conservation. the daily losses of iron, 1 - 2 mg in adults, represent < 0.1% of total iron in the human body, this loss occurs predominantly through desquamation of epithelial cells in the intestine and the skin, through minor bleeding and must be replaced from dietary sources to maintain iron balance. psoriasis is a hyperproliferative skin disease with a markedly increased (5 - 6 times normal) rate of epidermal turnover. accelerated loss of nutrients in psoriasis through the hyperproliferation and desquamation of the epidermal layer of skin and decreased serum iron concentration has been reported. there is strong evidence that supports the important role of hepcidin in the etiology of the anemia of chronic disease. we predicted that serum hepcidin levels in patients with psoriasis would differ from the healthy subjects with similar age and sex. also, there is no study in the literature about the hepcidin in patients with psoriasis. a total of 46 psoriasis patients (21 females, 25 males, mean age 36.89 6.76 years) consulting the department of dermatology, during september 2011 and february 2012 were included in the study. the control group consisted of healthy volunteers (n = 32, 15 females, 17 males, mean age 37.50 7.11) having no significant medical illness. persons with history of diabetes, hypertension, psychiatric disorders, cardiac problems, respiratory problems, endocrine diseases and other chronic skin disorders (other than psoriasis) were excluded. the duration of psoriasis varied from 4 years to 16 years. psoriasis was graded according to the psoriasis area severity index (pasi), presenting at the time of blood collection. patients who have the high pasi score were included study to investigate the relationship with hepcidin. peripheral blood cell counts of patients and controls were studied using automated hematology analyzer (cell - dyn ruby - abbott diagnostics). analysis of serum fe was carried out using iron assay on the aeroset system (abbott diagnostics). serum ferritin was measured by chemiluminescent microparticle immunoassay method using automated analyzer (abbott diagnostics). interleukin (il-6) level in serum was measured by enzyme - linked immunosorbent assay (elisa) test kit (ebioscience - human il-6 platinum elisa, austria) according to the manufacturer 's protocol. serum hepcidin was measured using elisa test kit (drg instruments gmbh, germany). according to the manufacturer 's protocol ; 10 l sample buffer added to each of 96-well plates, which were coated with a monoclonal antibody directed toward the antigenic site of the bioactive hepcidin 25 molecule. 20 l of each standard, control and samples dispensed into appropriate wells and incubated for 30 min at room temperature on a plate shaker at 500 rpm. following incubation, 150 l assay buffer and 100 l enzyme conjugate added to each of wells and incubated for 180 min at room temperature on a plate shaker at 500 rpm. following incubation, wells rinsed for five times with distilled water (400 l / well) and 100 l enzyme complex dispensed into each well and incubated for 45 min at room temperature. after this incubation, wells rinsed for five times with distilled water again and 100 l of substrate solution dispensed into each well and incubated for 30 min at room temperature, after incubation the enzymatic reaction stopped by adding 100 l of stop solution and the absorbance (optical density) of each well determined at 450 10 nm with a microtiter plate reader (multiskan go, thermo scientific) within 10 min. comparisons between the psoriasis and the control group were carried out by independent t - test. mean pasi score of patients who participated in the study was high (mean score of pasi was 14.6 2.7). when compared with the control group, no significant difference was seen in the ferritin level between the psoriasis and the control group [table 1 ]. additionally, a gender - related statistically significant difference was not detected between the groups. iron (fe), ferritin, il-6 and hepcidin levels in psoriasis patients and controls il-6 and hepcidin levels (respectively 24.33 0.87 pg / ml and 17.59 0.33 ng / ml) in the psoriasis patients were found to be higher than the control group (10.59 0.66 pg / ml and 12.97 0.64 ng / ml) [figures 1 and 2 ]. box plots represent the mean levels of interleukin-6 in the patient and control group box plots represent the mean levels of hepcidin in the patient and control group this requirement is mainly provided by macrophages recycling iron from senescent erythrocytes and in a small amount by intestinal iron absorption. during critical illness anemia the most important of these are the redistribution of body iron out of the circulation and into reticuloendothelial macrophages. recently, lee and beutler isolated a peptide from human urine and named it hepcidin (hepatic bactericidal protein). hepcidin is encoded by the hepcidin antimicrobial peptide gene located on chromosome 19 (19q13) and humans have only a single copy of the hepcidin gene. hepcidin is produced primarily in hepatocytes and transcriptionally regulated by il-6 through the stat-3 signaling pathway, enters the circulation and negatively regulates the export of iron in certain cell populations such as re macrophages (important for iron storage) and duodenal enterocytes (iron absorption). in addition, hepcidin expression has been reported in polymorphonuclear neutrophils, macrophages and peripheral blood mononuclear cells. hepcidin is synthesized as a preprohepcidin ; the signal peptide is cleaved leading to prohepcidin, which is further processed causing the 25 amino acids hepcidin. the predominant form is the 25 amino acid peptide, although shorter peptides with 20 and 22 amino acids are also detectable in human urine. while the iron store and inflammatory regulation activate hepcidin transcription in the hepatocytes, hypoxia, anemia, excessive iron burden and increased erythropoiesis all negatively regulate hepcidin expression. the interplay between positive and negative stimulus is critical in determining the net hepcidin levels. iron and ros are closely related and during the inflammation process, free iron is released from storage proteins such as ferritin. there is a relationship between ros formation and iron, iron promotes the ros formation responsible for frequent oxidative damage. psoriasis is considered a t helper-1 (th1) disease, because of significantly high levels of th1 cytokines (il-1 interferon-, tumor necrosis factor - alpha [tnf- ]) have been reported in serum. in a study, pro - inflammatory cytokines (il-1 and tnf-) injected mice developed hypoferraemia and anemia. il-6 is a small glycoprotein and produced in a broad spectrum of cells including immune cells (macrophages, dendritic cells and mast cells, b cells, t cells) and a variety of non - leukocytes cells (endothelial cells, fibroblasts, astrocytes, epithelial cells). il-6 has a wide variety of functions, acts not only on b cells but also on t cells and hepatocytes. as in our study, increased blood levels of il-6 has been shown in both the circulation and in lesional skin compared with controls in psoriasis patients. pasi score has been used for the assessment of severity of psoriasis and as a tool to compare with serum markers like iron, ferritin, il-6 and hepcidin in our study. patients who have only high pasi score enrolled the study for determining the accuracy of our hypothesis ; therefore, comparisons were not made with pasi score. considering the increased il-6 level, th1-mediated pathogenesis and accelerated loss of nutrients from the hyperproliferation and desquamation of the epidermal layer of skin in psoriasis, it can be speculated that hepcidin may play a role in regulation of iron and consequently in the pathogenesis of psoriasis so the results of our study supports this claim. as in our female patients, anemia is a common condition in women of reproductive age, but in our study, more than half of patients (58.7%) were 36 years old and over so that we think that this situation did not affect our results in the statistical size. it is known that a mild to moderate normocytic normochromic anemia is characterized by decreased serum iron and transferrin and increased iron stores in the form of ferritin in chronic inflammatory diseases. in a study, hepcidin was already found to be higher in 23 patients who have the anemia of chronic disease than in controls. considering the results of our study (no significant difference in the ferritin level, lower fe levels, higher il-6 and hepcidin levels), similar situation may have occurred in our patients who have a high - pass score. we think that studies on hepcidin expression in psoriatic plaques will contribute to our understanding the role of iron and hepcidin in the pathogenesis of psoriasis.	background : iron is an essential nutrient for mammals. accelerated loss of nutrients through hyperproliferation and desquamation from the skin in psoriasis is known. hepcidin is an important and recently discovered regulator of iron homeostasis.aims and objectives : the present study was undertaken to investigate the hepcidin expression in psoriasis patients.materials and methods : we examined peripheral blood cell counts, serum fe, ferritin, interleukin-6 (il-6) and hepcidin levels using respectively automated hematology analyzer, iron assay on the aeroset system, chemiluminescent microparticle immunoassay with automated analyzer, and enzyme - linked immunosorbent assay.results:the independent comparison of fe, ferritin, il-6 and hepcidin levels in psoriasis patients and control group (healthy volunteers) revealed lower fe and higher il-6, hepcidin levels in psoriasis patients. no significant difference was seen in the ferritin level between the psoriasis and the control group.conclusions:we think that studies on hepcidin expression in psoriatic plaques will contribute to our understanding the role of iron and hepcidin in the pathogenesis of psoriasis.
the aim of nursing assessments is to collect objective and subjective data to determine the health status of the patients to achieve a professional clinical judgment. the nursing assessments are the determinants of the nursing interventions which have direct and indirect influence on the health status of the patients.this process increases the ability of the nurses in monitoring and determining the changes in the patients ' condition and allows nurses to use their theoretical knowledge in different clinical situations.developing health assessment skills can help the nurses to make more accurate diagnosis.many nursing faculties in the u.s believe that health assessment training is an important and essential part of nursing students ' education and according to professional nursing performance standards, it is critical.therefore, choosing the right method to achieve successful learning is crucial for the students. the lecture is the most common educational method in medical groups.but, this method is not suitable for all educational purposes.limiting the capabilities of the students in participating and answering the questions, loss of concentration of the students over time, reduction of absorption, stability and remembering the subjects, inactive role of students, and boringlecture are the some reasons which have made lecture among the least effective teaching methods.the search for some solutions for these deficits resulted in development of new methods for active learningthat different approaches are proposed in their application. team - based learning (tbl) is an active learning approach which has been designed in order to help the students achieve the goals of a training course and the manner of working in a team.this accelerates the change in the teaching method and can be considered as a replacement for the traditional lecture method. in this new approach, students are first given the educational information and then, they are divided into smaller groups in classes, each group is given a problem - based learning (pbl) scenario to stimulate the discussion among the students. this help students to exchange the information about the topic and use previously given information to solve the problem through practical way.according to the approach developed by hunt., the primary goal of a team - based learning is to build student self - confidence by giving them a chance to practice using the concepts that they have learned during the previous courses. this approach results in spending the majority of the class time on team working by using the practical assignments. team - based learning in nursing results in decreasing tension and study time and increasing preparation time for the class among nursing students, which make it possible to allocate more time for discussion about complicated nursing topics. other advantages of this method includes increasing class dynamics, students involvement in class discussions, increasing differential diagnosis skills in students and clinical experiences, decreasing the economic costs, enhancing self - learning, and stimulating higher levels of knowledge based on performance. moreover, team - based learning improves student performances in some healthcare educational courses.therefore, this approach is recognized as an effective educational method due to its high applicability. different studies have shown that using this new method could lead to improving students learning and increases students scores.pileggi and oneill 's showed that the performance of the students has been improved following team - based learning method. in this study, the mean score of the final exam in the team based education course had also been increased as compared with the traditional lecture based courses.vasan etal., and letassy etal. it has also been found through investigation of the students ' attitude toward this method that all students were satisfied with team - based learning and they had positive feedback about it. there are, however, conflicting data on whether tbl improves knowledge outcomes compared to other educational techniques. haidet.,did not find a significant difference in knowledge outcomes between tbl and lectures. but, some found improved examination scores in the tbl group while using the historical controls has made its interpretation difficult.others found improvement in some aspects. based on the aforementioned literature review, learning is basically a social activity, and considering the significance of health assessment in developing of clinical skills and professional capabilities of nursing students and according to the promising results of team - based learning, our aim in this study was to determine the effectiveness of using this active educational method on nervous system examination knowledge in nursing students of urmia nursing and midwifery faculty. this is a quasi - experimental study conducted with the aim of determining the impact of team - based learning in nervous system examination knowledge in nursing students at the bachelor of science in nursing (bsn) course from urmia nursing and midwifery faculty, a big urban city in the northwest of iran in 2013. all nursing students at 5th and 6th semester were invited to participate in this study and were selected through a census. fifth semester students (n=32) were assigned as the intervention group and sixth semester students (n=30) were considered as the control group. researchers obtained ethics approval from the ethics committee of the uremia medical university and nursing students filled out the informed consent form. based on the principles mentioned in previous researchesand obeying 3s proposed by michelson (same problem, specific case and simultaneously report), an adjusted tbl design was used in the intervention group and the traditional lecture method was used for the control group to provide educational content. to collect data a 40-question test in the format of multiple choice, matching, gap - filling, and descriptive questions with several modifications in writing questions as scenarios. the maximum score was 40 for pre - test and post - test sections., the demographic characterizations of the students including age, sex, score in health status assessment course which were passed in the 2 semester and grade point average (gpa) was also collected. thus, the data gathering instrument was prepared by using literature review and referring to related books and articles. then its content was given to 10 faculty members at urmia university of medical sciences for critical review. for investigating the reliability pilot study (10 nursing students in 7semester) was conducted and the cronbach alpha was 0.88. before starting the study, a pre - test was taken from all nursing students to determine their knowledge in the nervous system examination. after the pretest, at the " first phase ", the educational materials in the form of booklets and educational slides as a module about nervous system examination skills were given to all students. the students in the intervention group were given one week to study these materials. at the second phase, intervention was performed as an 8-hour workshop with tbl group. as soon as starting the workshop, the students answered individual readiness assurance test (rat) to assess their perception of the learned knowledge and concepts in the first phase. rat consisted of 40 questions in various formats from the educational module. giving correct answers to these questions required using topics that the students should have learned in the first phase. immediately, after finishing the rat and collecting the papers, the students were randomly assigned to 6 groups of 5 or 6 numbers and the same test was taken from the teams in a different format. this step called group readiness assurance test (grat), each team should come to an agreement by all team members to choose one answer to each question. after answering by all groups and collecting the grat papers, each group discussed and justified its own answers with other groups. the teacher, as a facilitator, discussed about all questions and answers with the students and clarified any concepts that the students might have problems with, as shown in figure 1. the conduction pattern of the research in intervention group the students in the control group received the educational content through traditional lecture method. finally, five weeks after performing the research, a post - test was taken individually and with the changes in writing of questions from the intervention and control groups. post - tests consisted of the same scenarios as the pre - test, but the order of scenarios and questions randomly scrambled. data we reanalyzed by spss software ver. 13 using descriptive and inferential statistical tests (chi square, paired - sample t - test and independent t - test). in this study, 40% and 50% of the subjects in control group and intervention group were male, respectively. moreover, independent t - test result indicated that there was no significant difference statistically between two groups in terms of variables such as age, the mean score of health status assessment and gpa (table 1). mean (sd),n (%), independent t - test result, chi square test result based on paired t - test result, there was no significant difference in the post - test mean score of health status assessment knowledge among the students in the control group (p=0.14). but, after performing team - based learning, the mean score of health status assessment knowledge among the students in the intervention group significantly increased compared with pre - test score (p=<0.001) (table 2). the results showed that the mean score of nursing students on nervous system examination knowledge in team - based learning was greater than control group, and this increase was statistically significant (p<0.001). the independent t - test showed no statistically significant difference in the scores of nervous system examination knowledge between the two groups at pre - test mean score, but there was a significant difference at post - test scores between two groups (p<0.001) (table 3). other findings showed that in the intervention group the mean and standard deviation of the scores in nursing students in individual readiness assurance test (rat) was 25.05 (3.36), while it increased to 31.68 (1.33) in group readiness assurance test (grat). paired t - test showed a statistically significant difference between the mean scores in the rat and grat in the intervention group (p<0.001). the present study results showed that using the team- based learning in comparing with traditional lecture - based learning leaded to significant improvement in knowledge of nursing students as the primary outcome of the study. arisen a significant difference between two groups in the post - test that was held 5 weeks following tbl performance, showed a significant improvement in students learning with the tbl method in comparison with the traditional lecture method. consistent with the results of this study, the majority of the previous studies which have dealt with the comparison of the knowledge scores of students using tbl and lecture based methods, show that the scores in the team - based learning course are higher. the obtained improvement in students ' performance in this project is consistent with the results achieved by koles.,and wiener., in which the students showed greater dominance on the contents in the tbl group compared with other traditional approaches and non - tbl methods. increased the examination scores with tbl was found in other studies which had compared it with other approaches such as self - studying. the results of another crossover study with the aim of comparing active learning through tbl with inactive learning by self - studying showed that the post - test mean scores in the intervention group was significantly higher than control group. this finding is supported through numerous other studies in which the score improvement has been reported by tbl. based on the results of the previous researches, there are few contradictory data about the further improvement of learning outcomes with tbl compared with the other educational techniques which are contrary to our findings. haidet., compared the tbl - like active learning method with inactive lecture method and there was no significant difference was found in scores between the two groups. the reason for this can be attributed to the difference in the way tbl was performed or the heterogeneity of participants (as mentioned in tan 's., study).what is more apparent in the previous studies about disadvantages of the tbl related to its effectivness to various topics presented in the research, using tbl led to improve some of the topics, not all of them. in this study, the post - test score in tbl compared with pre - test had been surprisingly increased ; such that the mean score in the class had reached from 13 to 31 point from total 40 point. this difference is more important to know that the post - test was taken 5 weeks after performing tbl while students were unaware of the post - test and most likely resulted in their lack of study ; therefore, it could be concluded that this high mean score in class and remembering the contents by students can be a net effect of tbl method. the results of a study with the aim of evaluating effectiveness and efficiency of tbl showed that the students in the pre - test was in the least knowledge level (the mean score of 36.9%) but this reached to 63.4% in grat which had been considered as a post - test that showed a significant improvement in the students ' performance with tbl. however, considering grat as a post - test can not be a proper criteria to be compared with pretest in this study, because it is clear that the group readiness assurance test score which is obtained from individuals in the group will be higher compared to the individual scores. in general, reason for increasing tbl scores can be related to factors such as reinforcing and utilizing the concepts, increasing students ' involvement in class discussions, and creating meaningful learning. although, we did nt intend to assess retaining the contents with tbl but performing post - test 5 weeks after conducting the research in the intervention group retained the contents much more than control group. from this viewpoint, our study finding is consistent with other studies that show that the knowledge can be retained better following tbl. on the other hand, the student in the control group had nt been able to achieve significant improvement in retaining the contents which shows that the presented topics through lecture are forgotten faster. our findings in this research showed that the mean score of the students in the intervention group in grat was significantly higher than rat. our result was similar to these in the pileggi., and o'neal., studies, the mean of missed questions (unanswered) in rat and grat was 8.29 and 2.98 respectively, which this had led to a higher score of grat compared with an individual rat score. study, the score of rat and grat was 86% and 97%, respectively. in the present study, there was no significant correlation between students ' total gpa and the amount of progress in scores by tbl. this means that the weak and strong students were benefited to the same amount from the tbl education. this finding is inconsistent with other study results that indicate tbl has more effect on weaker students. the strength of the current study is the delayed post - test that had measured retaining the contents in mind addition to the effect of tbl on knowledge improvement. also not using true or false questions on the taken tests, is another advantage of this study. because this type of question has inherent limitations.. full performance of tbl consists of three major phases (preparation before the class, individual and group readiness assurance tests and finally using the obtained knowledge from first and second phases in clinical problems) that it had been focused on the first two phases in this study. the main reason for expressing this limitation is that the lack of full performance of tbl may lead to negative results regarding the efficiency of this approach. although, this had nt happened in this study, it was possible that if fully performed, more positive results could be achieved and caused no change in the main aspects and the nature of the findings ; a point that other researchers have also mentioned. moreover, this is tbl features that in some parts because of its flexibility allows teachers to choose one or more phases depend on the concept and the course 's demand. also, several other studies have been used adjusted tbl design because of their research conditions. so, it is recommended that experimental studies to be conducted with a higher sample size to support the positive results about the efficiency of the tbl. using tbl method compared to the traditional lecture approach resulted in more improvement and stability in the knowledge of nursing students in the nervous system examination skills. therefore, using tbl can result in higher preparation of nursing students for the class and allocating more time for class discussions about complicated nursing topics. thus, according to the findings of the current study, it might be concluded that using tbl in nursing education could be used to promote the learning quality of nursing students. this article was derived from an approved research project at the urmia medical sciences university under registration number of 982, therefore, the authors feeling the need to appreciate from the university for its great cooperation.	introduction : team - based learning is one of the active learning approaches in which independent learning is combined with small group discussion in the class. this study aimed to determine the impact of team - based learning in nervous system examination knowledge of nursing students. methods : this quasi - experimental study was conducted on 3rd grade nursing students, including 5th semester (intervention group) and 6th semester (control group). the traditional lecture method and the team - based learning method were used for educating the examination of the nervous system for intervention and control groups, respectively. the data were collected by a test covering 40-questions (multiple choice, matching, gap - filling and descriptive questions) before and after intervention in both groups. individual readiness assurance test (rat) and group readiness assurance test (grat) used to collect data in the intervention group. in the end, the collected data were analyzed by spss ver. 13 using descriptive and inferential statistical tests. results : in team - based learning group, mean and standard deviation was 13.39 (4.52) before the intervention, which had been increased to 31.07 (3.20) after the intervention and this increase was statistically significant. also, there was a statistically significant difference between the scores of rat and grat in team - based learning group. conclusion : using team - based learning approach resulted in much better improvement and stability in the nervous system examination knowledge of nursing students compared to traditional lecture method ; therefore, this method could be efficiently used as an effective educational approach in nursing education.
the study was conducted at the centre hospitalier des armes bouffard, a french military hospital in djibouti serving military and civilian natives from the entire city, and at other public health facilities of djibouti. from 1997 to 2002, clinical malaria in the hospital shows the same temporal fluctuations as in dispensaries in the city (figure). the incidence of patients with p. falciparum malaria admitted to the hospital increased > 10-fold from march to may 1999 compared with the same period in 1997, 1998, and 20002002. in contrast, the number of admissions, consultations at the outpatient clinic, or blood counts performed for other causes than fever did not vary over the same period. the meteorologic station of the international airport of djibouti recorded heavy rainfall the month before the epidemic. however, similar rainfall in 1997 or autumn 1999 was not followed by such a dramatic increase in malaria incidence in the ensuing months (figure). when annual averages were compared, no particular variations in minimal or maximal mean air temperatures were found to occur during the months preceding the epidemic. rainfall (bars) and monthly incidence of plasmodium falciparum clinical malaria cases (curve) at the french military hospital cha bouffard (circle) and in the dispensaries of the city (department of epidemiology and public hygiene. forty - six blood samples were collected from september 14 to december 31, 1998 (period 1), 61 from april 12 to april 30, 1999 (period 2), and 32 from march 15 to may 15, 2002 (period 3), from patients with p. falciparum clinical cases who had not travelled outside the city of djibouti during the preceding month and declared not having taken any antimalarial drug before the blood sampling. venous blood was collected before treatment administration in vacutainer edta tubes (becton dickinson, rutherford, nj, usa). thin blood smears were stained with an ral kit (ractifs ral, paris, france). p. falciparum genetic diversity was investigated by using msp1 and msp2 encoding highly polymorphic loci from merozoite surface protein genes. msp1 and msp2 were genotyped by using nested polymerase chain reaction (pcr), as described (7), except that family - specific fluorescent primers were used in the nested pcr for assignment to the k1-, mad20-, or ro33-type msp1 family and to the 3d7- or fc27-type msp2 family. the mean multiplicity of infection, i.e., the number of genotypes present in the blood sample, was 1.5 concurrent p. falciparum infections per person, with a decreasing tendency over the study period (table 1). for each locus we identified 9 msp1 alleles in 83 isolates and 17 msp2 alleles in 108 isolates. the genetic diversity estimated by the unbiased expected heterozygocity (8), i.e., the probability that 2 randomly chosen genotypes are different in the sample, before, during, and after the 1999 outbreak was 0.79 (n = 23), 0.37 (n = 39), and 0.64 (n = 21) at the msp1 locus and 0.83 (n = 31), 0.34 (n = 47) and 0.63 (n = 30) at the msp2 locus, respectively. during the epidemic, ro33 - 131 accounted for 79% of the msp1 allele and fc27 - 408 accounted for 81% of the msp2 alleles. both alleles were present before and after the epidemic but with a much lower prevalence. they accounted for 26% of the msp1 and 35% of the msp2 alleles in 1998 and 14% of the msp1 and 10% of the msp2 alleles in 2002 (table 2). sd, standard deviation ; the genotypes at the pf dhfr, pf dhps, and pf crt locus refer to the one - letter symbolized amino acids coded by the codons. a, alanine ; c, cysteine ; f, phenylalanine ; g, glycine ; i, isoleucine ; k, lysine ; n, asparagine ; r, arginine ; s, serine ; t, threonine. isolates collected in 1998 (msp1 : n = 23 ; msp2 : n = 31), 1999 (msp1 : n = 39 ; msp2 : n = 47), and 2002 (msp1 : n = 21 ; msp2 : n = 30). to look for resistance - associated point mutations and haplotypes, the complete coding region of pfdhfr (dihydrofolate reductase) and pfdhps (dihydropteroate synthase) was amplified and sequenced (abi 3100 genetic analyser, applied biosystems, courtaboeuf, france) as described (9). we focused the analysis on point mutations of pfdhfr codons 16, 51, 59, 108, and 164 and pfdhps codons 436, 437, 540, 581, and 613, which have been associated with resistance to pyrimethamine and proguanil metabolite and to sulfadoxine, respectively (10). no mutant was detected for pfdhfr codons 16 and 164 and pfdhps codon 581. a single isolate collected in period 2 harbored the pfdhps a613s mutation. no isolate harbored the quintuple mutant haplotype (pfdhfr s108n, n51i, and c59r and pfdhps k540e and a437 g) or the pfdhfr c59r and pfdhps k540e combination that predicts sulfadoxine - pyrimethamine clinical failure (9). one isolate containing at least 2 p. falciparum populations harbored 3 pfdhfr mutations (s108n, n51i, and c59r) and the pfdhps k540e mutation. from 1998 to 1999, the frequency of isolates with mutated pfdhfr codons 51, 59, and 108 decreased (not significantly), and pfdhps allelic frequency did not differ significantly. the prevalence of isolates harboring the pfdhfr n51i, pfdhfr s108n, pfdhps a437 g, and pfdhps k540e mutations increased from 19981999 to 2002 (fisher exact test, p < 0.001 each). presence of the chloroquine resistance associated k76 t mutation of pfcrt (chloroquine - resistance transporter) (11) was analyzed by nested allele specific pcr. over the study period, 93% of the isolates harbored the pfcrt k76 t mutation (table 1), without any significant temporal variation. twenty seven p. falciparum isolates collected during the 1999 epidemic with a 0.05%5.0% parasitemia were transported at 4c to our laboratory in marseille, france, and analyzed for in vitro drug sensitivity by using an isotopic microtest (12). among them, 93% were classified as resistant to chloroquine (table 3). no isolate was resistant to amodiaquine. in vitro resistance was 4% for both pyrimethamine and cycloguanil. the 50% inhibitory concentration (ic50) of chloroquine diphosphate, amodiaquine, pyrimethamine dihydrochloride, and cycloguanil, i.e., the drug concentration corresponding to 50% of the uptake of 3h - hypoxanthine by the parasites in drug - free control wells, was determined by nonlinear regression analysis of log - dose / response curves. mean ic50 and proportion of resistant isolates according to cut - off values are indicated. before and after the 1999 epidemic, p. falciparum genetic diversity in djibouti was large, with 80% and 63% heterozygocity. this finding is somewhat surprising for an area where disease endemicity is low (13) and probably reflects importation of strains from neighboring areas such as ethiopia or somalia (1,5). falciparum genetic diversity was diminished during the epidemic, reflecting the circulation of a restricted number of strains during that period. most of these strains harbored an msp1 and msp2 genotype that was detected before the epidemic. the prevalence of pfcrt, pfdhfr, and pfdhps mutant genotypes did not vary significantly from 1998 to 1999. thus, our data do not support the hypothesis of a sudden increase in the drug resistance of the local p. falciparum population as causing the epidemic. our data are also not consistent with massive invasion by a single strain / genotype but rather suggest expansion during the epidemic of a few strains that were already prevalent. further genotyping is needed to establish how many strains were circulating and their possible origin. unfortunately, no vectors were captured at that time, and this hypothesis is difficult to explore retrospectively. the low prevalence of pfdhfr and pfdhps resistance mutations in 1998 and 1999 and of proguanil or pyrimethamine in vitro resistance in 1999 may explain the very low incidence of clinical malaria among the french soldiers stationed in djibouti who were taking chloroquine - proguanil chemoprophylaxis. however, the sharp increase of pfdhfr and pfdhps resistance mutations observed in 2002 threatens sulfadoxine - pyrimethamine efficacy in the near future, even more so since the limited acquired immunity is unlikely to contribute to sustained drug efficacy (14). this finding calls for an urgent in vivo assessment of the antimalarials presently used in djibouti in order to consider a rapid change in first - line treatment policy.	analysis of plasmodium falciparum isolates collected before, during, and after a 1999 malaria epidemic in djibouti shows that, despite a high prevalence of resistance to chloroquine, the epidemic can not be attributed to a sudden increase in drug resistance of local parasite populations.
gastrojejunocolic fistula (gjf) is a rare and late complication of gastrojejunostomy applied for recurrent peptic ulcer disease. it was thought to be induced by a stomal ulcer, due to inadequate gastrectomy or incomplete vagotomy. the symptoms in these situations include upper abdominal pain, weight loss, diarrhea, gastrointestinal bleeding, and fecal vomiting. these patients are cachectic and dehydrated, with labs showing malnutrition. barium upper gastrointestinal series (ugis), gastroscopy, and colonoscopy are used for diagnosis. although the occurrence of gjf has decreased remarkably as a result of better treatment, the modern management of this condition and the generally accepted surgical treatment strategies must be discussed. a 55-year - old man was admitted to the medical center at dankook university hospital in december 2011. the patient had complaints of chronic watery diarrhea that occurred immediately after meals for 6 months, general weakness, and a weight loss of 15 kg during a 3-month period. past medical history revealed primary repair for duodenal ulcer perforation in 1982 and truncal vagotomy and gastrojejunostomy in 1996 due to gastric outlet obstruction. laboratory parameters revealed malnutrition with albumin level of 2.6 g / dl (range, 3.4 to 4.8), protein level of 4.3 g / dl (range, 6.4 to 8.3), and cholesterol of 72 mg / dl (range, 120 to 239). the infusion of contrast into the colon was identified near the gastrojejunostomy, according to a thin barium ugis double study (fig. copious fecal fluid was evident in the stomach through a gastroscopy, and a canal was identified in the previous gastrojejunostomy site and low body posterior wall connected directly to the colon (fig. 2). a colonscopy revealed an anastomosis site was seen in the transverse colon stricture and t - colon connected to the stomach. after improving the patient 's state of malnutrition and his weight through total parenteral nutrition (tpn), the patient was taken into the operation room and an exploratory laparotomy was undertaken. in surgery, the gastrojejunostomy site and distal gastrectomy, roux - en - y anastomosis, t - colon segmental resection and anastomosis were performed. three month after discharge, there was no ulcer, inflammation or passage disturbance at anastomosis site on endoscopic examination and the patient improved his symptoms. along with the development of medical treatment, such as h2 receptor blocker, proton pump inhibitors, and eradication of helicobacter pylori, the need for an operative treatment for peptic ulcer disease decreased, dramatically. for such a reason, the incidence rate of these fistulas has been remarkably decreased. the symptoms of gjf are diarrhea with food containing stools, upper abdominal pain or discomfort, gastrointestinal bleeding, fecal vomiting, weight loss, and general weakness. computed tomography is used to determine this complex fistula and to exclude extra - abdominal diseases that may define underlying etiology. gastroscopy and colonoscopy is a diagnostic tool that can identify the orifice of the fistula, find intraluminal diseases, and rule out malignancy thorough an endoscopic biopsy. the conventional treatment for gjf includes improving the nutritional status and two - to - three - phased operations with colostomy to minimize mortality. today, however, due to improved parenteral and enteral support treatment, single - stage procedures can be applied and these have been favored to minimize mortality. in our case, truncal vagotomy was performed in previous operation on medical record, therefore we did n't perform truncal vagotomy. if truncal vagotomy has not previously been completed, it is advisable to perform it nonetheless. in conclusion, the development of the peptic ulcer agents including those capable of eradicating h. pylori, gjf incidence has decreased dramatically. but, since the fistula formation needs a 20 to 30 years latent period after surgery, this complication can occur. today, one - stage resection is preferred over the staged operation if the general status of the patient is suitable. tpn is a crucial factor for recovering the patient 's malnutrition status and so should precede surgery.	gastrojejunocolic fistula is a rare condition after gastrojejunostomy. it is severe complications of gastrojejunostomy, which results an inadequate resection or incomplete vagotomy during peptic ulcer surgery. the symptoms are diarrhea, upper abdominal pain, bleeding, vomiting and weight loss. a 55-year - old man with chronic diarrhea and weight loss for 6 months visited dankook university hospital. the patient had received a truncal vagotomy and gastrojejunostomy for duodenal ulcer obstruction 15 years previously. the patient underwent gastroscopy and upper gastrointestinal series evaluations, which detected the gastrojejunocolic fistula. after improving of malnutrition, an exploratory laparotomy was undertaken, which revealed that the gastrojejunostomy site and the t - colon formed adhesion and fistula. en block resection of the distal stomach and t - colon included the gastrojejunocolic fistula, and roux - en - y gastrojejunostomy was performed. recovery was uneventful and the patient remained well at the follow - up. we report a gastrojejunocolic fistula, which is a rare case after gastrojejunostomy.
the goals of endodontic instrumentation include thorough debridement and disinfection of the root canal system, in addition, to create a suitable shape to achieve a complete three - dimensional obturation. the concept of thorough instrumentation of the apical region has long been considered to be an essential component in the cleaning and shaping process by grove. in an effort to obtain these goals, debris such as dentinal shavings, necrotic pulp tissue, bacteria and their byproducts or irrigants may be extruded into the periradicular tissues which can lead to an inflammatory response, postoperative pain and possible delayed healing. vande visse and brilliant have shown that in the absence of irrigant, no significant extrusion of debris was observed while a thick worm of debris extruded when an irrigant was used. shaping and cleaning of the root canal system more recently advanced instrument designs such as radial lands, noncutting tips, different flute depth, variable tapers and cross - sections and the use of different operational principles have been developed to improve working safety, to shorten the working time and to create a greater flare within the preparations. the newer systems also provide a cleaner and smoother preparation to receive the final obturation. a common finding of the studies examining the amount of apically extruded debris and irrigant was that the instrumentation techniques using a push - pull motion tend to produce a more apical extrusion of debris than instrumentation techniques using a rotational motion. various instrumentation techniques have been used in the past for evaluating the apical extrusion of debris, but very few studies are reported in the literature comparing both debris and irrigant utilizing the reciprocating waveone instrumentation design. the purpose of this ex vivo study was to compare and evaluate the amount of apically extruded debris and irrigant in freshly extracted human mandibular premolars with single canal by three different instrumentation techniques using protaper hand, m - two and waveone primary. thirty freshly extracted mandibular premolars with a patent single root canal, and fully formed apices were selected for the study. teeth were immersed in 2.5% sodium hypochlorite for 2 h and then stored in 10% formalin for complete disinfection. the calculi and soft tissue remnants on the external surface were removed with a periodontal curette. the teeth were observed under an operating microscope to confirm that they had a single apical foramen. the teeth were digitally radiographed from buccal, lingual and proximal views to ensure that they had single canals and orifices. pulpal tissues were removed prior, making sure that the debris extruded was dentinal shavings and not pulpal remnants during the process of chemomechanical preparation. the buccal cusp edge of each tooth was flattened as a reference point, and coronal access was prepared conventionally using a high - speed bur. to achieve uniformity, the canal patency was controlled with a size 15 k - file (dentsply maillefer, ballaigues, switzerland). the working length (wl) of each canal was determined as 1 mm short of the length of a size 15 k - file that was visible at the major diameter of the apical foramen. the teeth were randomly divided into three groups according to the file used for the preparation of root canals. the groups were as follows : group 1 : protaper hand files (dentsply maillefer, ballaigues, switzerland) were used according to manufacturer 's instructions using a gentle in and out motion. the instrumentation sequence was sx at two - third of the wl, s1 and s2 at 1 mm short of wl, and f1 and f2 at the wl already determined.group 2 : m - two ni - ti rotary instruments (vdw, munich, germany) were used in an endodontic motor (x - mart plus, dentsply maillefer, ballaigues, switzerland) at 300 rpm. the standard set for this system includes number 10 to number 25, and tapers ranging from 0.04 to 0.06 (size 10/0.04 taper, size 15/0.05 taper, size 20/0.06 taper, size 25/0.06 taper). a glide path was established with a number 10 stainless steel k - type file, instruments taken at the wl already determined with light apical pressure. m - two instruments were used in a simultaneous technique without any early coronal enlargement.group 3 : the waveone primary file, tip size iso 25 (dentsply maillefer, ballaigues, switzerland) was used to prepare the canals. the waveone is a single file reciprocating system which works on balanced force technique and requires no change of file. the instrumentation was done in pecking motion and was completed up to the wl already determined. group 1 : protaper hand files (dentsply maillefer, ballaigues, switzerland) were used according to manufacturer 's instructions using a gentle in and out motion. the instrumentation sequence was sx at two - third of the wl, s1 and s2 at 1 mm short of wl, and f1 and f2 at the wl already determined. group 2 : m - two ni - ti rotary instruments (vdw, munich, germany) were used in an endodontic motor (x - mart plus, dentsply maillefer, ballaigues, switzerland) at 300 rpm. the standard set for this system includes number 10 to number 25, and tapers ranging from 0.04 to 0.06 (size 10/0.04 taper, size 15/0.05 taper, size 20/0.06 taper, size 25/0.06 taper). a glide path was established with a number 10 stainless steel k - type file, instruments taken at the wl already determined with light apical pressure. m - two instruments were used in a simultaneous technique without any early coronal enlargement. group 3 : the waveone primary file, tip size iso 25 (dentsply maillefer, ballaigues, switzerland) was used to prepare the canals. the waveone is a single file reciprocating system which works on balanced force technique and requires no change of file. the instrumentation was done in pecking motion and was completed up to the wl already determined. in this study, the experimental model described by myers and montgomery was used. eppendorf tubes were used in the experiment and stoppers were separated from them. an analytical balance with an accuracy of 10 three consecutive weights were obtained for each tube, and the mean value was calculated. each tooth was cemented up to the cementoenamel junction, and a 27-g needle was placed alongside the stopper which helped as a drainage canula and maintained the air pressure inside and outside the tube. then, each stopper with the tooth and the needle was attached to its eppendorf tube, and the tubes were fitted into vials. the eppendorf tube was shielded by a rubber dam sheet to eliminate bias during chemomechanical preparation. in each sample, a total of 4 ml distilled water was used as an irrigant between the files (groups 1 and 2), between pecking sequences in (group 3). immediately after canal instrumentation, the eppendorf tube was removed from the vial and volume of irrigant was measured. calibrated eppendorf tube was filled with saline and compared with experimental tube following the same method given by myers and montgomery. after the chemomechanical preparation was completed, the stopper, needle, and tooth were separated from the tube and debris adhering to the root surface was collected by washing the root with 0.5 ml of distilled water in the tube. the tubes were then stored in an incubator at 70c for 2 days to evaporate the distilled water before weighing the dried debris. the tubes were weighed using the same analytical balance to obtain the final weight of tubes including the extruded debris. the dry weight of the extruded debris was calculated by subtracting the weight of the empty tube from that of the tube containing debris. the mean dry weights of extruded debris were analyzed statistically using spss version 20.0 software (spss inc., the mean extrusion values and standard deviation for each group, median values and the range of extrusion (minimum and maximum) were evaluated. multiple group comparisons were analyzed by one - way analysis of variance followed by student 's t - test (unpaired) for pairwise comparisons. the waveone reciprocating system showed the maximum amount of apical extrusion of debris and irrigant among all the groups. the p value for each group was > 0.05 showing that no significant difference existed among the groups. the mean values and standard deviations for all groups are listed in table 1 along with the volume of irrigants in table 2. mean weights and standard deviations of groups volume of irrigant solution using myers and montgomery 's method (10 ml precision) an acute inflammatory response may develop in the periradicular tissues as a result of insults from the root canal system, which can be mechanical, chemical, or microbial in origin. mechanical and chemical injuries are usually associated with iatrogenic factors, such as over - instrumentation, apical extrusion of debris or irrigant, perforations, etc. apical extrusion of contaminated debris into the periradicular tissues is one of the principal cause of mid treatment flare - up and postoperative pain. the results have shown that preparation up to the apex, the diameter of apical patency, the amount of irrigant used, formation of a dentin plug, the use of step - back versus crown - down technique, and the use of conventional hand filing versus rotary motion, all have a correlation to the amount of extruded debris. irrigation or chemical debridement is accepted as being a necessary aid in the chemomechanical cleansing of the root canal as irrigation assists in debris removal. furthermore, the proximity of the irrigating needle to the apex plays an important role in removing the canal debris. a common finding of nearly all the studies in endodontic literature led to a generalized view that the crown - down technique extrudes less debris and irrigants apically as compared to the step - back technique and that a linear filing motion extrudes more debris when compared to instruments used in rotational motion. in this study, we are comparing the volume of irrigant and debris extrusion by using hand and rotary file system with single and multiple files (hand protaper, rotary waveone and m - two). it must be emphasized that the results of this study directly focuses on a comparison between the different generations of new file system leading emphasis on the periapical extrusion by same. beeson. reported that, when the instrumentation was performed to the apical foramen, significantly more debris was forced apically than when instrumentation was done 1 mm short. reported an accumulation of 0.00153 g of apical debris using protaper system (protaper f3) with decoronated mandibular incisors. the higher amount of collected debris compared with our data may be caused by the difference in teeth used and final finishing file, that is, mandibular premolars and protaper f2. m - two is used in single length method in which all the instruments were used till wl and very small hand files are used for initial glide path. in m - two files, the distance between the cutting blades increases from the instrument tip to the shaft, the progressive pitch and absence of radial lands produce less dentinal debris. the waveone files are manufactured with m - wire niti alloy, composed of 508 nitinol, under specific tensions and heat treatments at various temperatures. these instruments work on the reciprocating action and simulate the balanced force technique, as theorized by roane and sabala reciprocating motion provides the advantage of preserving complex canal anatomies as stated by berutti. evaluated the incidence of postoperative pain when tf15, waveone, and tf adaptive systems were used for chemomechanical preparation for root canals. they measured that the incidence of postoperative pain was significantly higher with the waveone single file reciprocating system. the present study agrees with the above literature as during comparative evaluation ; waveone single file system showed the maximum amount of apical debris and irrigant extrusion when compared to protaper hand and m - two rotary. the reciprocation movement in waveone system is formed by a wider cutting angle and smaller release angle. while rotating in the release angle the flutes will not remove debris but push them apically. moreover, waveone file is quite big, rigid with an increased taper (in the study 0.08 taper primary file is used which corresponds to iso size 25) which is directed to reach the apex. based on the results of this study, independent of the systems used, all instrumentation techniques produced debris and irrigant extrusion. further studies should evaluate the behavior of newly introduced niti systems in complicated root canals. with the given data and observations obtained in the sample size and limitations of the study, there was no statistical significant difference between the apical extrusion of debris and irrigant by all the instrumentation techniques. there was moderately lesser amount of debris and irrigant extrusion by protaper hand as compared to m - two rotary. although all instrumentation techniques produced debris and irrigant, waveone single file reciprocating system showed the maximum amount of apical debris and irrigant extruded when compared to protaper hand and m - two rotary.	aims : the purpose of this ex vivo study was to evaluate and compare the weight of debris and volume of irrigant extruded apically from teeth using different preparation techniques.subjects and methods : thirty extracted human mandibular premolars with single canals and similar lengths were instrumented using hand protaper f2 (25, 0.08 ; dentsply maillefer, ballaigues, switzerland), m - two (25, 0.06 ; vdw, munich, germany) and waveone primary (25, 0.08 ; dentsply maillefer, ballaigues, switzerland). debris and irrigant extruded during instrumentation were collected into preweighed eppendorf tubes. the volume of the irrigant was measured, and then the tubes were stored in an incubator at 70c for 2 days. the eppendorf tubes were weighed to obtain the final weight when the extruded debris was included. three consecutive weights were obtained for each tube.statistical analysis used : data were statistically analyzed by one - way analysis of variance and student 's t-test.results:there were no statistically significant differences among the groups. the waveone reciprocating system showed the maximum amount of apical extrusion of debris and irrigant among all the groups. the least amount of debris and irrigant was observed in protaper hand instrument (p > 0.05).conclusion : all instrumentation techniques were associated with debris and irrigant extrusion.
skeletal class ii malocclusion as a result of mandibular deficiency is a problem commonly diagnosed in the orthodontic department. functional appliances for the treatment of class ii malocclusion have been used since the 1930s. mainly, functional appliances have been divided into two groups : fixed functional appliances and removable appliances. functional appliance treatment among adolescent patients has the power to treat mandibular retrognathia by increasing condylar growth and mandibular advancement. these appliances affect the functional muscles, and also influence the glenoid fossa and mandibular condyle position. the orthopedic treatment of class ii malocclusions increases upward and backward condyle growth, and this mechanism has been found to have an important role in the forward movement of the mandible. experimental animal studies have shown bone remodeling in the condyle in response to mandibular advancement. the results of many studies indicate that condyle growth and the mandible advancement can be increased in growing rats through the use of bite - jumping devices. produced a mandibular protrusion with a bite - jumping appliance, and found that the length of condylar process increased significantly over a 30 day experimental period. taira. reported that mandibular growth is enhanced in growing rats through mandibular advancement promoted by the use of fixed functional appliance. found a significant increase in vascularization in condyle and mandibular bone growth in growing rats, through the use of bite - jumping appliance. results in mandibular morphological and condylar growth studies are typically analyzed with lateral radiography, which is commonly used to evaluate mandibular morphology changes in orthodontic patients. the primary objective of this study was to investigate the effect of mandibular advancement appliance on mandibular growth in growing rats, through the use of lateral radiography. experimental procedures were approved by the inn university experimental animal ethical committee, with a 2013/a-38 code. twenty - four male, 8-week - old wistar albino rats were divided randomly into two groups : group i (n = 12) was the control group, and group ii (n = 12) was the bite - jumping appliance group [table 1 ]. the rats were caged in a 12-h light - dark cycle, within a 23c and ideal - humidity environment. they were fed ad libitum a soft diet, to prevent any damage to the functional bite - jumping appliances. linear and angular anatomical parameters in mandible and condyle growth were determined with cephalometric radiography during the study period. intramuscular injections of 80% xylazine (rompun - bayer, germany) and 20% ketamine hydrochloride (ketalar - eczacba, turkiye) were used as anesthesia prior to cephalometric radiography and bite - jumping appliance application. groups and experimental protocol cephalometric radiographs of experimental animals were taken before (t0) and after (t1) the study process with the same digital machine (dx3000 ; dexcowin, seoul, south korea). the right side of the head of each rat was placed in contact with digital phosphor plate films (digora imaging plates, size 2 ; soredex, finland). the distance between the film and the radiographic focus of the device was 10 cm, and x - ray images were obtained (60 kvp, 1 ma ve 1.30 s). the criteria for radiograph selection were that the condylar area, the posterior border of the ramus, and the lower border of the mandible all had to be clearly readable on the cephalometric radiographs, to make it possible to define mandibular and condylar landmarks. each digital radiograph was traced with a digital tracing program (universal desktop ruler, avp soft) by the same investigator (r.o). in what follows, the most posterosuperior point of the condyle is called the a point, the midpoint of the mandibular foramen is called the b point, the most anterior point of the lingual alveolar bone is called the c point, the menton is called the d point, the most inferior part of the lower border angular process is called the e point, and the mandibular plane is called the mp point [figure 1 ]. skeletal landmarks, linear and angular measurements the length of the condylar process is referred to as a - b ; the mandibular length, a - c ; the distance from point a to menton, a - d ; the length of mandibular base, b - c ; and the perpendicular distance from a to mp, a - mp [figure 1 ]. the angle of the condylar process axis to the mandibular plane is called the stutzman angle [figure 1 ]. the bite - jumping appliance used in this research is similar to that used in previous studies to improve rat mandibular incisors. functional bite - jumping appliances were fitted to the mandibular incisors of rats in the experimental group and bonded to mandibular incisors with self - etching composite (transbond plus ; 3 m, monrovia, ca, usa). this bite - jumping appliance produced 2.5 - 3 mm forward mandibular advancement in group ii [figure 2 ]. bite - jumping appliance a nonparametrical friedman test and a mann whitney u - test were executed to examine intra- and inter - group significant differences in angular and linear measurements [tables 2 and 3 ]. chicago, il, usa ; windows version 20.0) was used in statistical analysis. the mean values and intragroup differences in angular and linear measurements in two groups on days 0 and 30 comparisons of linear and angular measurements between groups in different time - points in what follows, the most posterosuperior point of the condyle is called the a point, the midpoint of the mandibular foramen is called the b point, the most anterior point of the lingual alveolar bone is called the c point, the menton is called the d point, the most inferior part of the lower border angular process is called the e point, and the mandibular plane is called the mp point [figure 1 ]. the length of the condylar process is referred to as a - b ; the mandibular length, a - c ; the distance from point a to menton, a - d ; the length of mandibular base, b - c ; and the perpendicular distance from a to mp, a - mp [figure 1 ]. the angle of the condylar process axis to the mandibular plane is called the stutzman angle [figure 1 ]. the bite - jumping appliance used in this research is similar to that used in previous studies to improve rat mandibular incisors. functional bite - jumping appliances were fitted to the mandibular incisors of rats in the experimental group and bonded to mandibular incisors with self - etching composite (transbond plus ; 3 m, monrovia, ca, usa). this bite - jumping appliance produced 2.5 - 3 mm forward mandibular advancement in group ii [figure 2 ]. a nonparametrical friedman test and a mann whitney u - test were executed to examine intra- and inter - group significant differences in angular and linear measurements [tables 2 and 3 ]. chicago, il, usa ; windows version 20.0) was used in statistical analysis. the mean values and intragroup differences in angular and linear measurements in two groups on days 0 and 30 comparisons of linear and angular measurements between groups in different time - points there were statistically significant changes in mandibular morphology in the control and appliance groups within the study period. at the t1 time - point, it was found that a - b, a - d, and b - c had each increased significantly (p < 0.05). in addition, in group ii, the a - b, a - c, and a - d parameters had increased significantly (p < 0.05) during the study period. according to the mann whitney u statistical test results, the growth of a - b between t0 and t1 in group ii was significantly greater than that in the control group (p < 0.05). the dimension change in a - d at 30 days within group ii was significantly greater than that in the control group (p < 0.05). the growth of the b - c length in group ii during the study period was significantly lower than that in the control group (p < 0.05). in addittion, there were no statistically significant changes in the a - mp or stutzman angle parameters (p < 0.05). the body weight of the experimental animals in both groups did not decline during the study period. during the study, the increase in weight in the control group was greater than that in group ii [table 4 ] the effects of skeletal class ii treatment with mandibular retrognathia depend upon the capacity of bite - jumping appliances with regard to condylar growth. many functional bite - jumping appliances have been used to enhance condylar growth and mandibular advancement. in this experimental study, functional bite - jumping appliances were used to promote condylar changes and mandibular advancement among growing rats over a 30 days period. due to the morphological, anatomical, and structural similarities to human condyles and mandibles, the findings of this study with respect to three parameters (a - b, a - d, and b - c) indicate that group i showed significant mandibular and condylar growth without any external stimulus during t0-t1. group ii showed statistically significant growth with respect to three sagittal mandibular growth variables during t0-t1. a number of studies concluded that mandibular growth was increased, and that functional bite - jumping appliances enhance mandibular growth in growing rats. similar to our findings, ghafari and degroote found that continuous anterior and vertical displacement produce an acceleration of ossification and maturation. according to our experimental animal study results, the growth of the condylar process (a - b) in the bite - jumping group between t0 and t1 were significantly greater than that in the control group. our study results indicate that condyle growth among growing rats could be increased through the use of bite - jumping appliances over a 30 days period ; these findings align with those of previous studies, as mentioned. although group ii showed greater mandibular length (a - c) growth than did the control group, there were no statistically significant differences between the two groups. this condition may be due to the brevity of the functional appliance treatment time - period, and also to the fact that the anatomical landmark (c point) may be affected by bite - jumping appliance therapy. found there to be a significant mandibular length increase in their appliance group versus their control group. a possible reason may be that the bite - jumping appliance used in their study produced 3.5 mm forward and 3 mm downward movement. mandibular growth in this length within the bite - jumping appliance group (between t0 and t1) was significantly greater than that in the control group. at the 30 days period, taira. showed that functional appliances enhance the growth of this effective mandibular length parameter in rats. a functional bite - jumping appliance may not enhance this length, because the application area for functional devices is far from the distance between the b and c points. in addition to this, the c point is very near the behind - mandibular incisor to which the appliance is fitted. within mandibular advancement research, growth in the perpendicular distance from a to mp charlier. studied the effects of mandibular functional appliance use with respect to young rat condyle, and they found that growth was more horizontal in the appliance group. mandible advancement and condyle growth in rats has also been determined by the opening of the stutzman angle. there were no statistically significant differences between the two groups, but the bite - jumping appliance group nonetheless showed a greater increase in the stutzman angle than did the control group. the limitations of this study include the absence of histological evaluation of condylar cartilage growth, and also the relatively short experimental time of 30 days. our experimental study results indicate that the mandibular advancement promoted through the use of a functional appliance increases mandibular and condylar growth in rats. furthermore, the use of a bite - jumping appliance does not affect the vertical dimension of the mandible. further investigations are suggested to evaluate the effects of bite - jumping appliances on mandibular morphological changes over a longer time - horizon and with a larger study sample.	objective : the aim was to evaluate the effects of the use of mandibular advancement appliances on mandibular growth in growing rats.materials and methods : twenty - four 8-week - old male wistar albino rats were randomly divided into two experimental groups (12 rats each) : group i was a control group, and group ii was the mandibular advancement appliance group. a functional bite - jumping appliance was used in group ii to promote mandibular advancement. anatomical changes in the condyle and mandible were evaluated by comparing radiographic results from before and after the study, with angular and linear measurements. friedman and mann - whitney u - tests were used in statistical analysis.results:according to the radiographic results, the growth of mandibles and condyles in group ii was significantly greater than with the length of the condylar process (a - b) and distance from condyle to menton (a - d) variables (p < 0.05). in addition, group i showed greater mandibular base growth than did group ii (p < 0.05).conclusions : we conclude that the use of an intraoral bite - jumping appliance can stimulate condylar growth and increase sagittal mandibular advancement in growing rats.
at present, implants have become a standard means of replacing missing teeth since they have the advantage of allowing preservation of the integrity of sound teeth adjacent to the edentulous area. the most widely used material for dental implants is titanium owing to its superior properties such as biocompatibility and corrosion resistant. after placement and osseointegration of implants, it is the responsibility of the dentist to maintain properly designed and fitted prosthesis. clinical decisions are not limited to the selection of the type of implant but are also dependent on the material of prosthesis. regardless of the implant system used, a restoration is selected to emerge from the tissues to imitate a natural tooth. the quest for predictable long - term results has raised several questions concerning the materials used as well as the techniques followed in clinical practice. implant supported restorations can be fabricated by nonprecious alloys or all ceramic materials. in the recent scenario of advanced technology, recent progress in casting techniques as well as the introduction of computer aided design / computer aided machined (cad / cam) has made it possible to make the prostheses using titanium also. cad / cam technology, all ceramic systems, and high strength ceramic materials have become integral parts of modern dentistry. optimal esthetics and characteristics such as color stability, high wear resistance, and low thermal conductivity make all ceramic materials ideal for the fabrication of dental prostheses. the main advantage of all ceramic systems lies in the absence of metal, especially in cases with very thin tissue thickness and shallow implant depths allowing a more esthetically pleasing restoration. dental implant restorations can be cement retained, screw retained or a combination of both. because it is difficult to achieve passive fit with screw - retained implant restorations, some dentists prefer cemented prosthesis. on the other hand, cement retained prosthesis has the disadvantage of compromised stability in case of less interocclusal distance, as the abutment lacks the height and taper for cement retention. retrievability also serves as the safety mechanism to allow the prosthesis to be removed without causing harm to underlying implant superstructures. retention certainly influences the lack of complications as well as the longevity of implant prostheses. over the years, those engaged in implant supported restorations have debated as to which type of mechanism is preferable for increase bonding of cemented restorations. the success of implant - supported restorations depends on the success of bonding between prosthesis and metal interface. retention of crown depends on cement used, surface preparation of the implant abutment, and other variables such as internal surface characteristics of coping and the height and taper of implant abutment. for better simulation of clinical conditions, investigation of the bonding or retentive strength numerous studies investigating the bonding strength of different luting agents on retention of cement - retained prosthesis and also effect of airborne - particle abrasion on zirconia copings are available, but there is a paucity of studies having direct comparison of different copings to the best of our knowledge. due to the above concerns, there is a need for comparing the shear bond strength of abutment copings of different materials, made by different techniques as well as effect of surface conditioning on bonding. the null hypothesis was that there was no difference in bond strength of abutment copings made of different materials and also no effect of surface conditioning on bonding. the alternate hypothesis states that there was difference in bond strength of abutment copings, and also surface conditioning affected the bonding strength. the present research is a cross - sectional in vitro study aimed to compare the shear bond strength of different copings and effect of surface conditioning on shear bond strength of three different types of abutment copings made on titanium implant abutment (indident dental implant system, drdo inmas, new delhi, india). in the present study, three different types of abutment copings were fabricated on titanium implant abutment (indident dental implant system, drdo inmas, new delhi, india). the sample size was estimated to be 30 in each group based on previous studies. for group a, 30 cad / cam zirconia copings were fabricated on one implant abutment [figure 1a ] using cercon cad / cam system (dentsply, germany). optical scanning was done with cercon eye. once the scanning was completed with cercon eye, cement gap, wall thickness, and occlusal geometry were adjusted for the coping with the help of cercon art program. a luting gap of 30 m and wall thickness of 0.8 mm were chosen for the coping fabrication. presintered zirconia blank of 38 size was inserted in cercon brain to complete the milling. after milling was completed, the frameworks of the coping were separated from the blank with the help of micromotor and sintered in furnace, according to manufacturer 's instructions. dimensions of cad / cam coping was measured with digital caliper (shenzhen yks technology ltd., (a - c) abutment copings made of computer aided design / computer aided machined (left), pressable ceramic (middle), and milled metal (right) for group b, 30 pressable ceramic copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [figure 1b ]. implant abutment was screwed on implant analog, and two coatings of die spacer (durolan, dfs) were applied on implant abutment to attain the same cement space. to attain the similar dimension as cad / cam coping, silicon index was fabricated of cad / cam coping extending on the predetermined groove on implant analog. molten inlay wax (geo - dip, renfert, germany) was poured in silicon index, and mounted abutment was inserted in that index to the level of predetermined groove. investment of wax pattern was done in silicone ring with the phosphate bonded investment material (ips pressvest speed). investment ring was inserted in the center of hot press furnace (multimat 2 touch + press) using investment tongs ; selected program was started. after cooling of the ring, the sprue and reaction layer on the copings were removed, and copings were retrieved. dimensions of pressable ceramic coping were verified with a digital caliper. for group c, 30 milled metal copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [figure 1c ]. the cad volume data was transferred to the multi - axis metal milling machine (tds cutter, turbodent system, taiwan). metal milling of titanium blank for abutment copings copings of cad / cam, pressable ceramic and metal millings were divided into two halves. to maintain standardization, one - half (15 out of 30 samples in each group) was surface conditioned by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s at a distance of 10 mm in the sandblaster [figure 3 ], and other half were left nonconditioned. thus, three groups with two subgroups of each of the copings were prepared : surface conditioning done by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s group a (n = 30) : copings made by computer - aided technique subgroup a1 (n = 15) : copings made by computer - aided technique that were surface conditionedsubgroup a2 (n = 15) : copings made by computer - aided technique that were nonconditioned group b (n = 30) : copings made by pressable ceramic technique subgroup b1 (n = 15) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 (n = 15) : copings made by pressable ceramic technique that were nonconditioned group c (n = 30) : copings made by metal milling group a (n = 30) : copings made by computer - aided technique subgroup a1 (n = 15) : copings made by computer - aided technique that were surface conditionedsubgroup a2 (n = 15) : copings made by computer - aided technique that were nonconditioned subgroup a1 (n = 15) : copings made by computer - aided technique that were surface conditioned subgroup a2 (n = 15) : copings made by computer - aided technique that were nonconditioned group b (n = 30) : copings made by pressable ceramic technique subgroup b1 (n = 15) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 (n = 15) : copings made by pressable ceramic technique that were nonconditioned subgroup b1 (n = 15) : copings made by pressable ceramic technique that were surface conditioned subgroup b2 (n = 15) : copings made by pressable ceramic technique that were nonconditioned group c (n = 30) : copings made by metal milling subgroup c1 (n = 15) : milled metal copings that were surface conditionedsubgroup c2 (n = 15) : milled metal copings that were nonconditioned. subgroup c1 (n = 15) : milled metal copings that were surface conditioned subgroup c2 (n = 15) : milled metal copings that were nonconditioned. all specimens were cemented to titanium abutment using glass ionomer cement (gic) (meron, voco gmbh, germany) to make standardization, which was mixed according to manufacturers recommendations. an alignment apparatus [figure 4 ] was used that applied a weight of 750 g to the bonded specimens. extra cement was scraped out, and after initial set (8 min), specimens were stored in distilled water for 24 h to simulate the moist oral environment. alignment apparatus applying a weight of 750 g to the bonded specimens in the present study, shear bond strength was evaluated by universal testing machine [figure 5 ] (22.5 k instron, model 4482). forces were applied axially on coping cemented on abutment at a cross - head speed of 1.0 mm / min. a computer connected with the instron machine recorded the result of each test. the nonadhesive chemical bond strength () values (expressed in effects of mpa) were calculated using the formula : = l / a. where l is load (in n) and a is the adhesive area (in m). pull - off test for determination of bonding strength the shear bond strength was evaluated by pull - off test for all the abutment copings. all calculations were performed using the spss (version 16) for windows (spss inc., the present research is a cross - sectional in vitro study aimed to compare the shear bond strength of different copings and effect of surface conditioning on shear bond strength of three different types of abutment copings made on titanium implant abutment (indident dental implant system, drdo inmas, new delhi, india). in the present study, three different types of abutment copings were fabricated on titanium implant abutment (indident dental implant system, drdo inmas, new delhi, india). the sample size was estimated to be 30 in each group based on previous studies. for group a, 30 cad / cam zirconia copings were fabricated on one implant abutment [figure 1a ] using cercon cad / cam system (dentsply, germany). optical scanning was done with cercon eye. once the scanning was completed with cercon eye, cement gap, wall thickness, and occlusal geometry were adjusted for the coping with the help of cercon art program. a luting gap of 30 m and wall thickness of 0.8 mm were chosen for the coping fabrication. presintered zirconia blank of 38 size was inserted in cercon brain to complete the milling. after milling was completed, the frameworks of the coping were separated from the blank with the help of micromotor and sintered in furnace, according to manufacturer 's instructions. dimensions of cad / cam coping was measured with digital caliper (shenzhen yks technology ltd., (a - c) abutment copings made of computer aided design / computer aided machined (left), pressable ceramic (middle), and milled metal (right) for group b, 30 pressable ceramic copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [figure 1b ]. implant abutment was screwed on implant analog, and two coatings of die spacer (durolan, dfs) were applied on implant abutment to attain the same cement space. to attain the similar dimension as cad / cam coping, silicon index was fabricated of cad / cam coping extending on the predetermined groove on implant analog. molten inlay wax (geo - dip, renfert, germany) was poured in silicon index, and mounted abutment was inserted in that index to the level of predetermined groove. investment of wax pattern was done in silicone ring with the phosphate bonded investment material (ips pressvest speed). investment ring was inserted in the center of hot press furnace (multimat 2 touch + press) using investment tongs ; selected program was started. after cooling of the ring, the sprue and reaction layer on the copings were removed, and copings were retrieved. dimensions of pressable ceramic coping were verified with a digital caliper. for group c, 30 milled metal copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [figure 1c ]. the cad volume data was transferred to the multi - axis metal milling machine (tds cutter, turbodent system, taiwan). copings of cad / cam, pressable ceramic and metal millings were divided into two halves. to maintain standardization, one - half (15 out of 30 samples in each group) was surface conditioned by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s at a distance of 10 mm in the sandblaster [figure 3 ], and other half were left nonconditioned. thus, three groups with two subgroups of each of the copings were prepared : surface conditioning done by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s group a (n = 30) : copings made by computer - aided technique subgroup a1 (n = 15) : copings made by computer - aided technique that were surface conditionedsubgroup a2 (n = 15) : copings made by computer - aided technique that were nonconditioned group b (n = 30) : copings made by pressable ceramic technique subgroup b1 (n = 15) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 (n = 15) : copings made by pressable ceramic technique that were nonconditioned group c (n = 30) : copings made by metal milling group a (n = 30) : copings made by computer - aided technique subgroup a1 (n = 15) : copings made by computer - aided technique that were surface conditionedsubgroup a2 (n = 15) : copings made by computer - aided technique that were nonconditioned subgroup a1 (n = 15) : copings made by computer - aided technique that were surface conditioned subgroup a2 (n = 15) : copings made by computer - aided technique that were nonconditioned group b (n = 30) : copings made by pressable ceramic technique subgroup b1 (n = 15) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 (n = 15) : copings made by pressable ceramic technique that were nonconditioned subgroup b1 (n = 15) : copings made by pressable ceramic technique that were surface conditioned subgroup b2 (n = 15) : copings made by pressable ceramic technique that were nonconditioned group c (n = 30) : copings made by metal milling subgroup c1 (n = 15) : milled metal copings that were surface conditionedsubgroup c2 (n = 15) : milled metal copings that were nonconditioned. subgroup c1 (n = 15) : milled metal copings that were surface conditioned subgroup c2 (n = 15) : milled metal copings that were nonconditioned. all specimens were cemented to titanium abutment using glass ionomer cement (gic) (meron, voco gmbh, germany) to make standardization, which was mixed according to manufacturers recommendations. an alignment apparatus [figure 4 ] was used that applied a weight of 750 g to the bonded specimens. extra cement was scraped out, and after initial set (8 min), specimens were stored in distilled water for 24 h to simulate the moist oral environment. in the present study, shear bond strength was evaluated by universal testing machine [figure 5 ] (22.5 k instron, model 4482). forces were applied axially on coping cemented on abutment at a cross - head speed of 1.0 mm / min. a computer connected with the instron machine recorded the result of each test. the nonadhesive chemical bond strength () values (expressed in effects of mpa) were calculated using the formula : = l / a. where l is load (in n) and a is the adhesive area (in m). the shear bond strength was evaluated by pull - off test for all the abutment copings. all calculations were performed using the spss (version 16) for windows (spss inc., chicago, il, usa). a one - way analysis of variance was computed for statistical significance at p = 0.05 for individual groups with post - hoc (tukey 's honest significant difference) and intragroup comparison was done by unpaired t - test. the mean difference for cad / cam surface the mean difference for pressable surface conditioned group was 1.18 0.04, and for nonconditioned group was 0.75 0.28. the mean difference for milled metal surface conditioned group was 2.57 0.58, and for nonconditioned group was 1.49 0.15 [graph 1 and table 1 ]. mean and standard deviation of bonding strength (in mpa) between surface conditioned (c) and nonconditioned specimens (nc) mean bonding strength (in mpa) of different specimens on intragroup comparison, results showed that pressable ceramic and milled metal group had the statistically significant difference (p = 0.05) in conditioned and nonconditioned specimens [table 2 ]. on intergroup comparisons, cad / cam, pressable, and milled metal surface conditioned specimens had statistically significant difference, and similar results were found for nonconditioned specimens [table 3 ]. mean comparison and standard deviation of bonding strength(in mpa) between different abutment copings mean comparison of bonding strength (in mpa) between surface conditioned and nonconditioned specimens this study assessed the bonding strength of copings made out of different materials and also evaluated the effect of surface conditioning on bonding strength. the results of this study led to the rejection of null hypothesis that there was no significant difference in the bond strength of tested groups. metal copings fabricated by milling technique provided much stronger retention as compared to other materials and also surface conditioning increases the bonding strength. two all ceramic materials and titanium metal were used in this study, and titanium metal was not casted because milling of titanium metal overcomes the problem of production of reactive surface layer on its surface when cast in thin and fragile sections. over the years, questions have been put on bonding of implant supported restorations. in order to improve bonding, the internal surface of restoration chemical surface treatments include the use of various reactive agents while mechanical procedures include airborne - particle abrasion with alumina particles and abrasion with a mounted stone. investigated the factors which determine the retentiveness of copings made out of co - cr alloy and zirconia luted with permanent and provisional luting cements. the conclusion of this study was that copings made of metal alloy and zirconia showed no different level of retentiveness when set onto titanium abutments fixed with permanent or provisional cements. as concluded in this study, surface conditioning with airborne particle abrasion enhances the bonding of metallic as well as ceramic - based restorations. tashkandi evaluated the effect of surface treatment on micro - shear bond strength of zirconia frameworks. ebert. in 2007 found similar results when evaluating the effect of two surface conditioning methods and two luting gap sizes on the bonding of zirconia copings. airborne - particle abrasion has also been recommended as the best method of pretreatment in previous studies to improve the bond strength to oxide ceramics. sadig and al harbi compared the effect of different surface conditions on the retentiveness of titanium crowns cemented abutments using two types of cement. shahin and kern in 2010 also concluded that air - abrasion significantly increases crown retention. in the present study, although resin cements has got the maximum retention for all ceramic restorations, previous studies assessed that gic has also got the adequate retention. compared the retentive strength of metal copings on prefabricated abutments with five different luting agents. gic was used for luting all the specimens to make standardization, and moreover different resin cements require different pretreatment which could alter results. gic show high water solubility and require sufficient time for complete setting reaction to maximize their retention. manufacturer of gic states that the material will reach its full set in 47 min so 8 min setting period that was allowed in the previous study should prove satisfactory. the limitations of this study include that all copings were produced and tested under ideal conditions, which may not reflect conditions in daily clinical practice. thermal cycling and long - term water storage are the factors that can have effects on the durability of bond strength, and they are important parameters to simulate the oral conditions. in terms of in vivo loading, the masticatory cycle consists of combination of vertical and lateral forces, subjecting the restoration to a variety of off - axis loading. in the current investigation, hence, future studies should attempt to simulate intraoral conditions, as well as utilize additional mechanical tests, which can provide a more reliable assessment of retention. within the conditions and limitations of this in vitro study, milled metal copings had the highest bond strength as compared to cad / cam and pressable ceramic copings. surface conditioning with air - abrasion achieved better bond strength of all type of copings. further studies are needed to evaluate optimal surface conditioning in order to enhance the longevity of restorations clinically.	background / purpose : the aim of this study was to compare the shear bond strength of computer aided design / computer aided machined ceramic (cad / cam), pressable ceramic, and milled metal implant copings on abutment and the effect of surface conditioning on bonding strength.materials and methods : a total of 90 test samples were fabricated on three titanium abutments. among 90 test samples, 30 copings were fabricated by cad / cam, 30 by pressable, and 30 by milling of titanium metal. these 30 test samples in each group were further subdivided equally for surface treatment. fifteen out of 30 test samples in each group were surface conditioned with airborne particle abrasion. all the 90 test samples were luted on abutment with glass ionomer cement. bonding strength was evaluated for all the samples using universal testing machine at a crosshead speed of 5 mm / min. the results obtained were compared and evaluated using one - way anova with post - hoc and unpaired t - test at a significance level of 0.05.results:the mean difference for cad / cam surface conditioned subgroup was 1.28 0.12, for nonconditioned subgroup was 1.20 0.11. the mean difference for pressable surface conditioned subgroup was 1.18 0.04, and for nonconditioned subgroup was 0.75 0.28. the mean difference for milled metal surface conditioned subgroup was 2.57 0.58, and for nonconditioned subgroup was 1.49 0.15.conclusions:on comparison of bonding strength, milled metal copings had an edge over the other two materials, and surface conditioning increased the bond strength.
frictional dermal melanosis, also known as lifa disease, is a common dermatological problem causing great cosmetic disfigurement. it is characterised by hyperpigmentation over bony prominences, secondary to the friction associated during bathing. various modalities like q - switched lasers, dermabrasion and cryosurgery have been tried, but they have not been satisfactory. chances of developing complications like hyper and hypopigmentaion and permanent scarring are also more with these procedures. chemical peeling is a popular, relatively inexpensive, and generally safe method for the treatment of cutaneous pigmentation. chemical peels are used to create an injury at a specific skin depth with the goal of stimulating new skin growth and improving surface, texture and appearance. the exfoliative effect of chemical peels stimulates new epidermal growth and collagen with more evenly distributed melanin thus improving dermal melanosis. there is a wide array of peeling agents available in the market today, with different formulations and combinations. those most commonly used include alpha hydroxyacids (ahas), beta hydroxyacids and trichloroacetic acid (tca). chemical peels are very effective for treating epidermal pigmentation like melasma, solar lentigines and post - inflammatory hyperpigmentation. chemical peels have been tried for dermal pigmentation but the results are not very satisfactory. in a study by sharquie., a chemical peeling agent lactic acid has been found effective in frictional melanosis. however, till now there have been no studies to determine the efficacy of other peels in this condition. hence, this open randomised pilot study was undertaken to establish the efficacy of two most commonly used peels tca and glycolic acid (ga) in frictional melanosis. forty patients attending the dermatology outpatient department, with typical frictional dermal melanosis, clinically seen as pigmentary changes over the extensor aspect of the hands, and had not received any systemic or topical treatment in the last 6 months were included in the study. patients with frictional melanosis in the other areas like back and clavicle were not included for the ease of comparison of the two agents. patients with a history of taking oral contraceptive pills, isotretinoin, pregnancy, lactation, history of keloids or hypertrophic scars, active dermatitis at the site, concomitant systemic or skin disease and those with unrealistic expectations were excluded from the study. a complete history of the patient with regard to the age, sex, onset of the disease, total duration, and any previous treatment for the disease was taken. a biopsy was not done on any patient as most patients were unwilling to undergo an invasive procedure since they considered the condition as a simple cosmetic concern. patients selected were randomly allocated into two groups : group a and group b. pre - peel priming was carried out in both groups with 12% glycolic acid cream and sunscreen for 2 weeks before the peel. group a was treated with trichloroacetic acid (tca, 15%) and group b with glycolic acid (ga 50%). before application of the peeling agent, the involved area was degreased with 70% alcohol. tca 15% and ga 50% were applied over the lesion carefully with cotton buds. after a contact period of 3 - 5 minutes if the patient developed erythema in case of ga peel and frosting in case of tca peel, it was neutralised immediately even before 3 minutes. four peels were carried out at serial intervals of 2 weeks each. after the completion of the fourth peel, patients were advised to continue strict sun protection and topical 12% ga at night. colour photographs were taken of all patients at baseline and 30 days after the last peel. all the treated patients were evaluated objectively and subjectively regarding their response to the treatment. the darkness of the hyperpigmentation was assessed according to a special colour score chart that was invented by sharquie. according to this score, the darkness or the colour of pigmentation graded from 0 to 4 as follows : score 0 : similar to the surrounding skin colour subjective improvement, i.e., patient satisfaction was graded as : grade 0 : not satisfied grade 1 : moderately satisfied grade 2 : greatly but not fully satisfied grade 3 : fully satisfied patients were evaluated every month for a period of 3 months after the last peel to detect any side effects and relapse. a total of 40 patients, with 34 females and 6 males were included in the study. the age at presentation ranged from 15 to 60 years with a mean age of 30.7 years. the disease duration varied between 3 months to 10 years with a mean of 3 years. in four patients, other areas like back all patients showed improvement with regard to hyperpigmentation and overall appearance. at the end of four peels, 6 out of 20 patients in group a (15% tca peel) improved from grade 4 to grade 1 ; 3 upgraded from grade 4 to grade 3 ; 8 from grade 3 to grade 1 and 2 from grade 3 to grade 2 [table 1 ]. objective assessment - tca in group b (50% ga peel) after four peels, 1 out of 20 patients improved from grade 4 to grade 1 ; 2 patients from grade 4 to grade 3 ; 5 patients improved from grade 4 to grade 2 ; 6 from grade 3 to grade 2 and 6 from grade 3 to grade 1 [table 2 ]. objective assessment - ga the difference in the pre - peel levels in both the groups was not statistically significant and thus they were found to be comparable [table 3 ]. pre - peeling scores of both groups both the groups showed improvement after the peeling [figures 1 and 2 ], which was evident by comparing pre peel and post peel score using wilcoxon test and the improvement was statistically significant (p 0.05) difference in the efficacy between the two groups the mean value of tca group is less than ga group indicating that tca peel is better [table 5 ]. (a and b) pre and post treatment photographs after tca peeling (a and b) pre and post treatment photographs after ga peeling comparison of pre - test and post test scores in each group post peeling values of both groups in group a (tca 15%), six patients were fully satisfied (grade 3), nine patients were greatly but not fully satisfied (grade 2) and five patients were moderately satisfied (grade 1) [figure 3 ]. subjective assessment in group b (ga 50%), 1 patient was fully satisfied (grade 3), 13 patients were greatly but not fully satisfied (grade 2) and 6 patients were moderately satisfied (grade 1). on comparing the subjective scores, while group a received a higher score (higher score means more satisfaction), the difference between the two groups was not statistically significant (p = 0.18) [table 6 ]. this means that the patients in the tca group were more satisfied than the ga group but this difference was not statistically significant. comparison of subjective scores among the two groups during the study, none of the patients had any severe or permanent side effects. there was mild to moderate burning sensation in seven patients of group a and eight patients of group b. mild erythema was seen in three patients of group a and six patients of group b. mild frosting was seen in 10 patients of group a and none in group b. post inflammatory pigmentation was seen in one patient each of group a and group b. postpeel cracking was reported in nine patients of group a but in none of the patients in group b. it was not very troublesome for the patient and improved after applying emollient. hypopigmentation [figure 4 ] was seen in one patient in group a and none in group b. no allergic reaction or scarring or infection was observed in either group. hypopigmentation seen in a patient no relapse was seen in any of the patients during the 3-month follow - up period. at the end of four peels, 6 out of 20 patients in group a (15% tca peel) improved from grade 4 to grade 1 ; 3 upgraded from grade 4 to grade 3 ; 8 from grade 3 to grade 1 and 2 from grade 3 to grade 2 [table 1 ]. objective assessment - tca in group b (50% ga peel) after four peels, 1 out of 20 patients improved from grade 4 to grade 1 ; 2 patients from grade 4 to grade 3 ; 5 patients improved from grade 4 to grade 2 ; 6 from grade 3 to grade 2 and 6 from grade 3 to grade 1 [table 2 ]. objective assessment - ga the difference in the pre - peel levels in both the groups was not statistically significant and thus they were found to be comparable [table 3 ]. pre - peeling scores of both groups both the groups showed improvement after the peeling [figures 1 and 2 ], which was evident by comparing pre peel and post peel score using wilcoxon test and the improvement was statistically significant (p 0.05) difference in the efficacy between the two groups the mean value of tca group is less than ga group indicating that tca peel is better [table 5 ]. (a and b) pre and post treatment photographs after tca peeling (a and b) pre and post treatment photographs after ga peeling comparison of pre - test and post test scores in each group post peeling values of both groups in group a (tca 15%), six patients were fully satisfied (grade 3), nine patients were greatly but not fully satisfied (grade 2) and five patients were moderately satisfied (grade 1) [figure 3 ]. subjective assessment in group b (ga 50%), 1 patient was fully satisfied (grade 3), 13 patients were greatly but not fully satisfied (grade 2) and 6 patients were moderately satisfied (grade 1). on comparing the subjective scores, while group a received a higher score (higher score means more satisfaction), the difference between the two groups was not statistically significant (p = 0.18) [table 6 ]. this means that the patients in the tca group were more satisfied than the ga group but this difference was not statistically significant. comparison of subjective scores among the two groups during the study, none of the patients had any severe or permanent side effects. there was mild to moderate burning sensation in seven patients of group a and eight patients of group b. mild erythema was seen in three patients of group a and six patients of group b. mild frosting was seen in 10 patients of group a and none in group b. post inflammatory pigmentation was seen in one patient each of group a and group b. postpeel cracking was reported in nine patients of group a but in none of the patients in group b. it was not very troublesome for the patient and improved after applying emollient. hypopigmentation [figure 4 ] was seen in one patient in group a and none in group b. no allergic reaction or scarring or infection was observed in either group. hypopigmentation seen in a patient no relapse was seen in any of the patients during the 3-month follow - up period. it is characterised by hyperpigmentation over bony prominences, secondary to the use of a rough washing agent during showering. the washing agents commonly implicated are nylon towels, sponges, cotton towels, brushes and back scratchers etc. in india, coconut coir and sometimes even washing stone have been used. it is speculated that the pigmentation is a response to repeated damage of the basal layer of the epidermis as a result of squeezing the epidermis between the washing brush above and the bone below. superficial bony prominences and areas like clavicular zones, trunk (mainly over scapular areas) and the extensor aspect of the hands are most commonly affected. the histology of the disease is characterised by a striking degree of pigmentary incontinence and the presence of melanophages in the dermis. firstly, the patients should avoid rubbing the skin with nylon towels, sponges, brushes and back scratchers etc. treatments found useful for dermal melanosis like dermabrasion or laser therapy like q switched nd : yag laser have been found to be useful in frictional melanosis as well but the chances of complications like scarring and hypo and hyperpigmentation are more. chemical peels have become an increasingly popular method to treat a myriad of benign skin disorders. chemical peeling or chemical rejuvenation is a procedure where a chemical agent or combination of agents of defined strength is applied to the skin, causing a controlled destruction of the layers of the skin. this is followed by regeneration and remodelling of the skin with improvement of texture and surface abnormalities. both the agents used in this study, tca 15% and ga 50%, are cost effective and easily available. application of tca to the skin causes precipitation of proteins and coagulative necrosis of cells in epidermis. this leads to re - epithelialisation with replacement of smoother skin with an even skin tone. ga acts by decreasing corneocyte cohesion leading to sloughing of dead cells and stimulation of new cell growth in the basal cell layer. it increases synthesis of number of connective tissue components like collagen, elastin, glycosaminoglycans and mucopolysaccharides. in addition to this, it also decreases melanin production by direct inhibition of tyrosinase. in our study this is comparable to the study conducted by sharquie. where the mean age was 24 years. in the study by sharquie, frictional melanosis was more common in female, the mean duration of the disease was 2.7 years and 16% of the patients gave family history which was comparable with our study. in our study also, it was more common in females, the mean duration of disease was 3 years and 22.5% of the patients gave family history of frictional melanosis. a study done by sharquie. showed lactic acid peel as an effective mode of therapy in treating patients with frictional dermal melanosis. our study was done to assess the efficacy of tca and ga, in the treatment of frictional melanosis. to our knowledge, no other study has compared these two modalities for the treatment of frictional melanosis. both these agents showed a good therapeutic response which was statistically very significant. in a study by sachdeva, which compared the efficacy and side effects of tca and ga in facial pigmentation, ga was found to be the superior peeling agent with better patient tolerance and lesser side effects compared to tca, but the difference was not statistically significant. in our study, tca was more efficacious than ga peel, but it was statistically not significant. even though post peel cracking of the skin and frosting were seen only in the tca group it did not cause any distress to the patient. this may be aided by the strict sun protection and continuous use of glycolic 12% cream even after the four sessions of peel were completed. from the present study, it can be concluded that both 15% trichloroacetic acid (tca) and 50% glycolic acid are equally effective peeling agents in the treatment of frictional melanosis of the forearm. however, the response with tca was better than glycolic acid, though statistically not significant. both the peels are very safe with minimal side effects and the beneficial results achieved can be maintained with topical application of sunscreen and glycolic acid 12%. further studies using glycolic and tca peels in a larger sample size along with histopathological work up are required to substantiate the results of the present work.	background : frictional dermal melanosis is aesthetically displeasing. various modalities ranging from depigmenting agents to lasers have been tried but it continues to be a difficult problem to treat.objective:to study and compare the efficacy of 15% trichloroacetic acid (tca) and 50% glycolic acid in the treatment of frictional melanosis of the forearm.materials and methods:40 patients of frictional melanosis of the forearm were included in the study. patients were randomly divided into two equal groups a and b. pre - peel priming was carried out with 12% glycolic acid and sunscreen for 2 weeks. group a was treated with trichloroacetic acid (tca-15%) peel and group b with glycolic acid (ga-50%) peel. four peels were done one every 15 days. clinical photographs were taken to assess the response. response to therapy was evaluated by both objective and subjective methods. the patients were followed up for 3 months after the last peel to note any relapse.results:both tca and glycolic acid peels were effective in frictional melanosis. tca showed better response compared to glycolic acid at the end of the treatment, both by subjective and objective methods. however, this difference was not statistically significant (p > 0.05). no permanent side effects were seen in any of the treated patients and the improvement was sustained without any relapse at 3 months.conclusion:chemical peeling with both tricholoroacetic acid (15%) and glycolic acid (50%) is safe and effective for the treatment of frictional dermal melanosis. tricholoroacetic acid was found to be marginally superior to glycolic acid.
joint hypermobility syndrome (jhs) is a hereditary connective tissue disorder, characterized by musculoskeletal pain and an excessive range of motion in joints (2). as there are no laboratory tests to indicate jhs (3), it is usually subjectively assessed using the brighton criteria (4), which include the beighton score (5) for joint hypermobility. it has been reported that symptomatic joint hypermobility affects around 5% of women and 0.6% of men (6). the prevalence of jhs amongst those attending rheumatology and physiotherapy clinics has been estimated to be between 30% and 60% and is higher in non - caucasian populations (7,8). however, the diverse and fluctuating symptoms associated with jhs may easily be attributed to other causes and the true prevalence of jhs may be much higher than previously estimated. it has been suggested that many patients presenting with painful non - inflammatory musculoskeletal problems may have unrecognized jhs (9). although most individuals exhibiting joint hypermobility do not experience problems, a diagnosis of jhs may be given when symptoms such as arthralgia, proprioception difficulties, fatigue, soft tissue injury and joint instability are observed in the absence of genetic markers to indicate disorders such as osteogenesis imperfecta or marfan syndrome (10). jhs, osteogenesis imperfecta, marfan syndrome and ehlers danlos syndrome share many symptoms and many experts now consider jhs to be indistinguishable from ehlers danlos syndrome, hypermobility type (11). in this article primary - care practitioners are usually the patients first point of contact on entering the health - care system. they can do much to assist individuals to effectively self - manage their condition (12) and refer patients for appropriate secondary care such as physiotherapy, although the effectiveness of physiotherapy has yet to be established due to the lack of high - quality research in this area (13). the aim of the current investigation is to examine patients lived experience of jhs, their views and experiences of jhs diagnosis and management. four focus groups were conducted between january and february 2013 in four locations in the uk. participants were recruited via physiotherapy services at two national health service (nhs) trusts, local members of the hypermobility syndromes association (hmsa) and patients who had previously expressed an interest in assisting with research activity at two university locations. eligible participants were aged 18 years or over, had previously received a diagnosis of jhs (including ehlers danlos syndrome, hypermobility type), had attended physiotherapy within the preceding 12 months and were able to speak english. individuals with other known musculoskeletal pathology causing pain, particularly osteoarthritis and inflammatory musculoskeletal disease such as rheumatoid arthritis, were excluded. the purposive sampling strategy aimed for diversity with regard to age, gender, socio - economic situation and geographical location to capture maximum variation in views and experiences. ethical approval was obtained from the north east nhs research ethics committee (12/ne/0307) and all participants gave written consent. data reported in this article were collected from focus groups with individuals with jhs, aimed at developing a physiotherapy intervention for jhs management. topic guides used to facilitate discussions covered issues of living with jhs, day - to - day self - management and provision of support for symptom management. in line with an inductive approach, topic guides were revised in the light of emerging findings. the focus groups were conducted in non - clinical settings, facilitated by two researchers (stp and jph), and open - ended questioning techniques were used to elicit participants own experiences and views. all focus groups were audio - recorded, fully transcribed, anonymized, checked for accuracy and imported into a qualitative software package (nvivo 10) to aid data analysis. thematic analysis, using the constant comparison technique (14), was used to scrutinize the data to identify and analyse patterns across the dataset and the data were scrutinized for negative cases. transcripts were examined on a line - by - line basis, with codes being assigned to segments of the data and an initial coding frame developed. rht and jph independently coded transcripts and any discrepancies were discussed to achieve a coding consensus and maximize rigour. the emergent themes were discussed by the multi - disciplinary research team to ensure credibility and confirmability. scrutiny of the data showed that data saturation had been reached at the end of the analysis, such that no new themes were arising from the data (15). data reported in this article were collected from focus groups with individuals with jhs, aimed at developing a physiotherapy intervention for jhs management. topic guides used to facilitate discussions covered issues of living with jhs, day - to - day self - management and provision of support for symptom management. in line with an inductive approach, topic guides were revised in the light of emerging findings. the focus groups were conducted in non - clinical settings, facilitated by two researchers (stp and jph), and open - ended questioning techniques were used to elicit participants own experiences and views. all focus groups were audio - recorded, fully transcribed, anonymized, checked for accuracy and imported into a qualitative software package (nvivo 10) to aid data analysis. thematic analysis, using the constant comparison technique (14), was used to scrutinize the data to identify and analyse patterns across the dataset and the data were scrutinized for negative cases. transcripts were examined on a line - by - line basis, with codes being assigned to segments of the data and an initial coding frame developed. rht and jph independently coded transcripts and any discrepancies were discussed to achieve a coding consensus and maximize rigour. the emergent themes were discussed by the multi - disciplinary research team to ensure credibility and confirmability. scrutiny of the data showed that data saturation had been reached at the end of the analysis, such that no new themes were arising from the data (15). twenty - five individuals with jhs participated in the focus groups, aged 1966 years (mean : 38.2 years), 22 were female and 23 were of the analysis led to the development of four key interrelated themes : the impact of jhs, jhs as a poorly understood condition, receiving a diagnosis and jhs management and self - care. verbatim extracts are provided to illustrate the findings. participants characteristics and demographic information relating to the participants of the four focus groups, carried out between january and february 2013 (total n = 25) ses was measured as index of multiple deprivation (imd) quintile from home postcode. participants described in detail the impact of jhs, which included fatigue, pain and proprioception problems : day in day out you re managing your pain and it s a lot of pain, it s a dull ache and it makes you sleepy and it makes you tired and you re exhausted (female g, age 30, fg1). it s on your mind the whole time because i m constantly thinking about where my hands and feet are (female g, age 48, fg2). recurring joint dislocation and cycles of injury and recovery were common and participants frequently talked about the need to modify or restrict behaviours and activities.... it s just difficult to know how much to push yourself because then you are worried about injuring and then you re setting yourself back, it s a vicious cycle really (female b, age 27, fg3). however, participants acknowledged that the impact and consequences of these varied symptoms were different for each patient, with one participant noting, all of us are probably so different yet we re categorised as the same (female d, age 21, fg1). thus, some participants found living with jhs symptoms to be very debilitating, (male e, age 36, fg3), whilst others were determined to persevere with their chosen activities, in spite of their symptoms : i teach like rock - climbing, surfing, body boarding and all of that stuff, like, and i m not going to stop doing it because i m in pain like you ca nt live your whole life with pain dictating what you can and ca nt do (female g, age 45, fg3). although jhs is characterized by pain, participants alluded to the complexity of their pain experience, for example, describing difficulties in distinguishing between chronic and acute pain, and in recognizing how or ifinjuries had occurred : how do we know whether we ve injured something ? because we ve got pain all the time (female c, age 40, fg1). participants also described how prior experiences of repeated injuries led to heightened levels of anxiety and catastrophizing about future injuries : i feel like i m in a constant state of anxiety, waiting for the next injury and trying to pre - empt anything that s going to cause it (female g, age 48, fg2). oh my god is this going to be like this for the next 60 years of my life? (female b, age 27, fg3). participants perceived jhs to be a condition that was poorly understood by health professionals, and within society more widely. i think i was described as a biomechanical conundrum by one of the physiotherapists i saw [] and this is what i found repeated over and over again, that hypermobility should nt be causing pain, it s just the way you are (female c, age 53, fg2). participants described feeling stigmatized and fraudulent : i m really struggling, but because of people s expectations and their perceptions and you do nt want to ruin that, you do nt want people to start thinking oh well, you know, [unclear 1.02.47 ], we do nt employ people with disabilities because this is what happens (female c, age 40, fg1). (female d, age 21, fg1). and felt they had been blamed for their symptoms : when i was at school i just had to sit at the side while they were doing all the games, they sort of almost, i felt they were blaming it on me the odd one out (female b, age 34, fg2) and tried to hide their experiences and appear normal : it s so exhausting mentally and physically to try and appear to be normal and do normal things throughout the day with everybody and pretend it s alright (female g, age 48, fg2). participants felt that the unpredictable, diverse, evolving and fluctuating nature of their symptoms exacerbated others misunderstanding of the nature of jhs and contributed to a lack of social support : if you re inconsistent as well, they sort of go, she was alright with that last week, why is it this week she s saying that, you know, that s going to be difficult for her today (female c, age 53, fg2). many participants reported lengthy diagnosis trajectories, and being treated for individual symptoms (e.g. pain) rather than jhs : we ve all been passed from pillar to post where people do nt recognise it or they just attribute a pain to something else, when a snap kind of diagnosis just comes out of the air and you know, you progress from there (female g, age 45, fg3). often, obtaining a correct diagnosis was a coincidental occurrence : it was only because a locum happened to be in the day i went in because my gp was off sick, and he just started saying, well, to start bending everything but if had nt been for him i would nt have been put on the right track, because otherwise what other route do you really have if it s not through your gp ? (female b, age 27, fg3). receiving a diagnosis was considered necessary in order to access appropriate care pathways, for example, referral for physiotherapy for jhs rather than for an acute single joint problem : i was originally seen by a physio who had nt diagnosed with the hypermobility and then went back to a musculo - skeletal specialist who then put me forward to specialist hypermobility physiotherapist and since then it s been amazing ; i feel like it s been worthwhile and it felt like the right thing to do and i ve been really enjoying it (female b, age 27, fg3). in addition, a diagnosis helped to validate participants experiences and was psychologically helpful : getting a diagnosis on paper, this is what s wrong with me ? i mean that helped me hugely psychologically (female a, age 60, fg2). a diagnosis in itself, however, still did not guarantee beneficial treatment : when i was first diagnosed, i was nt, i really felt i was nt given that much information about the condition [] and it just seemed to be all exercises they d given me at the time seemed to make me worse (female b, age 27, fg3). similarly, a diagnosis did not necessarily lead to greater understanding and support from others in the participants social networks : there are people who do nt feel it s a genuine diagnosis, that it s something psychological, that you just need to be a bit braver (female a, age 21, fg2). education for health professionals was a key issue for participants, to facilitate timely diagnosis of, and referral for, jhs. thus, education for health professionals was a prerequisite for diagnosis, and diagnosis was a prerequisite for participants to access education. participants recognized that individuals with jhs also need education, in order to find ways to self - manage their condition and to understand and engage with prescribed treatments. i suppose it s where someone who does nt really know about it, they ve got to learn about it first because you ca nt tell someone to do it [ie engage with a particular treatment ] if they do nt understand it (female d, age 21, fg1). participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [. ] there s lots of things i still need to know about hypermobility, but on the flip side i do think it s the health professionals that need to know more (female g, age 42, fg3). participants described learning and understanding about jhs as a two - way process : so i think i get a huge amount of enlightenment from her [the physiotherapist ], and i lend her books and she lends me books about hypermobility and all that helps (female d, age 54, fg2). participants accepted that treatment should aim to manage symptoms rather than provide a cure : it s all about them helping you to manage your pain, rather than cure it (female, age 44, fg1). participants recognized the importance of self - care activities such as appropriate exercise, once a diagnosis had been received and a clear understanding of jhs had been developed. participants also recognised that activity pacing is really key to managing jhs symptoms (female b, aged 32) : i have this balancing act, if i do too much it all hurts, do nt do enough, it all hurts, do it just right, i m okay (male a, age 50, fg1). participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises (female e, age 44, fg1). it s got to be holistic, it really has to be (female d, age 21, fg1). participants also felt that education about jhs should be provided at an early age, and therefore, early diagnosis was necessary : i m only 19 now but if you d have said that when i was 16, i might not be in as much pain as i am now [] so if like if they d have told me more about how to treat it back then it probably would nt be as bad as it is now (female f, age 19, fg3). participants described in detail the impact of jhs, which included fatigue, pain and proprioception problems : day in day out you re managing your pain and it s a lot of pain, it s a dull ache and it makes you sleepy and it makes you tired and you re exhausted (female g, age 30, fg1). it s on your mind the whole time because i m constantly thinking about where my hands and feet are (female g, age 48, fg2). recurring joint dislocation and cycles of injury and recovery were common and participants frequently talked about the need to modify or restrict behaviours and activities.... it s just difficult to know how much to push yourself because then you are worried about injuring and then you re setting yourself back, it s a vicious cycle really (female b, age 27, fg3). however, participants acknowledged that the impact and consequences of these varied symptoms were different for each patient, with one participant noting, all of us are probably so different yet we re categorised as the same (female d, age 21, fg1). thus, some participants found living with jhs symptoms to be very debilitating, (male e, age 36, fg3), whilst others were determined to persevere with their chosen activities, in spite of their symptoms : i teach like rock - climbing, surfing, body boarding and all of that stuff, like, and i m not going to stop doing it because i m in pain like you ca nt live your whole life with pain dictating what you can and ca nt do (female g, age 45, fg3). although jhs is characterized by pain, participants alluded to the complexity of their pain experience, for example, describing difficulties in distinguishing between chronic and acute pain, and in recognizing how or ifinjuries had occurred : how do we know whether we ve injured something ? because we ve got pain all the time (female c, age 40, fg1). participants also described how prior experiences of repeated injuries led to heightened levels of anxiety and catastrophizing about future injuries : i feel like i m in a constant state of anxiety, waiting for the next injury and trying to pre - empt anything that s going to cause it (female g, age 48, fg2). oh my god is this going to be like this for the next 60 years of my life? (female b, age 27, fg3). participants perceived jhs to be a condition that was poorly understood by health professionals, and within society more widely. i think i was described as a biomechanical conundrum by one of the physiotherapists i saw [] and this is what i found repeated over and over again, that hypermobility should nt be causing pain, it s just the way you are (female c, age 53, fg2). i m really struggling, but because of people s expectations and their perceptions and you do nt want to ruin that, you do nt want people to start thinking oh well, you know, [unclear 1.02.47 ], we do nt employ people with disabilities because this is what happens (female c, age 40, fg1). they think you re trying to cheat out of doing something (female d, age 21, fg1). and felt they had been blamed for their symptoms : when i was at school i just had to sit at the side while they were doing all the games, they sort of almost, i felt they were blaming it on me the odd one out (female b, age 34, fg2) and tried to hide their experiences and appear normal : it s so exhausting mentally and physically to try and appear to be normal and do normal things throughout the day with everybody and pretend it s alright (female g, age 48, fg2). participants felt that the unpredictable, diverse, evolving and fluctuating nature of their symptoms exacerbated others misunderstanding of the nature of jhs and contributed to a lack of social support : if you re inconsistent as well, they sort of go, she was alright with that last week, why is it this week she s saying that, you know, that s going to be difficult for her today (female c, age 53, fg2). many participants reported lengthy diagnosis trajectories, and being treated for individual symptoms (e.g. pain) rather than jhs : we ve all been passed from pillar to post where people do nt recognise it or they just attribute a pain to something else, when a snap kind of diagnosis just comes out of the air and you know, you progress from there (female g, age 45, fg3). often, obtaining a correct diagnosis was a coincidental occurrence : it was only because a locum happened to be in the day i went in because my gp was off sick, and he just started saying, well, to start bending everything but if had nt been for him i would nt have been put on the right track, because otherwise what other route do you really have if it s not through your gp ? (female b, age 27, fg3). receiving a diagnosis was considered necessary in order to access appropriate care pathways, for example, referral for physiotherapy for jhs rather than for an acute single joint problem : i was originally seen by a physio who had nt diagnosed with the hypermobility and then went back to a musculo - skeletal specialist who then put me forward to specialist hypermobility physiotherapist and since then it s been amazing ; i feel like it s been worthwhile and it felt like the right thing to do and i ve been really enjoying it (female b, age 27, fg3). in addition, a diagnosis helped to validate participants experiences and was psychologically helpful : getting a diagnosis on paper, this is what s wrong with me ? i mean that helped me hugely psychologically (female a, age 60, fg2). a diagnosis in itself, however, still did not guarantee beneficial treatment : when i was first diagnosed, i was nt, i really felt i was nt given that much information about the condition [] and it just seemed to be all exercises they d given me at the time seemed to make me worse (female b, age 27, fg3). similarly, a diagnosis did not necessarily lead to greater understanding and support from others in the participants social networks : there are people who do nt feel it s a genuine diagnosis, that it s something psychological, that you just need to be a bit braver (female a, age 21, fg2). education for health professionals was a key issue for participants, to facilitate timely diagnosis of, and referral for, jhs. thus, education for health professionals was a prerequisite for diagnosis, and diagnosis was a prerequisite for participants to access education. participants recognized that individuals with jhs also need education, in order to find ways to self - manage their condition and to understand and engage with prescribed treatments. i suppose it s where someone who does nt really know about it, they ve got to learn about it first because you ca nt tell someone to do it [ie engage with a particular treatment ] if they do nt understand it (female d, age 21, fg1). participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [. ] there s lots of things i still need to know about hypermobility, but on the flip side i do think it s the health professionals that need to know more (female g, age 42, fg3). participants described learning and understanding about jhs as a two - way process : so i think i get a huge amount of enlightenment from her [the physiotherapist ], and i lend her books and she lends me books about hypermobility and all that helps (female d, age 54, fg2). participants accepted that treatment should aim to manage symptoms rather than provide a cure : it s all about them helping you to manage your pain, rather than cure it (female, age 44, fg1). participants recognized the importance of self - care activities such as appropriate exercise, once a diagnosis had been received and a clear understanding of jhs had been developed. participants also recognised that activity pacing is really key to managing jhs symptoms (female b, aged 32) : i have this balancing act, if i do too much it all hurts, do nt do enough, it all hurts, do it just right, i m okay (male a, age 50, fg1). participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises (female e, age 44, fg1). it s got to be holistic, it really has to be (female d, age 21, fg1). participants also felt that education about jhs should be provided at an early age, and therefore, early diagnosis was necessary : i m only 19 now but if you d have said that when i was 16, i might not be in as much pain as i am now [] so if like if they d have told me more about how to treat it back then it probably would nt be as bad as it is now (female f, age 19, fg3). education for health professionals was a key issue for participants, to facilitate timely diagnosis of, and referral for, jhs. thus, education for health professionals was a prerequisite for diagnosis, and diagnosis was a prerequisite for participants to access education. participants recognized that individuals with jhs also need education, in order to find ways to self - manage their condition and to understand and engage with prescribed treatments. i suppose it s where someone who does nt really know about it, they ve got to learn about it first because you ca nt tell someone to do it [ie engage with a particular treatment ] if they do nt understand it (female d, age 21, fg1). participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [. ] there s lots of things i still need to know about hypermobility, but on the flip side i do think it s the health professionals that need to know more (female g, age 42, fg3). participants described learning and understanding about jhs as a two - way process : so i think i get a huge amount of enlightenment from her [the physiotherapist ], and i lend her books and she lends me books about hypermobility and all that helps (female d, age 54, fg2). participants accepted that treatment should aim to manage symptoms rather than provide a cure : it s all about them helping you to manage your pain, rather than cure it (female, age 44, fg1). participants recognized the importance of self - care activities such as appropriate exercise, once a diagnosis had been received and a clear understanding of jhs had been developed. participants also recognised that activity pacing is really key to managing jhs symptoms (female b, aged 32) : i have this balancing act, if i do too much it all hurts, do nt do enough, it all hurts, do it just right, i m okay (male a, age 50, fg1). participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises (female e, age 44, fg1). it s got to be holistic, it really has to be (female d, age 21, fg1). participants also felt that education about jhs should be provided at an early age, and therefore, early diagnosis was necessary : i m only 19 now but if you d have said that when i was 16, i might not be in as much pain as i am now [] so if like if they d have told me more about how to treat it back then it probably would nt be as bad as it is now (female f, age 19, fg3). pain, fatigue and repeated episodes of injury were particularly challenging features of the wide - ranging symptoms of jhs. bury (16) describes how chronic illness brings about biographical disruption, and that the illness or disease requires the individual to make certain changes to their lives (17,18). in our study, participants lives were disrupted repeatedly by fluctuating symptoms and cycles of injury and recovery. participants described the complex and individual nature of their pain experience and their responses to unpredictable symptoms. for some, previous episodes of pain or injury led to heightened levels of anxiety and catastrophizing about the future and their symptoms required them to modify or restrict their activities, while others refused to be dictated to by their symptoms. patients perceived jhs to be often poorly understood by health professionals and those in their wider social environment and reported feeling fraudulent and blamed for their symptoms. participants felt stigmatized, marked out as different (19) and alien to others in society and found it exhausting to try to appear normal. stigma can have wide - ranging negative biopsychosocial consequences including reduced participation in activities and an exacerbation of disability and disease, for example, through delayed diagnosis and treatment (20). a lack of awareness of jhs amongst health professionals meant that obtaining a diagnosis of jhs was often difficult. participants had often been misdiagnosed or treated for symptoms (e.g. pain) rather than the condition itself and passed from pillar to post often until a serendipitous or coincidental diagnosis of jhs was made. previous studies have reported similar findings (9) and others have emphasized that being understood and believed by health professionals and significant others, along with social support, is instrumental in long - term pain management by facilitating (or inhibiting) pain acceptance (21,22). participants highlighted the importance of a correct diagnosis in facilitating access to appropriate health care, support and education and helping to validate participants experiences. having lived with problematic symptoms of jhs sometimes for long periods of time, it is possible that the receipt of a diagnosis represented the beginning of a process during which they were able to understand and make sense of their symptoms, obtain appropriate treatment and subsequently find ways to self - manage the condition. participants experiences resonate with williams (23) who uses the term narrative re - construction to describe how individuals with chronic illness re - establish order and meaning in their lives. primary - care practitioners play an important role in helping patients to understand and self - manage long - term health conditions. a prerequisite of being able to provide support for patients is that primary - care practitioners are able to recognize and diagnose jhs and to refer patients to jhs - trained specialists. without a correct diagnosis, unsuitable treatments or information currently, primary - care practitioners and other health professionals such as physiotherapists do not routinely receive training related to jhs (9) and the validity of diagnostic criteria (such as the beighton score) has recently been questioned (25). education and access to information was important for participants to allow them to make informed health - care choices. participants described the value of collaborating with health professionals and reciprocal learning between practitioners and patients in order to develop effective self - care strategies, and for holistic long - term management. recognizing that jhs would not be cured, participants felt that health professionals and those with jhs could potentially develop a deeper understanding of jhs management by learning from each other as a teachable dyad (26, p. 682). to ensure validity, participants were recruited from four different geographical locations in the uk and had the experience of different health - care services. participants provided rich personal narratives of the day - to - day experiences of living with and managing jhs from patients perspectives and data analysis demonstrated general consensus and shared experiences. a limitation of this research is that participants were recruited through jhs support groups or via physiotherapy services for jhs and may therefore have been more active or aware of their condition than, for example, newly diagnosed individuals. the focus groups formed one phase of a study to develop a physiotherapy intervention to manage jhs. it was clear that the lived experience of those with jhs that emerged from the data analysis was an important story, which to date has received little attention. the issues raised in these focus groups highlight the need for more in - depth research in this area. future research could conduct interviews to provide a more detailed investigation of personal accounts of living with jhs, in particular those newly diagnosed with jhs. individuals with jhs experience diverse, fluctuating and often debilitating symptoms and diagnosis is often slow. without a correct diagnosis, following diagnosis, access to jhs - trained health professionals could help patients to effectively manage their condition over a life course and receive psychological support when needed. patients and practitioners may be able to learn from one another and so assist in developing a deeper understanding of a currently poorly understood condition. to ensure validity, participants were recruited from four different geographical locations in the uk and had the experience of different health - care services. participants provided rich personal narratives of the day - to - day experiences of living with and managing jhs from patients perspectives and data analysis demonstrated general consensus and shared experiences. a limitation of this research is that participants were recruited through jhs support groups or via physiotherapy services for jhs and may therefore have been more active or the focus groups formed one phase of a study to develop a physiotherapy intervention to manage jhs. it was clear that the lived experience of those with jhs that emerged from the data analysis was an important story, which to date has received little attention. the issues raised in these focus groups highlight the need for more in - depth research in this area. future research could conduct interviews to provide a more detailed investigation of personal accounts of living with jhs, in particular those newly diagnosed with jhs. individuals with jhs experience diverse, fluctuating and often debilitating symptoms and diagnosis is often slow. without a correct diagnosis, following diagnosis, access to jhs - trained health professionals could help patients to effectively manage their condition over a life course and receive psychological support when needed. patients and practitioners may be able to learn from one another and so assist in developing a deeper understanding of a currently poorly understood condition. funding : this project was funded by the national institute for health research health technology assessment programme (project number 10/98/05). the views and opinions expressed therein are those of the authors and do not necessarily reflect those of the health technology assessment programme, nihr, nhs or the department of health. ethical approval : this study received ethical approval from the north east nhs research ethics committee (12/ne/0307).	background.musculoskeletal problems are common reasons for seeking primary health care. it has been suggested that many people with everyday non - inflammatory musculoskeletal problems may have undiagnosed joint hypermobility syndrome (jhs), a complex multi - systemic condition. jhs is characterized by joint laxity, pain, fatigue and a wide range of other symptoms. physiotherapy is usually the preferred treatment option for jhs, although diagnosis can be difficult. the lived experience of those with jhs requires investigation.objective.the aim of the study was to examine patients lived experience of jhs, their views and experiences of jhs diagnosis and management.methods.focus groups in four locations in the uk were convened, involving 25 participants with a prior diagnosis of jhs. the focus groups were audio recorded, fully transcribed and analysed using the constant comparative method to inductively derive a thematic account of the data.results.pain, fatigue, proprioception difficulties and repeated cycles of injury were among the most challenging features of living with jhs. participants perceived a lack of awareness of jhs from health professionals and more widely in society and described how diagnosis and access to appropriate health - care services was often slow and convoluted. education for patients and health professionals was considered to be essential.conclusions.timely diagnosis, raising awareness and access to health professionals who understand jhs may be particularly instrumental in helping to ameliorate symptoms and help patients to self - manage their condition. physiotherapists and other health professionals should receive training to provide biopsychosocial support for people with this condition.
poorly differentiated cancers usually behave more aggressively and are associated with worse survival rates than well - differentiated cancers. pdtc lies on the spectrum between well - differentiated and anaplastic thyroid carcinoma, it accounts for only 47% of thyroid cancers worldwide and is frequently advanced or metastatic at the time of diagnosis. with less differentiation, the expression of the sodium iodide symporter is lost and therefore, the utility of radioiodine as a therapeutic option is reduced as the tumour becomes iodide non - avid. the evidence for external beam radiotherapy is less robust and standard chemotherapy agents are not useful. new targeted therapies are needed for patients with pdtc, as these patients typically develop advanced iodine refractory disease. ret mutations in pdtc and undifferentiated thyroid and lung cancers are rare [2, 3 ]. sorafenib is a multikinase inhibitor targeting ras, braf / mek / erk signaling pathways, ligand - independent ret / ptc receptor tyrosine kinase activation, vegf and platelet - derived growth factor (pdgf) pathways. phase 3 data from the decision trial, recently presented at asco and published in lancet, has led to sorafenib becoming the standard 1st line medication for the treatment of iodine refractory thyroid cancer. this trial compared sorafenib versus placebo in iodine refractory thyroid cancer and the results demonstrated a progression free survival (pfs) advantage of 5 months in the sorafenib group (10.8 months in the sorafenib cohort vs. 5.8 months in the placebo group). combrestatin a-4 phosphate, also known as fosbretabulin (ca4p), is a vascular disrupting agent that acts by binding to the beta - subunit of tubulin. this trial was a prospective randomised controlled phase 2/3 trial assessing the safety and the efficacy of carboplatin / paclitaxel with ca4p versus without ca4p. epidermal growth factor (egfr) mutations as therapeutic targets are well - established in the treatment of metastatic lung adenocarcinoma. erlotinib was licensed in 2011 for the 1st line treatment in patients with metastatic lung adenocarcinoma who harbour egfr mutations. the battle trial, a phase 3 trial exploring the use of sorafenib in the 3rd line setting for nsclc patients, did not demonstrate any improvement in overall survival and has therefore not been published. however, subgroup analysis presented at esmo in 2013 suggested that patients with egfr mutations might benefit from sorafenib. the incidence of egfr mutations in thyroid carcinoma was previously thought to be low. however, more recently, it has been suggested that egfr mutations may be in the region of 30%. a retrospective examination of thyroid tissue from a series of 23 patients with papillary thyroid carcinoma was striking : 7 were found to harbour drug - sensitising mutations and 1 patient had egfr amplification. this suggests that egfr mutations may occur within a certain subset of thyroid carcinoma patients, just as egfr mutations in lung carcinoma occur within subsets of patients (adenocarcinoma, never - smokers, young, female, asian). additionally, intratumoral heterogeneity may influence the frequency that these mutations are detected and may explain why initial studies suggested that egfr mutations were rare [7, 8 ]. phase 2 trials evaluating the use of gefitinib in patients with advanced thyroid cancer did not select patients based on egfr mutations [9, 10 ]. however, although these phase 2 trials did not show positive results, 1 patient within a trial responded for more than 12 months [9, 10 ]. a literature search has revealed fewer than 6 cases in the literature of patients with egfr - mutated thyroid carcinoma, or lung cancer with thyroid metastases, who have been treated with egfr tkis (erlotinib or gefitinib). all of these reported cases responded to egfr tki treatment [7, 9, 11, 12, 13, 14 ]. presented the case of a patient with an oncocytic, focally anaplastic thyroid cancer treated with a therapeutic trial of erlotinib. interestingly, in lung adenocarcinoma, exon 18 and 20 mutations confer relative resistance to tki therapy. here, we present the case of a 79-year - old male with metastatic pdtc with an egfr mutation who was treated with the tki erlotinib. our patient had a good partial response to treatment with a pfs of 11 months. we suggest that targeted therapy with an egfr tki is a possible targeted treatment pathway for patients with metastatic thyroid carcinoma who harbour an egfr mutation. biopsy of this lesion showed metastatic ttf1-positive adenocarcinoma of either lung or thyroid origin, therefore the patient was referred to the lung cancer team at the royal marsden hospital. he was a previous light smoker with a 3-pack - per - year history in total. an fdg - pet ct scan showed a strongly fdg - positive (suv 16.57) lesion in the right inferior pubic ramus, consistent with a metastatic deposit. there was high - grade irregular increased fdg uptake involving both lobes of the thyroid gland (suv max 16.95). there were multiple fdg positive lymph nodes involving the right supraclavicular, right thoracic inlet, paratracheal, subcarinal, right hilar, anterior mediastinal and posterior mediastinal regions in keeping with nodal metastases. there was some small pleural - based nodularity within the apical segment of the right lower lobe, but no discrete pulmonary lesions were identified. overall, these pet ct appearances were highly suggestive of stage iv metastatic thyroid cancer (bilateral) with extensive nodal and solitary bone metastases. the patient was initially treated with zolendronic acid and palliative radiotherapy (20 gray in 5 fractions) to the symptomatic metastasis in the right pubic ramus. the royal marsden hospital histopathology review of the pubic ramus biopsy showed adenocarcinoma positive for ttf1 and ck7, and negative for ck20 and psa. mutational analysis revealed 2 mutations within the egfr gene : c.2573t > g ; p.l858r and egfr exon 20 insertion. the patient 's low thyroglobulin value despite the large - volume disease may reflect the patient 's poorly differentiated disease that no longer expresses thyroglobulin. this would fit with the recognised flip - flop phenomenon : as dtc becomes pdtc, the fdg uptake on pet imaging increases, whilst the radioiodine uptake decreases. the proposed management was total thyroidectomy followed by radioiodine (rai) therapy in the first instance. prior to the planned thyroidectomy, the patient was found to have a dvt and pes and was commenced on therapeutic low - molecular - weight heparin. thyroidectomy was postponed whilst anticoagulation was established. in the interim, in view of the known egfr mutation, the patient was commenced on systemic treatment with the egfr tki erlotinib at a dose of 150 mg once a day. six weeks later, he underwent total thryroidectomy with right level 4/5 (supraclavicular) and left level 3 neck dissection. final histopathology demonstrated a poorly differentiated carcinoma with sarcomatoid areas, staged as pt4a pn1b cm1 (fig. mutational analysis of the thyroid specimen showed an egfr mutation c.2573t > g ; p.leu858arg (p.l858r) (a sensitising mutation) and did not demonstrate an egfr exon 20 insertion or mutation. post - operatively, the patient continued on 150 mg of erlotinib once a day. a repeat fdg pet scan performed 9 months after initial diagnosis demonstrated an ongoing good partial response (fig. an iodine uptake scan performed 11 months after diagnosis showed ongoing pfs, with no iodine-131 uptake in the mediastinal nodal or bony disease. the patient has not been treated with rai in view of the response to erlotinib, the tolerability of the full dose (150 mg once a day), the minimal side effects from erlotinib and the patient 's good quality of life. after 12 months of erlotinib therapy, pet imaging suggests that the patient 's disease is beginning to progress. we have presented the case of a 79-year - old male with metastatic pdtc with an egfr mutation who responded to treatment with the egfr tki erlotinib. the clinical decision not to treat him with rai was based upon the evidence that there was high fdg uptake on pet, low serum thyroglobulin and a negative diagnostic iodine scan. taken together, this suggested that this patient 's disease would be less likely to respond to rai. additionally, the patient was already responding to erlotinib at that stage, and had minimal side effects from treatment. our patients bone metastasis contained 2 egfr mutations : c.2573t > g ; p.leu858arg (p.l858r) and p.exon 20 insertion. interestingly, mutational analysis on the thyroid specimen post - thyroidectomy revealed only a single egfr mutation : c.2573t > g ; p.leu858arg (p.l858r). the literature suggests that exon 20 mutations may confer resistance to egfr tkis, and the discrepancy between these 2 sites, plus the fact that the patient clinically responded to erlotinib, supports intratumoral heterogeneity. our patient 's pfs of 11 months on erlotinib was comparable to the decision data showing a pfs of 10.8 months on sorafenib. on further review of comparable literature, we found that there are only 6 similar clinical cases reported : 5 of these are cases of patients with disease in both the lung and the thyroid, and 1 of these is thyroid - only disease [7, 9, 11, 12, 13, 14 ]. different authors drew different conclusions regarding whether the primary is thyroid or lung ; this will be discussed in detail below. some of the authors concluded that distant metastases were lung primary with thyroid metastases (instead of thyroid primary with lung metastases) based on the presence of egfr mutations [11, 13 ]. their patient was originally thought to have lung adenocarcinoma, but was subsequently found to have advanced papillary thyroid carcinoma. this patient had an activating egfr mutation (exon 19 mutation) and responded to the egfr inhibitor gefitinib. another case presented by the same group at asco in 2011 demonstrated a similar clinical picture : a patient with metastatic anaplastic thyroid carcinoma with an exon 21 mutation who responded to first line erlotinib and survived for more than 6 months. interestingly, this patient 's egfr mutation was present at the metastatic sites (transbronchial and cervical lymph node biopsies), but not in the thyroid primary. this further supports the case for intratumoural heterogeneity, with mutations occurring at the metastatic sites, but not the primary. a similar picture of mutational heterogeneity was found in our patient. in 2011, albany. presented the case of a patient who presented with synchronous lung and thyroid masses, the thyroid biopsy was ttf1 and ck7 positive, and the authors state that the lung biopsy revealed adenocarcinoma of the lung. the authors felt that the thyroid biopsy could not distinguish whether the thyroid mass was a lung metastasis or a thyroid primary. however, on the basis of the presence of an egfr exon 21 mutation in the lung biopsy, albany. concluded that this must be a lung primary with a thyroid metastasis. the patient was treated with first line erlotinib and achieved a response which lasted for more than 8 months. this case may therefore possibly represent a case of metastatic thyroid carcinoma harbouring an egfr mutation responding to erlotinib. describe a 56-year - old female previously radically treated for lung cancer who, a few months later, was found to have a distant lung metastasis in addition to a papillary thyroid carcinoma (detected synchronously). egfr mutational analysis was performed on the lung biopsy obtained from the new lung metastasis, and the patient was found to have an exon 19 mutation. the patient was treated systemically with gefitinib and demonstrated a complete response in both the lung and the thyroid. the authors do not state whether egfr mutational analysis was performed on the thyroid again describe a 60-year - old female with adenocarcinoma in both the lung and the thyroid. within the thyroid one area was found to harbour an egfr exon 21 mutation whilst the other area was not. the group concluded that a lung adenocarcinoma had metastasised to the thyroid and the patient was treated with a tki targeting egfr. this case bears similarity to the cases presented by matsumoto. and albany. [11, 12 ]. here we have presented the case of a caucasian male with egfr - mutated metastatic thyroid carcinoma, treated with 150 mg of erlotinib. he had a sustained response for 11 months, experienced minimal side effects from treatment, and maintained an excellent quality of life. in summary, our case, taken together with the available literature, suggests that egfr mutations can and do occur in thyroid carcinomas. in some of the examined case reports discussed here, some authors concluded that patients had primary lung carcinoma with thyroid metastases based on the presence of egfr mutations. we suggest that some of these cases may represent primary thyroid carcinoma with metastatic sites harbouring egfr mutations. in view of intratumoural heterogeneity, it may be beneficial to perform mutational analysis using biopsies from both primary and metastatic sites. in the era of targeted therapy, targeting the egfr receptor in metastatic thyroid carcinoma represents a change in the way this cancer can be treated. we suggest that mutational analysis is performed at diagnosis for patients with poorly differentiated carcinomas. we conclude that treatment with an egfr tki in egfr - mutant metastatic thyroid carcinoma may be a valuable treatment pathway.	poorly differentiated cancers are a diagnostic and therapeutic challenge in oncology. new therapies are needed for patients with poorly differentiated thyroid carcinoma (pdtc) or anaplastic thyroid cancer, as these patients often present with advanced disease and effective systemic treatment options are currently limited. epidermal growth factor (egfr) mutations may occur in pdtc more often than previously thought. however, there are fewer than 6 cases reported in the literature where egfr tyrosine kinase inhibitors (tkis) (such as erlotinib or gefitinib) were used to target egfr mutations in pdtc. here, we present the case of a 79-year - old male with metastatic pdtc with an egfr mutation who responded to treatment with the selective egfr tki erlotinib, with a progression - free survival of more than 11 months. a lung primary rather than a thyroid primary was initially detected. we suggest that the egfr status should be analysed at diagnosis in any patient with a poorly differentiated tumour. the presence of an egfr mutation may provide an effective therapeutic pathway for these patients. this pathway requires further investigation and consideration in the future.
similar to other countries, the age structure in brazil is changing, with an increase of the elderly population. currently, life expectancy at birth is 74.6 years,1 compared with 66 years in 1991,2 which is a red flag to health care professionals, who must be prepared to satisfactorily attend to this population. in the elderly population this is an age - related degenerative change,3 which causes sensorineural, bilateral, symmetrical, and progressive hearing loss.4 5 it is a common misfortune among the elderly, and it starts approximately between the third and fifth decade of life ; because it is more pronounced in the higher frequencies, it causes difficulties to understand speech, especially in the concomitant presence of noise and speech.6 7 these difficulties may be accentuated when there is impairment of auditory information processing or when cognitive disorders occur.8 on average, 1 db of hearing threshold is lost per year in people aged over 60 years.9 despite the extensive knowledge of presbycusis, there is no clinical method to predict its appearance in advance. there are indications that some factors, such as age, sex, race, genetic and environmental factors, and comorbidities (hypertension, diabetes, etc.) can influence the presence and degree of hearing loss.10 in addition to problems directly related to communication, hearing loss in the elderly can contribute to problems such as depression, isolation, and possible dementia11 12 13 ; in middle - aged adults, it is one more change in the midst of a phase in which subjects already face several biopsychosocial changes.14 treatment for presbycusis includes use of hearing aids, but despite the high prevalence of presbycusis in the elderly population, the use of prosthesis is not yet significant, regardless of the country surveyed. a u.s. there was an association with the degree of loss, education, income, and social activities.15 another north american study showed that 76.4% of the surveyed elderly saw a doctor because of hearing disorders, but only 34% started using hearing aids ; in contrast, 98.6% of elderly people with visual impairment sought medical advice and 93.0% of them wore glasses.16 after 5 years of monitoring, researchers of the blue mountains hearing study found that 8.1% of the subjects aged 55 to 99 used hearing aids.17 in a study on health problems of the elderly, the authors found that 63.1% of the subjects in the sample self - reported hearing loss. although prevalence was high, the use of hearing aids was reported by only 1.47% of subjects.18 in a research study conducted with 7,315 elderly people in 59 cities in the brazilian state of rio grande do sul, 4,300 (58.78%) of the subjects considered their hearing to be fair, poor, or very poor. nevertheless, only 3.8% of all assessed patients used hearing aids.19 these statistics reinforce the idea that, although age - related hearing problems are well known, adherence to treatment should be further researched. therefore, it is relevant to understand whether self - assessment of hearing could be a predictor for purchase of a hearing aid. a u.s. study questioned patients to see if they were able to proceed to amplification through their self - assessment of hearing.20 there was a relationship between patients ' opinions about their hearing and their decision to use a hearing aid. thus, disadvantages of hearing loss in the elderly and the stages of treatment are already known. there is a need, however, to understand the determinants for the purchase of hearing aids, considering the data presented. considering that there are few studies in the literature on predictors for the purchase of a hearing aid, especially by the brazilian population, the aim of this study was to determine whether self - assessment of hearing is a predictor for purchase of hearing aids. this study was approved by the research committee and by the research ethics committee of the institute (protocol 24401). the sample consisted of middle - aged and elderly adults, interested in having hearing aid tests, seen at a private hearing center located in the city of porto alegre (rio grande do sul, brazil). inclusion criteria were diagnosis of hearing loss, medical prescription of hearing aid use, signing of the informed consent form, and collaboration with the study procedures. the study excluded elderly people who refused to participate voluntarily, who did not follow any of the study procedures, or who had a history of cognitive, psychiatric, or neurologic impairment (checked during the diagnostic interview). patients who met the inclusion criteria continued in the study. during the diagnostic interview, patients answered the following question : on a scale of 1 to 10, with 1 being the worst and 10 the best, how would you rate your overall hearing ability?20 the subsequent steps were pure tone audiometry, selection and home trial of hearing aids, and decision to purchase the hearing aid or not. within 2 to 3 weeks after the diagnostic interview, a query to the auditory center database was made to verify whether or not the patient had purchased the prosthesis. for analysis of audiometric data, hearing loss were classified as conductive, mixed, or sensorineural.21 for measuring degree of loss, the classification by the world health organization was used,22 considering the average of the hearing thresholds at 500, 1,000, 2,000, and 4,000 hz. under this classification, average values between 10 and 25 dbhl indicate normal hearing ; between 26d and 40 dbhl, mild hearing loss ; between 41 and 60 dbhl, moderate hearing loss ; between 61 and 80 dbhl, severe hearing loss ; and values higher than 81 dbhl, profound hearing loss.22 initially, the values obtained in the collection of data were analyzed using descriptive statistics, through absolute (n) and relative (%) distribution, and the mean, standard deviation, and median were analyzed with the study of symmetry by the shapiro - wilk test. in the comparison of continuous variables between the purchase and nonpurchase of prosthesis, the mann - whitney test was used. when the comparison was made with categorical variables, analyses were performed using software spss (statistical package for social sciences, ibm, usa) version 2.0. for statistical decision criteria, the results shown refer to a sample of 32 subjects, whose characteristics and audiological results are shown in tables 1 and 2, respectively. mean age was 71.41 12.14 years ; there was a higher number of women (59.4%) and of new users of hearing aids (68.75%). most of the assessed patients had bilateral, sensorineural, and moderate hearing loss in both ears. considering the scores awarded by the subjects in their self - assessment of hearing and the number of subjects in the sample, the values were grouped for carrying out the analysis, similar to what was done by palmer.20 groups were sorted as follows : values of up to 5 points, between 6 and 7 points, and between 8 and 10 points. the results showed that 64.7% of the individuals who scored their hearing with values between 2 and 5 purchased the hearing aid, and 76.09% of those who scored their hearing between 6 and 7 purchased the hearing aid. purchase of the hearing aid was 50% for those who self - assessed their hearing with scores between 8 and 9. still, there was no association between self - assessment of hearing and purchase of hearing aids (p = 0.263 ; table 3). percentages obtained based on the total number of cases in each category of the acquisition of prosthesis. because the analysis showed no association between the categories we created, we decided to analyze the data continuously. after comparing the scores of the question with the purchase of the hearing aids (table 4), again there was no statistically significant difference (p = 0.688), which is indicative that the variations between the elderly subjects who purchased the hearing aids (5.3 1.6) and those who did not purchase it (5.0 1.8) can not be associated with purchase of hearing aid in the study sample. data analysis showed that the mean age of the individuals was 71.41 12.14 years. considering that age - related hearing loss begins in the third decade of life and is progressive,4 5 7 it is likely that subjects in the sample had already been experiencing hearing difficulties for some time. among the participants of the beaver dam eye study, 20% of people who were monitored for 10 years purchased hearing aids 5 to 10 years after detection of hearing loss.23 the present research found that the majority of participants were new users, but 9 subjects (28.13%) had been using prosthesis prior to joining the study ; a significant portion of the sample had already become aware of the effects of hearing loss. a higher number of women (59.4%) was also found, although the literature asserts that presbycusis is more pronounced in men,24 which is believed to be due to the fact that men look for health care services less often than women.25 analysis of audiological data showed that the average of hearing thresholds was 51.72 dbhl in the right ear and 51.72 dbhl in the left ear, which characterizes moderate hearing loss. the most prevalent type of hearing loss was sensorineural, as expected by the researchers of the present study. the type and degree of hearing loss observed are typical of age - related hearing loss.26 27 bilateral hearing loss was prevalent in 30 subjects (93.74%) ; one subject had unilateral loss in the right ear and another in the left ear. this also was expected, as it is one of the characteristics of presbycusis.4 5 although there was no significant association between self - assessment and purchase of hearing aid (p = 0.263), most purchases were made by subjects whose self - assessments scored 2 to 7 points. of the 32 subjects of the sample, 30 scored between 2 and 7, and 21 of them (70%) purchased hearing aids. only 2 subjects scored between 8 and 9 ; one of them purchased the hearing aid and the other one did not. subjects whose self - assessment score range between 2 and 7 are 2.3 times more likely to purchase a hearing aid than otherwise. this finding corroborates previous findings in which self - perception of hearing loss increases more than three times the rate of purchase of a hearing aid.23 in a study that gave rise to the research question, it was found that 0% of those who scored between 9 and 10 continued to amplification compared with 18% of those who scored 8 ; 50% continued to amplification for scores between 6 and 7 ; and 78 continued to amplification to 82% for scores between 3 and 5, reinforcing the idea that self - assessment can be an important predictor.20 a longitudinal study found that in a period of 5 years, 9.5% of people who had rated their hearing as good purchased a hearing aid, and 36% of those who had rated it as bad purchased the aid. between 5 and 10 years, 41% of those who reported hearing difficulties purchased hearing aids, and 13% did not report that, but purchased it anyway.23 these data also confirm that one 's opinion about their own hearing is a decisive factor for purchase of hearing aids. table 4 show the respondents ' scores for the question on a scale of 1 to 10, with 1 being the worst and 10 the best, how would you rate your overall hearing ability ? none of the subjects rated their hearing at the worst and best scores of the self - assessment scale (1 and 10) ; furthermore, the median was 5 for both subjects who purchased and those who did not purchase a hearing aid. the mean values for those who purchased and those who did not purchase a hearing aid were 5.3 and 5 points, respectively, thus showing no significant differences (p = 0.68). other authors also found the score 5 as median in the portion of the sample for the reliability test of the question on the self - assessment of hearing, as well as in the research sample. in addition, in the sample that was studied separately to ascertain the reliability of the study, no extreme values (1 and 10) were found, either.20 subjects ' decision not to purchase hearing aids is likely to be influenced by self - assessment. other factors, however, can not be neglected and should be evaluated by speech therapists working in the field. in a research study with elderly subjects on the use of hearing aids for improving quality of life, aspects such finance, independence, and health were considered for assessment of quality of life by both users and nonusers of hearing aids. only one of the six respondents in the elderly group of nonusers of hearing aids rated their quality of life as regular because of hearing difficulty. the author of the study pointed out that the elderly rated their quality of life while considering their experiences, and information is required for construction of such experiences. elderly people usually access information through the radio, television, and the newspaper, which are media that overtly link quality of life to food, physical exercise, and social life. the role of hearing for quality of life is explicit in the literature, but it appears to be inaccessible to most people.28 thus, the impact of hearing loss is not always linked to quality of life because there is no information on the implications of the former. older people may feel that they do not need a hearing aid because they lack information on its benefits and fail to understand the auditory treatment for presbycusis and the related improvements. they may even deny that they have a hearing loss.29 corroborating these data, another study found that only 20% of individuals seek a hearing center on their own initiative, and the remaining 80% are equally divided into those who seek help upon medical advice or family counseling. after performing the prosthesis test, 75% of subjects purchased hearing aids.30 thus, again it appears that middle - aged and elderly adults may be unaware of the effects of hearing loss and may not realize the need to use a hearing aid, but after they undergo the necessary tests, they experience the benefit of the hearing aid and are advised to purchase the device. other reasons elderly people do not purchase a hearing aid were found by researchers of the blue mountains hearing study. they found that the third main reason for the elderly not to purchase a hearing aid is that they do not feel they need one (9%) ; the first and the second reasons were, respectively, no prescription for use (8%) and high cost of hearing aids (1.7%). in addition, they found that 1 out of 10 elderly adults with hearing loss had purchased hearing aids. they pointed out, however, that not all elderly subjects actually used the aids. the predictors for purchase were the presence of hearing loss, self - report of this impairment, and restricted social life.17 many people who would benefit from audiological evaluation and a hearing aid are unwilling to do so. for decades, the image of the deaf old lady was widespread by the media as one of the marks of old age. current social and cultural venues for elderly people are places and situations that favor the young elderly, dynamic retirees, that is, places and situations where there is a denial of the concept of aging : physical, mental, and social decadence. among this image of the elderly, being identified as deaf is to be considered as old. denial of deafness and denial of aging are commonly observed together in this population.31 the new concept of aging positively affects the elderly in society. however, those who fail to meet this ideal goal of active elderly feel even more stigmatized. most elderly people seek to conceal their communication difficulty and an alternative to fight the stigma of being old is to hide the hearing loss by means of strategies. however, these attempts result in poor communication and isolation.31 thus, rating their hearing with low scores in self - assessments of hearing means recognizing that hearing loss interferes extensively in life and that something has to be done (i.e., a hearing aid has to be used). finally, the survey showed that there was no association between self - assessment of hearing and purchase of hearing aids. it is believed, however, that financial and aesthetic factors, stigma of hearing loss associated with aging, patient 's knowledge on the subject, among other factors, have a significant impact on the final decision to purchase a hearing aid. the present study showed that although there is evidence that low scores of self - assessment lead to the purchase of hearing aids, this association was not confirmed in this sample.	introduction presbycusis is a consequence of aging. prescription of hearing aids is part of the treatment, although the prevalence of use by elderly people is still small. objective to verify whether or not self - assessment of hearing is a predictor for purchase of hearing aids. methods quantitative, cross - sectional, descriptive, and observational study. participants were subjects who sought a private hearing center for selection of hearing aids. during the diagnostic interview, subjects answered the following question : on a scale of 1 to 10, with 1 being the worst and 10 the best, how would you rate your overall hearing ability ? after that, subjects underwent audiometry, selected a hearing aid, performed a home trial, and decided whether or not to purchase the hearing aid. the variables were associated and analyzed statistically. results the sample was comprised of 32 subjects, both men and women, with a higher number of women. mean age was 71.41 12.14 years. self - assessment of hearing ranged from 2 to 9 points. overall, 71.9% of the subjects purchased hearing aids. there was no association between scores in the self - assessment and the purchase of hearing aids (p = 0.263). among those who scored between 2 and 5 points, 64.7% purchased the device ; between 6 and 7 points, 76.09% purchased the device ; and between 8 and 9 points, 50% purchased the device, respectively. conclusion there is evidence that low self - assessment scores lead to the purchase of hearing aids, although no significant association was observed in the sample.
prepare two stock solutions of tyrode 's solution (16x) in advance in deionized water and store them at 4c : stock i (119.872 g / l nacl, 3.056 g / l cacl2 (2h2o), 5.6 g / l kcl, 2.6274 g / l nah2po4, 3.408 g / l mgcl2 (6h2o), (fisher scientific, fair lawn, nj));stock ii (26.88 g / l nahco3, (fisher scientific, fair lawn, nj)). stock i (119.872 g / l nacl, 3.056 g / l cacl2 (2h2o), 5.6 g / l kcl, 2.6274 g / l nah2po4, 3.408 g / l mgcl2 (6h2o), (fisher scientific, fair lawn, nj)) ; stock ii (26.88 g / l nahco3, (fisher scientific, fair lawn, nj)). prepare stock solutions of fluorescent dyes. to avoid repeated freezing and thawing, we store 30 l aliquots of both dyes at -20c, which is sufficient for one experiment : voltage - sensitive dye rh237 (invitrogen, carlsbad, ca) stock solution, 1.25 mg / ml solution in dimethyl sulfoxide (dmso, sigma, st. louis, mo);calcium indicator rhod-2am (invitrogen, carlsbad, ca) stock solution, 1 mg / ml solution in dmso. voltage - sensitive dye rh237 (invitrogen, carlsbad, ca) stock solution, 1.25 mg / ml solution in dimethyl sulfoxide (dmso, sigma, st. louis, mo) ; calcium indicator rhod-2am (invitrogen, carlsbad, ca) stock solution, 1 mg / ml solution in dmso. louis, mo, 2 mg / ml solution in dmso) in advance and store the dissolved blebbistatin at 4c. freshly prepare 2l tyrode 's solution (128.2 mm nacl, 1.3 mm cacl2 (2h2o), 4.7 mm kcl, 1.05 mm mgcl2 (6h2o), 1.19 mm nah2po4, 20 mm nahco3, 11.1 mm d - glucose in deionized water, ph=7.350.05). if stock solution is being used to make 2l of tyrode 's solution (sufficient for one experiment) take 1750 ml of deionized water and mix in 125 ml of stock i, 125 ml of stock ii, and 4 g of glucose. turn on the two pumps of the perfusion systems. set the peristaltic pump (peri - star, wpi, sarasota, usa) that is used for retrograde perfusion to 40 ml / min. set the other peristaltic pump (cole - parmer masterflex peristalic pump l / s, cole - parmer instrument company, vernon hills, illinois) that is used for superfusion and to return the perfusate back to the holding reservoir to 80 ml / min. wash the perfusion system with 70% ethanol for 30 min and then with 2l deionized water. once all the deionized water is evacuated from the chamber, circulate the tyrode 's solution and pass it through a 5-m filter (millipore, billerica, ma, usa). warm the perfusate to + 37c with a water jacket and circulator (thermoneslab ex7, newtown, usa) and oxygenate the perfusate by bubbling o2/co2 (95% / 5%) gas into the solution. monitor the ph of the solution with a ph meter (oakton instruments, vernon hills, il) and adjust the rate of o2/co2 bubbling to keep the ph at 7.35.05. the dual optical mapping apparatus consists of two micam ultima - l cmos cameras (scimedia, costa mesa, ca) that have high spatial (100x100 pixels, 23020 m per pixel) and temporal (1,000 - 3,000 frames / sec) resolution. fix a band - pass filter (59015 nm, thorlabs, newton, nj) in front of the designated calcium imaging camera ; while, a long - pass filter (> 700 nm, thorlabs, newton, nj) needs to be positioned in front of the designated voltage imaging camera. the cameras are arranged perpendicular to one another by a holder, which contains a dichroic mirror (635 nm cutoff, omega optical, brattleboro, vt). immediately below the dual camera holder there is a lens (nikon nikkor 55 mm 1:1.4 235052), which focuses the emission light coming from the heart onto the dichroic mirror. the excitation light is generated by a halogen lamp (newport oriel instruments, stratford, ct ; scimedia, costa mesa, ca) and is passed through a heat filter, shutter, and band - pass filter (52045 nm). a flexible light guide directs the band - pass filtered light onto the preparation, and a shutter is used to ensure that the preparation is exposed to light only during image acquisition to avoid photobleaching of the dyes. prepare ag / agcl2 electrodes for pacing and sensing in advance and install them in the chamber prior to placing the heart. anesthetize the mouse with ketamine / xylazine (ketamin, 80mg / kg bodyweight ; xylazine, 10 mg / kg bodyweight) and heparin (100 units) by intraperitoneal injection. after a mid - sternal incision, quickly remove the heart and wash it in oxygenated (95% o2, 5% co2), constant - temperature (371c) tyrode 's solution. using a dissecting microscope, rapidly identify the aorta and make a clean cut across the ascending aorta below the right subclavian artery. a short section of aorta is then attached to a custom made 21-gauge cannula with a flared tip. 4 - 0 black - braided silk suture (surgical specialties corporation, reading, pa) is used to fix the heart onto the cannula. the retrograde perfusion rate is adjusted in the range of 2 - 5 ml / min to keep the aortic pressure between 60 and 80 mmhg (pressure transducer, world precision instruments inc (wpi), sarasota, usa ; bridge amplifier tbm4 m, wpi, sarasota, usa). after the heart is cannulated, lung, thymus, and fat tissue are then dissected and removed. the isolated heart is pinned (fine science tools) at the apex to the bottom of the perfusion chamber (sylgard coated) to prevent stream - induced movement. the right and left atrial appendages are also stretched and pinned (fine science tools, inc, foster city, ca) to the bottom of the chamber, which provides maximal surface area for optical measurements of the atria. very important ! a small silicon tube is inserted into the left ventricle through the pulmonary veins and fixed by silk suture to nearby connective tissue. this prevents solution congestion and acidification of the perfusate trapped in the left ventricle, which is especially important after the suppression of ventricular contractions with an excitation - contraction uncoupler (see part 4 step 19). a custom made electrode is placed on the surface of the heart to conduct the pacing stimulations, which is generated by master-8 (a.m.p instruments ltd, jerusalem, israel) or powerlab 26 t (ad instruments, sydney, australia). a small cover glass is fixed on the surface of the solution over the heart to reduce motion artifact from the vibrating solution. in addition, adjust the distance between the dual camera apparatus and the heart so that maximum resolution will be obtained. turn off all lights in the room and commence electrical recordings using powerlab 26 t. dilute the remaining 0.1 ml of blebbistatin in 1 ml of tyrode 's solution and slowly inject it (over a 20 minute period) through a drug port located near the cannula. dilute 30 l of voltage sensitive dye rh237 stock solution in 1 ml tyrode 's solution and slowly inject over 5 - 7 minutes into the same injection port as blebbistatin. 30 l calcium indicator rhod-2 am is 1:1 mixed with pluronic f127 (invitrogen, carlsbad, ca, 20% solution in dmso) and then diluted in 1 ml tyrode 's solution and slowly applied over 5 - 10 min through the same injection port. wait for 5 - 10 minutes for blebbistatin and dyes to reach the cell membrane and cytosol. continuously monitor ecg recordings over the entire procedure to assure normal electrical function of the heart. commence fluorescent signal recordings using the scimedia custom software (scimedia, costa mesa, ca). em = emission ; lp = longpass figure 2. experimental preparation and signal examples recorded during ventricular pacing. left : ecg signals are collected from ag / agcl2 disk electrodes (top) and an example of s1s1 stimulation protocol is shown (bottom). center : langendorff mouse heart preparation. right : representative optical action potentials and calcium transient signals from atria (top) and ventricles (bottom) are shown. the yellow arrow (top) points to the fluorescent signal scattering coming from the ventricles, which is seen in the atrial recordings. lv = left ventricle ; rv = right ventricle ; la = left atrium ; ra = right atrium figure 3. representative optical recordings of vm and cat from the ventricles of a wild type mouse heart. a. the experimental preparation with an array of evenly spaced locations marked by black dots whose optical recordings can be seen in (c). b. example tracing of vm and cat from a central location on the array (see box in (c)). c. vm (blue) and cat (red) from the array of evenly spaced locations. a. an example activation map from a wild type mouse heart with transverse (t) and longitudinal (l) directions indicated by white arrows. b. vm signals (top) and dv / dt (bottom) corresponding to the three points seen in a (t1, action potential duration at 80% repolarization (apd80) and calcium transient duration at 80% relaxation (cad80) maps are shown from a heart under control conditions (left) and after 30 nm isoproterenol application (right). the yellow / green color in the ventricles (right) indicates isoproterenol shortened apd80 and cad80. b. example tracings of apd80 and cad80 from the wild type mouse ventricles (top) and atria (bottom). prepare two stock solutions of tyrode 's solution (16x) in advance in deionized water and store them at 4c : stock i (119.872 g / l nacl, 3.056 g / l cacl2 (2h2o), 5.6 g / l kcl, 2.6274 g / l nah2po4, 3.408 g / l mgcl2 (6h2o), (fisher scientific, fair lawn, nj));stock ii (26.88 g / l nahco3, (fisher scientific, fair lawn, nj)). stock i (119.872 g / l nacl, 3.056 g / l cacl2 (2h2o), 5.6 g / l kcl, 2.6274 g / l nah2po4, 3.408 g / l mgcl2 (6h2o), (fisher scientific, fair lawn, nj)) ; stock ii (26.88 g / l nahco3, (fisher scientific, fair lawn, nj)). prepare stock solutions of fluorescent dyes. to avoid repeated freezing and thawing, we store 30 l aliquots of both dyes at -20c, which is sufficient for one experiment : voltage - sensitive dye rh237 (invitrogen, carlsbad, ca) stock solution, 1.25 mg / ml solution in dimethyl sulfoxide (dmso, sigma, st. louis, mo);calcium indicator rhod-2am (invitrogen, carlsbad, ca) stock solution, 1 mg / ml solution in dmso. voltage - sensitive dye rh237 (invitrogen, carlsbad, ca) stock solution, 1.25 mg / ml solution in dimethyl sulfoxide (dmso, sigma, st. louis, mo) ; calcium indicator rhod-2am (invitrogen, carlsbad, ca) stock solution, 1 mg / ml solution in dmso. louis, mo, 2 mg / ml solution in dmso) in advance and store the dissolved blebbistatin at 4c. freshly prepare 2l tyrode 's solution (128.2 mm nacl, 1.3 mm cacl2 (2h2o), 4.7 mm kcl, 1.05 mm mgcl2 (6h2o), 1.19 mm nah2po4, 20 mm nahco3, 11.1 mm d - glucose in deionized water, ph=7.350.05). if stock solution is being used to make 2l of tyrode 's solution (sufficient for one experiment) take 1750 ml of deionized water and mix in 125 ml of stock i, 125 ml of stock ii, and 4 g of glucose. turn on the two pumps of the perfusion systems. set the peristaltic pump (peri - star, wpi, sarasota, usa) that is used for retrograde perfusion to 40 ml / min. set the other peristaltic pump (cole - parmer masterflex peristalic pump l / s, cole - parmer instrument company, vernon hills, illinois) that is used for superfusion and to return the perfusate back to the holding reservoir to 80 ml / min. wash the perfusion system with 70% ethanol for 30 min and then with 2l deionized water. once all the deionized water is evacuated from the chamber, circulate the tyrode 's solution and pass it through a 5-m filter (millipore, billerica, ma, usa). warm the perfusate to + 37c with a water jacket and circulator (thermoneslab ex7, newtown, usa) and oxygenate the perfusate by bubbling o2/co2 (95% / 5%) gas into the solution. monitor the ph of the solution with a ph meter (oakton instruments, vernon hills, il) and adjust the rate of o2/co2 bubbling to keep the ph at 7.35.05. the dual optical mapping apparatus consists of two micam ultima - l cmos cameras (scimedia, costa mesa, ca) that have high spatial (100x100 pixels, 23020 m per pixel) and temporal (1,000 - 3,000 frames / sec) resolution. fix a band - pass filter (59015 nm, thorlabs, newton, nj) in front of the designated calcium imaging camera ; while, a long - pass filter (> 700 nm, thorlabs, newton, nj) needs to be positioned in front of the designated voltage imaging camera. the cameras are arranged perpendicular to one another by a holder, which contains a dichroic mirror (635 nm cutoff, omega optical, brattleboro, vt). immediately below the dual camera holder there is a lens (nikon nikkor 55 mm 1:1.4 235052), which focuses the emission light coming from the heart onto the dichroic mirror. the excitation light is generated by a halogen lamp (newport oriel instruments, stratford, ct ; scimedia, costa mesa, ca) and is passed through a heat filter, shutter, and band - pass filter (52045 nm). a flexible light guide directs the band - pass filtered light onto the preparation, and a shutter is used to ensure that the preparation is exposed to light only during image acquisition to avoid photobleaching of the dyes. prepare ag / agcl2 electrodes for pacing and sensing in advance and install them in the chamber prior to placing the heart. anesthetize the mouse with ketamine / xylazine (ketamin, 80mg / kg bodyweight ; xylazine, 10 mg / kg bodyweight) and heparin (100 units) by intraperitoneal injection. after a mid - sternal incision, quickly remove the heart and wash it in oxygenated (95% o2, 5% co2), constant - temperature (371c) tyrode 's solution. using a dissecting microscope, rapidly identify the aorta and make a clean cut across the ascending aorta below the right subclavian artery. a short section of aorta is then attached to a custom made 21-gauge cannula with a flared tip. 4 - 0 black - braided silk suture (surgical specialties corporation, reading, pa) is used to fix the heart onto the cannula. the retrograde perfusion rate is adjusted in the range of 2 - 5 ml / min to keep the aortic pressure between 60 and 80 mmhg (pressure transducer, world precision instruments inc (wpi), sarasota, usa ; bridge amplifier tbm4 m, wpi, sarasota, usa). after the heart is cannulated, lung, thymus, and fat tissue are then dissected and removed. the isolated heart is pinned (fine science tools) at the apex to the bottom of the perfusion chamber (sylgard coated) to prevent stream - induced movement. the right and left atrial appendages are also stretched and pinned (fine science tools, inc, foster city, ca) to the bottom of the chamber, which provides maximal surface area for optical measurements of the atria. very important ! a small silicon tube is inserted into the left ventricle through the pulmonary veins and fixed by silk suture to nearby connective tissue. this prevents solution congestion and acidification of the perfusate trapped in the left ventricle, which is especially important after the suppression of ventricular contractions with an excitation - contraction uncoupler (see part 4 step 19). a custom made electrode is placed on the surface of the heart to conduct the pacing stimulations, which is generated by master-8 (a.m.p instruments ltd, jerusalem, israel) or powerlab 26 t (ad instruments, sydney, australia). a small cover glass is fixed on the surface of the solution over the heart to reduce motion artifact from the vibrating solution. in addition, adjust the distance between the dual camera apparatus and the heart so that maximum resolution will be obtained. turn off all lights in the room and commence electrical recordings using powerlab 26 t. dilute the remaining 0.1 ml of blebbistatin in 1 ml of tyrode 's solution and slowly inject it (over a 20 minute period) through a drug port located near the cannula. dilute 30 l of voltage sensitive dye rh237 stock solution in 1 ml tyrode 's solution and slowly inject over 5 - 7 minutes into the same injection port as blebbistatin. 30 l calcium indicator rhod-2 am is 1:1 mixed with pluronic f127 (invitrogen, carlsbad, ca, 20% solution in dmso) and then diluted in 1 ml tyrode 's solution and slowly applied over 5 - 10 min through the same injection port. wait for 5 - 10 minutes for blebbistatin and dyes to reach the cell membrane and cytosol. continuously monitor ecg recordings over the entire procedure to assure normal electrical function of the heart. commence fluorescent signal recordings using the scimedia custom software (scimedia, costa mesa, ca). em = emission ; lp = longpass figure 2. experimental preparation and signal examples recorded during ventricular pacing. left : ecg signals are collected from ag / agcl2 disk electrodes (top) and an example of s1s1 stimulation protocol is shown (bottom). right : representative optical action potentials and calcium transient signals from atria (top) and ventricles (bottom) are shown. the yellow arrow (top) points to the fluorescent signal scattering coming from the ventricles, which is seen in the atrial recordings. lv = left ventricle ; rv = right ventricle ; la = left atrium ; ra = right atrium figure 3. representative optical recordings of vm and cat from the ventricles of a wild type mouse heart. a. the experimental preparation with an array of evenly spaced locations marked by black dots whose optical recordings can be seen in (c). b. example tracing of vm and cat from a central location on the array (see box in (c)). c. vm (blue) and cat (red) from the array of evenly spaced locations. a. an example activation map from a wild type mouse heart with transverse (t) and longitudinal (l) directions indicated by white arrows. b. vm signals (top) and dv / dt (bottom) corresponding to the three points seen in a (t1, t2, t3 ; action potential duration at 80% repolarization (apd80) and calcium transient duration at 80% relaxation (cad80) maps are shown from a heart under control conditions (left) and after 30 nm isoproterenol application (right). the yellow / green color in the ventricles (right) indicates isoproterenol shortened apd80 and cad80. b. example tracings of apd80 and cad80 from the wild type mouse ventricles (top) and atria (bottom). in this experiment we modified the langendorff perfusion method by adding a small silicon tube, which is especially crucial after the suppression of ventricular contractions with an excitation - contraction uncoupler. the silicon tube is used to prevent solution congestion, acidification of the perfusion solution, and development of ischemia in the left ventricle. the mouse heart is very sensitive to hypothermia ; thus, temperature variations across the heart will cause artificial differences in action potential durations. consequently, a heating system was implemented in the perfusion chamber in order to maintain a constant temperature of 37c during the entirety of the experiment. since a langendorff model does not retain innervation of the heart, one needs to consider adding neurotransmitters to the perfusate in order to investigate physiological sympathetic and parasympathetic tone. besides retrograde perfusion, addition of superfusion of the heart helps to maintain suitable environmental parameters such as ph and temperature. in this method, the langendorff perfused heart was horizontally placed. a vertical langendorff perfusion setup can also be used, but may result in slightly different cardiac mechanics. in addition to cmos cameras, alternative detectors are also available and can be applied to map vm and cat simultaneously. application of cmos cameras of high spatio - temporal resolution assures the accuracy of the recordings ; however, optical mapping signals are not from a single cell. rather, each fluorescent signal comes from hundreds or thousands of cells, depending on optical magnification. the much larger ventricular fluorescence can distort atrial signals by optical scattering ; therefore, careful interpretation of the optically recorded signals is required. another limitation of the mouse preparation is the signal distortion and noise induced by the curvature of the surface due to the small size of the heart. conduction velocity measurements can be altered not only from the curvature of the mouse heart but also from electrode polarity and virtual electrodes. to achieve accuracy for conduction velocity, activation anisotropy, and repolarization maps, correct focusing of the camera at the surface of the heart is essential. in this method, voltage - sensitive dye (rh237) and calcium indicator (rhod-2am) are used in the protocol because of their fast response, similar excitation, and distinct emission spectra. there are alternative combinations of dyes that can be used to measure vm and cat other than rh237 and rhod-2am. a novel voltage - sensitive dye, pghi, with a large stoke 's shift (> 200 nm) was found to allow better vm and cat signals because of the greater separation of the emission wavelengths between pghi and rhod-2am. future improvements may focus on exploring novel fluorescent probes, development of new imaging detectors, and improved image processing software. higher resolution and novel optical imaging modalities for 3d optical mapping are also important future directions of optical mapping.	the mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (vm), calcium transients (cat), and other parameters. excitation - contraction coupling is characterized by action potential and intracellular calcium dynamics ; therefore, it is critically important to map both vm and cat simultaneously from the same location on the heart1 - 4. simultaneous optical mapping from langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites can not be achieved from cellular level investigation but is well solved by optical mapping1,5,6. in this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of vm and cat in mouse hearts with high spatio - temporal resolution using state - of - the - art cmos imaging technology. consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of langendorff perfused mouse hearts is both feasible and reliable.
	biochemical characterization of polyketide synthases (pkss) has relied on synthetic substrates functionalized as electrophilic esters to acylate the enzyme and initiate the catalytic cycle. in these efforts, n - acetylcysteamine thioesters have typically been employed for in vitro studies of full pks modules as well as excised domains. however, substrate engineering approaches to control the catalytic cycle of a full pks module harboring multiple domains remain underexplored. this study examines a series of alternatively activated native hexaketide substrates on the catalytic outcome of pikaiv, the sixth and final module of the pikromycin (pik) pathway. we demonstrate the ability to control product formation with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring. this outcome was achieved through modifying the type of hexaketide ester employed, demonstrating the utility of substrate engineering in pks functional studies and biocatalysis.
avian influenza (ai) is a devastating poultry disease with serious economic consequences to the commercial poultry industry. ai is also a significant public health concern because of recent highly pathogenic h5n1 avian influenza outbreaks causing also human deaths in asia, europe, and north africa. according to the world health organization (who) update, 2011, since 2003, 520 confirmed cases of human infection with h5n1 have been reported, of which 307 died due to disease complications. however, other avian influenza viruses including low - pathogenic avian influenza (lpai) can also be a risk to public health. for instance, the lpai subtype h9n2 infection in chickens is mild to asymptomatic and easily overlooked. however, it shares similar receptor binding epitopes with human influenza viruses and can infect humans. there is a risk for lpai subtypes h5 and h7 to become high - pathogenic avian influenza (hpai) viruses in chickens due to constant virus shedding and transmission to new birds within the flock or neighboring flocks [2, 3 ]. however, routine vaccination against ai has not been widely practiced throughout the world mainly for surveillance reasons [1, 2 ]. when there is the desire for routine vaccination, constant reformulation of ai vaccines is required according to the circulating field virus, which can be cumbersome in the case of an immediate outbreak. current vaccines against ai viruses can reduce mortality, clinical signs, shedding, and transmission of the virus in poultry, but they are not capable of preventing infection and virus replication. the design of a universal influenza vaccine has been the major focus of researchers in the influenza vaccinology field. the external domain of matrix protein 2 (m2e) has been one of the main interests for the generation of a universal ai vaccine. the m2e is encoded by a separate open reading frame of segment 7 of the influenza virus genome, is located in the viral envelope, and projects from the surface of the virus as tetramers [5, 6 ]. the m2 is composed of 97 amino acids which forms 3 domains : the external domain, the transmembrane domain, and the internal domain. the external domain of m2 (m2e) is recognized by the host 's immune system [79 ]. initially, vaccination of ferrets with whole m - or m2-expressing recombinant vaccinia virus showed no evidence of protection. however, later vaccine constructs using plasmid and recombinant salmonella expressing m or m2 induced significant protection in terms of reduction in virus growth and mortality in mice and chickens, respectively [1113 ]. a multiple antigenic peptide construct containing m2e (m2e - map) induced strong m2e - specific antibody titers in the serum of mice and resulted in significant protection against influenza virus challenge., 1994 showed the importance of cd4 t cells for nasal resistance and protection against the virus. it is assumed that m2e - specific memory th cells also may have an important role in protection against the virus in the nose and trachea of mice., 2005 used the hepatitis b virus core particle (hbc) as a carrier and fused m2e (conserved region of human influenza a virus) to either the c - terminus of hbc or inserted it in the immune - dominant loop of hbc. immunization of mice with this m2e - hbc vaccine was 100% protective against lethal challenge [1517 ]. therefore, usually a larger carrier protein, such as bovine serum albumin (bsa), keyhole limpet hemocyanin (klh), or a virus - like particle (vlp), is required for optimal immunogenicity. structural organization of the epitope on the carrier is critical for inducing stronger immune responses. denis., 2007 demonstrated that a monomeric form of m2e peptide was not immunogenic and huleatt., 2008 tried to solve that problem by adding 4 copies of the m2e peptide in their platform. here, we used peptide nanoparticles as a platform to display the m2e peptide to the host 's immune system. these nanoparticles represent a novel type of repetitive antigen display system which allows presenting the m2e peptide in high density in both, either in its monomeric or its tetrameric form. the monomeric peptide is composed of two coiled coils connected by a short linker region. the association between the coiled coils induces self - assembly of the monomers into spherical nanoparticles with either icosahedral or octahedral symmetry (figure 1) according to our computer models. the potential for these nanoparticles to serve as platforms for vaccines is apparent. as opposed to live attenuated vaccines, furthermore, the ease and speed of protein expression, purification, and self - assembly into nanoparticles reduce the cost and time of large - scale production. the concept has been successfully used for the design of malaria and sars vaccines prototypes. here, we present the biophysical characterization of the nanoparticles and an immunological profiling using chickens as test animals. the results suggest that the self - assembling polypeptide nanoparticle shows promise as a potential vaccine against ai. shortly, plasmids containing the peptide monomers (table 1) were constructed by cloning complementary oligonucleotides (cccgggggggcagcggcagcctgctgaccgaagtg - gaaaccccgacccgcaacggctgggaataatgaattc) encoding the avian m2e epitope with flanking residues (arggsgslltevetptrngwee) into the xmai / ecori restriction sites of the basic sapn expression construct to yield mono - m2e. to generate tetra - m2e, we first cloned the tetrameric oligomerization domain of tetrabrachion into the bamhi / bsshii restriction sites of ppep - t (figure 5), before cloning complementary oligonucleotides (atgcatccctggttccgcgtggaagcctgctgaccg - aagtggaaaccccgacccgcaacggctgggaatgca - aatgcagcgatagcagcggatcc) coding for the slightly longer avian m2e sequence (haslltevetptrngweckcsdssgs) including flanking residues into the n - terminal nsii / bamhi restriction sites. the m2e - gcn4 construct was made by replacing the nanoparticle fragment of tetra - m2e with the gcn4 sequence. the plasmids were transformed into escherichia coli bl21 (de3) cells, which were grown in luria broth with ampicillin at 37c. four hours after induction, cells were removed from 37c and harvested by centrifugation at 4,000 g for 15 min. the pellet was thawed on ice and suspended in a lysis buffer consisting of 9 m urea, 100 mm nah2po4, 10 mm tris ph 8, 20 mm imidazole, and 0.2 mm tris-2-carboxyethl phosphine (tcep). cells were lysed by sonication and the lysate was cleared by centrifuging at 30.500 g for 45 min. the cleared lysate was incubated with ni - nta agarose beads (qiagen, valencia, ca, usa) for at least 1 hour. the column was washed with lysis buffer and then a buffer containing 9 m urea, 500 mm nah2po4, 10 mm tris ph 8, 20 mm imidazole, and 0.2 mm tcep. protein was eluted with a ph gradient : 9 m urea, 100 mm nah2po4, 20 mm citrate, 20 mm imidazole, and 0.2 mm tcep. subsequent washes were done at ph 6.3, 5.9, and 4.3. following the ph gradient, a gradient of lysis buffer with increasing imidazole strength was used to further elute the protein. purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) as shown in figure 6. the protein solution was filtered with a 0.1 m polyvinylidene fluoride membrane filter (millipore billerica, ma, usa). nanoparticle self - assembly was performed by dialysis into buffer containing 8 m urea, 20 mm tris ph 7.5, 150 mm nacl, and 5% glycerol, at a protein concentration of 0.1 mg / ml. this was followed by dialysis into the same buffer containing decreasing concentrations of urea : 6 m, 4 m, 2 m, 1 m, and two changes of the same buffer without urea. following self - assembly dynamic light scattering experiments were carried out on a zetasizer nano s instrument (malvern, worcestershire, uk), with a 633 nm he - ne laser. all measurements were carried out at 25c in a buffer containing 20 mm tris ph 7.5, 150 mm nacl, and 5% glycerol. samples were negatively stained with 1% uranyl acetate (spi supplies, westchester, pa, usa) and observed with a fei tecnai t12 s / tem at an accelerating voltage of 80 kv (fei, hillsboro, oregon). the peptide concentration of the constructs was about 0.05 mg / ml. samples were dialyzed into 20 mm sodium phosphate ph 7.5, 150 mm nacl, and 5% glycerol and concentrated or diluted to a peptide concentration of about 0.13 mg / ml for mono - m2e and about 0.05 mg / ml for tetra - m2e. circular dichroism measurements were performed at room temperature using an applied photophysics (surrey, uk) pi star 180 spectropolarimeter, taking measurements from 200 to 250 nm. the influenza virus used in the direct challenge ai study was a / turkey / ca / d0208651-c/02 h5n2 low pathogenic. influenza a / turkey / wisconsin/1/1966 h9n2 low pathogenic was used for hyperimmune serum production provided by charles river avian vaccine services (storrs, ct). spf p2a line (b19/b19) white leghorn chickens eggs were obtained from cornell university, ithaca, ny. the eggs were hatched in the university of connecticut poultry farm and after the hatch, the chickens were moved to the office of animal research services (oars) facilities. after 2 weeks in the brooders with free access to water and a standard starter diet, the chickens were divided into groups, bled for baseline serology, transferred to isolators equipped with high - efficiency particulate air (hepa) filters, and were provided commercial diets and water ad libitum. a previously described plaque reduction assay was modified and used to evaluate the virus neutralization activity of collected sera after vaccination. an equal volume of a 1 : 10 dilution of pooled serum and lpai subtype h5n2 was mixed and incubated for 30 min at 37c. a commercially available anti - m2 antibody (prosci - inc, poway, ca) was used in a 1 : 1000 dilution as a control for antibody activity. chicken embryo kidney cell (cekc) monolayers in 6-well plates were washed twice with prewarmed pbs and 400 l of the above mixture was added to the cekc monolayer., the inoculums were removed and after 2 washes with prewarmed pbs, they were overlaid with 0.8% agar (university of connecticut cell culture facility) in minimum essential medium eagle (mem). after 72 h, the plates were checked for plaque formation and for further evaluation were fixed with 99% methanol and stained with crystal violet for plaque counting. the m2e epitopes, including the nanoparticle platforms with m2e epitopes (tetra - m2e and mono - m2e) and m2e linked to gcn4, (m2en - gcn4), were used for coating of the elisa plates. briefly, individual wells of the flat - bottom 96-well immulon 1b plates (nunc / thermo fisher scientific, rochester, ny) were coated with 5 g / ml of tetrameric m2e (m2en - gcn4) or the nanoparticle of interest. plates were rinsed with 2% tween 20 in phosphate - buffered saline (pbs) (pbs / tween 20 thermofisher) and blocked with a 3% bsa in pbs solution. plates were incubated at 37c for 3 - 4 h or 4c overnight (preliminary studies showed that there was no difference in the result). after incubation, plates were rinsed 4 times with pbs / tween 20 and incubated for 1 h at room temperature with the previously collected sera. briefly, 2-fold serial dilutions of each serum sample were prepared in a pbs solution containing 0.2 to 0.5% bsa. hyperimmune serum from previously infected birds with the lpai subtype h9n2 or commercial anti - m2e antibody were used as positive controls ; and sera from healthy, unvaccinated birds were used as a negative control. after appropriate washes, peroxidase - conjugated goat anti - chicken igy (sigma aldrich,) was prepared in a 1 : 10,000 dilution in pbs and was added to each well and plates were incubated for an additional hour at room temperature. after subsequent rinsing, the plates were developed using 3,3,5,5 tetramethylbenzidine (tmb) peroxidase substrate (thermo fisher scientific inc., rockford, il) followed by a room temperature incubation period of 15 to 30 min. the absorbance was read in a spectramax 250 microplate reader (molecular devices, sunnyvale, ca) at 450 nm. in order to generate a standard curve for real - time pcr, we transcribed standard rna in vitro using t7 ribomax express large - scale rna production system (promega, madison, wi). briefly, rna extraction was done by using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer. the coding region of the m gene from the lpai subtype h5n2 was amplified using previously described universal primers. rt - pcr was performed using a qiagen one - step rt - pcr kit (qiagen, valencia, ca) according to the standard manufacturer 's protocol. pcr products were visualized by electrophoresis through ethidium - bromide - stained (0.5 g / ml) 1.5% (40 mm tris - acetate ph 7.8, 0.1 mm edta) agarose gels under uv light. the amplified fragment was excised from the gel and cdna was recovered from agarose gel using a qiaquick gel extraction kit (qiagen, valencia, ca) according to the manufacturer 's protocol and the purified dna product was ligated with a pcr 2.1 vector (invitrogen, carlsland, ca) according to the manufacturer 's protocol to generate the pcr - m5 plasmid. for further confirmation, pcr positive plasmids six micrograms of plasmid dna was linearized using 6 units of the restriction enzyme bam hi for 4 h at 37c. then, linearized dna was used as a template in an in vitro transcription reaction with the t7 ribomax express large - scale rna production system (promega, madison, wi) according to the manufacturer 's recommendation. after the in vitro transcription reaction at 37c for 1 h, the possible remaining plasmid dna was digested by dnase i and purified rna was quantified with a spectrophotometer nanodrop nd-1000 (thermo fisher scientific, wilmington, de). the copy numbers of purified rna were determined using a previously described method and was used for the generation of a real time standard curve. in this study, real - time rt - pcr was performed using the previously published primers m+25 : aga tga gtc ttc taa ccg agg tcg and m-124 : tgc aaa aac atc ttc aag tct ctg for quantification of viral load. rna extraction was done on each swab sample followed by pcr in duplicate or triplicate using 5 l of rna per each pcr reaction. briefly, the power sybr green rna - to - ct 1-step kit (applied biosystem, foster city, ca) was used with a 20 l reaction mixture. for each pcr run, standards were designated for the plate and viral loads were calculated using fluorescence data acquired at the end of each annealing step. the amount of unknown sample was extrapolated based on the standard curve and was reported as viral copy number. prior to vaccination and challenge study, a pilot study was performed to evaluate pathogenicity of the virus and determine the peak of virus shedding by measuring viral copy number in tracheal and cloacal sample of chickens at various time points postinfection with subtype h5n2 of low - pathogenicity avian influenza virus (lpai). briefly, thirty 2-week - old spf chickens were divided into 3 groups of ten and were bled for baseline serology and transferred to isolators equipped with hepa filters. at 8 weeks of age, chickens were inoculated both intranasally and intraocularly with 0.21 ml diluted allantoic fluid depending on the treatment group. chickens in the low - dose challenge group received a 0.2 ml diluted allantoic fluid containing 10 eid50, whereas the high - dose challenge group received 1 ml of allantoic fluid containing 10 eid50. tracheal and cloacal swabs were collected at 2, 4, 6, 8, 10, and 14 days postinfection using bd universal viral transport (uvt) kits (becton, dickinson, nj) and universal viral transport polyester swabs (becton, dickinson, nj). upon determination of the peak of virus shedding and appropriate infectious dose in the pilot study, briefly, 42 spf chickens were divided into six groups of seven and received their first inoculation at 2 weeks of age followed by two boosters, two weeks apart, at 10 weeks and 12 weeks after hatch as described in table 2. preceding injection, nanoparticles were concentrated using amicon centrifugal filter units with a 100 kda mwco (millipore, billerica, ma). concentration was determined by absorbance at 280 nm and nanoparticle quality was assured by dls. the nanoparticle vaccine constructs were emulsified with either freund 's complete adjuvant (prime) or freund 's incomplete adjuvant (boosters) and injected into the pectoral muscle of each chicken. two weeks after the second booster, the birds, except for those in the negative control group, were challenged with 10 eid50 lpai subtype h5n2. briefly, each bird received 1 ml allantoic fluid containing 10 eid50 lpai subtype h5n2 divided among the eyes, nasal cavity, and oropharynx, while temporarily blocking the fresh air delivery to the isolator. fresh air was resumed after 510 min the following challenge of the last bird in the isolator. although the clinical signs associated with lpai viruses are rare, they were observed for possible clinical symptoms daily ; and the presence of the symptoms and their severity was recorded. tracheal and cloacal swabs were taken from each bird at days 2, 4, 6, and 8 after challenge and they were placed in a 3.0 ml uvt tube (becton, dickinson, nj). blood samples from each bird were collected before each booster as well as two weeks after the second booster prior to challenge. each blood sample was collected in a separating blood tube and serum was separated by placing the tubes at 37c for 1 h then at room temperature overnight followed by a 5 to 10 min centrifugation at 1000 rpm at 4c. the capsids of spherical viruses often have icosahedral symmetry, due to their need to build a large encapsulating structure from many copies of the same, or only few, capsid proteins. an icosahedron is the most efficient way to accomplish this. by utilizing pentameric and trimeric coiled coils, we have built a self - assembling nanoparticle which uses the threefold and fivefold symmetry of an icosahedrons. the pentameric coiledcoil motif of the monomer is taken from cartilage oligomeric matrix protein (comp) and the trimer is a de novo designed coiled coil. self - assembly occurs when the coiled - coil domains of different monomers associate, forming the icosahedral nanoparticle (figure 1). a nanoparticle with this sort of architecture can then be used as a vaccine platform by extending the ends of the monomer with an epitope sequence. the mono - m2e species of nanoparticle follows this plan (table 1). as a result, it repetitively displays a monomeric form of m2e on the surface of the nanoparticles. the m2e peptide on the icosahedral nanoparticles lacks its c - terminal five residues to avoid problems with disulfide crosslinking that presumably require the native tetrameric conformation for proper formation. although the simplest icosahedral particle with t1 icosahedral symmetry is made from 60 polypeptide chains, it may also be possible that mono - m2e particles possess higher triangulation numbers, resulting in particles with an even greater molecular mass. hence, to elicit conformationally specific antibodies, the m2e antigen displayed by a vaccine particle should ideally be tetrameric. with that in mind, we designed the tetra - m2e peptide (table 1). instead of a pentameric coiled coil, this polypeptide uses the tetrameric coiledcoil motif from the protein tetrabrachion. self - assembly using this peptide would result in a nanoparticle with threefold and fourfold symmetry axes or octahedral symmetry. as opposed to the larger icosahedral mono - m2e the formation of disulfide bridges between the cysteines of adjacent chains under oxidizing conditions is thought to stabilize their tetrameric conformation. the speed and ease of protein expression and purification, as well as of the self - assembly process, contribute to the overall viability of this technology as a vaccine platform. to facilitate purification, we have included polyhistidine tags at the n - terminal ends of the peptides. to enable detection of antibodies against tetrameric m2e, m2e is linked to a gcn4, a coiled coil whose oligomerization state can be determined by the identity of amino acid residues in key a and d positions of the coiled coil. in this case, the tetrameric version of gcn4 was used. by affixing m2e to a tetrameric protein, we can constrain it in its tetrameric conformation. the effect is similar to that experienced by the ends of the tetrameric coiled coil from tetrabrachion of the tetra - m2e nanoparticle. this guarantees that any antibodies bound to m2e - gcn4 are specific for the tetrameric version of m2e and not against the coiled coil or other parts of the nanoparticle. dynamic light scattering revealed that mono - m2e formed particles whose hydrodynamic diameters have a distribution which peaks at 34.5 nm, while the distribution of tetra - m2e peaks at 22.9 nm (figure 2). it is also noteworthy that the size distribution peak of mono - m2e is broader than that of tetra - m2e, suggesting that the former has a higher degree of polydispersity. we can see that nanoparticles were formed and that their diameters are comparable with those measured by dynamic light scattering. it can be seen from the micrographs that neither mono - m2e nor tetra - m2e form nanoparticles with perfectly spherical morphology. the double minima found by circular dichroism confirm the alpha helical structure of the nanoparticles (figure 4). it appears that tetra - m2e exhibits this behavior much less than mono - m2e. this may be partly due to the larger m2e epitope sequence in the tetra - m2e peptide as compared to that used in mono - m2e. the plaque reduction assay performed by using pooled serum from chickens inoculated with tetra - m2e did not show a significant (p > 0.05) difference compared to control nonvaccinated chicken serum and commercial anti - m2e antibody. the anti - m2e immune response was monitored by determining the titer of the m2e - specific igy at three different time points (2 weeks after each inoculation). chickens after each inoculation developed high levels of antibody against the injected construct and anamnestic response clearly was seen when the plates were coated with mono - m2e and tetra - m2e nanoparticles and m2e - gcn4 (tetrameric m2e), respectively (table 1, figures 7 and 8). for further investigation of the antibodies, the level of m2e specific antibody was measured using plates coated with tetrameric m2e - gnc4 peptide to evaluate the specific antibody against tetrameric m2e rather than the whole particle. the result of this study indicated that in chickens, after the second booster, the antibody levels are not at the same level as our previous results in mice with the same backbone but a different (malaria) epitope had been shown. the dose level was also higher than what was shown to be required in mice. this could be because of the lower haplotype - specific immunogenicity of the particles in chickens, the route of administration in mice (intraperitoneal and intranasally), the body weight of the mice compared with chickens, and different immune system repertoires of mammalian and avian species. in future studies, changing the administration route can be another approach to reducing the dose of vaccine construct. we also coated the plate with inactivated purified virus to observe seroconversion of the chickens after challenge with the virus at 2 weeks after the last boost. results indicated that whole virus response was higher as expected with hyperimmune serum (figure 9(a)), however, elisa response from chicken vaccinated with tetra - m2e and with whole virus reacted similarly on gcn - m2e coated plate (figure 9(b)). the protective efficacy of the anti - m2e antibody responses induced by different constructs was assessed by evaluation of viral shedding post challenge. to determine the peak of shedding, viral copy number was measured in tracheal and cloacal sample of chickens after infecting them with a lpai virus a / turkey / ca / d0208651-c/02 h5n2. briefly, in a pilot study chickens were infected with 10 eid50 and 10 eid50 of the virus and by using real time rt - pcr, tracheal and cloacal virus shedding was evaluated at 2, 4, 6, 8, 10, and 14 days postinfection. first, a significant (p < 0.05) rise in tracheal and cloacal virus shedding was observed at days 4 and 8 postinfection with a magnified peak of tracheal shedding at 8 days postinfection. taking into account these results, in the vaccination and challenge trial, individual tracheal and cloacal swabs were collected at 4, 6, and 8 days after challenge for better determination of protection. the results of the real - time rt - pcr testing of cloacal and tracheal swab samples taken on day 8 after vaccination and challenge study are shown in figure 10. we determined viral loads in tracheal and cloacal swabs samples on day 8 following challenge with 10 eid50 lpai subtype h5n2. reduction of cloacal and oropharyngeal shedding in vaccinated birds was significant in chickens vaccinated with tetra - m2e with freund 's adjuvant. virus shedding was evaluated at day 4 and day 6 after challenge ; the swabs were tested for virus load (figure 11). it is seen that virus shedding reduction starts at day 4 post infection with a significant decrease at day 8 post infection. currently available vaccines induce antibodies against specific field strains or closely related avian influenza strains. most of these vaccines are killed virus vaccines that induce short - lived immunity and are lacking a broad cross - reactive humoral immune response. recently, the generation of a universal influenza vaccine using conserved peptide regions among several influenza virus strains has been an area of interest in the human influenza vaccine field. m2e is a highly conserved region among influenza viruses and it has been studied as a possible universal vaccine candidate against human influenza virus infection [16, 17 ]. in the present study, protection efficiency of two different nanoparticle constructs harboring m2e was studied as possible vaccine candidates for low - pathogenicity avian influenza infection. biophysical analysis confirms that they are of relatively regular shape and size, but there is some degree of heterogeneity. though molecular weight measurements still remain to be carried out and we have no high resolution structural data, we assumed that nanoparticles assembled in a state close to what was expected, that is, icosahedral and octahedral nanoparticles, respectively. this will repetitively display m2e in both, either in its monomeric or its tetrameric form. there is speculation as to how the polyhistidine tag at the n - terminal end of the monomers may affect the self - assembly process, the final nanoparticle structure, or the immunogenicity of the vaccine, but attempts at producing his - tag free versions of the nanoparticle constructs either did not reliably provide pure protein or never adequately self - assembled. similarly, we attempted to include cd4 t cell epitopes to increase the immune response, but this also interfered with nanoparticle formation. since many variables can affect the host 's virus shedding and the course of disease [31, 32 ], prior to evaluation of vaccine constructs, the pathogenicity after challenge with the lpai subtype h5n2 virus was evaluated. a biphasic virus shedding was observed in this study. for the lpai subtype h5n2, the peaks for tracheal and cloacal shedding were at days 4 and 8 postinfection. the tetra - m2e vaccine construct provided a significant viral load reduction at the peak of viral shedding in immunized chickens. chickens immunized with tetra - m2e that harbors the tetrameric m2e with freund 's adjuvant showed a clear reduction in cloacal and tracheal excretion of lpai compared to challenge control groups. the results of immunization with mono - m2e with adjuvant and tetra - m2e without adjuvant were also promising and by improving both b- and t - cell epitopes of those constructs, desirable results may be obtained. in vaccine design, repetitive b - cell epitope display is considered a strategy for improving the humoral immune response [33, 34 ]. in addition to repetitive antigen display on the nanoparticle, we were able to present m2e in its native tetrameric conformation. the correlation between high protection and antibody response specific for tetrameric m2e elicited by tetra - m2e supports our assumption of tetrameric m2e presentation. the results of our studies show that tetrameric m2e stimulates a more specific immune response compared to the monomeric presentation and induces a significant protection against homologous virus challenge. the fact that a large portion of the antibody response is directed against the carrier and not only against the epitope(s) (figures 7 and 8) can be explained by the fact that significant portions of the core of the nanoparticles are also exposed to the immune system (compare figure 1) and hence, these portions will also induce a significant immune response. in this study, we showed that anti - m2e antibodies are not neutralizing antibodies ; however they are capable of binding to the m2 proteins that are abundantly presented on the surface of the infected cells (data not shown). these can describe an efficient delayed clearance of the virus in m2e vaccinated chickens based on the previously described nk cell involvement in adcc. significant improvement of virus clearance in vaccinated chickens with tetrameric m2e may be considered in a new vaccination strategy by vaccinating chickens with both a killed vaccine and a nanoparticle vaccine in order to provide robust protection, cross - reactive immunity, and clearance in case of emerging new strains of the virus. however, there remains the risk that such vaccination may cause a long - term persistence of hpai in poultry flocks, because the vaccine could not prevent the viral infection but rather suppresses the symptoms of hpai virus - infected chickens by reducing the virus shedding in chicken. thereby, especially in the case of hpai infection, the vaccination may make the infection less visible and the eradication of virus more difficult, and consequently it may provide a good opportunity for hpai virus to survive and persist in poultry flocks for a long time. for this reason, we plan to design new nanoparticle constructs that also contain fragments of hemagglutinin in addition to the m2e domain. immunization would then result in the generation of neutralizing hemagglutinin - specific antibodies in addition to the disease modulating m2e - specific antibodies. in this study, we evaluated a new approach to immunizing chickens against ai that uses a nanoparticle platform to carry an antigenic epitope. further designing and testing of new nanoparticle vaccines should demonstrate that they are effective tools for stimulation of an immune response against m2e and other b- or t - cell epitopes. therefore, application of the nanoparticle platform facilitates the development of a new generation of vaccines that harbor conserved epitopes of avian influenza viruses and would not be rendered ineffective by viral mutations such as antigenic shifts and drifts. the nanotechnology described here offers the opportunity to rapidly produce new vaccines according to the emergence of new strains of influenza virus without going through the time - consuming steps of production currently used in manufacturing commercial influenza vaccines. for future studies, the chicken 's lpai infection model needs to be improved to evaluate clinical signs and higher virus shedding. also, vaccination and hpai challenge may be used to evaluate the vaccine efficiency in protection against high - pathogenicity ai viruses.	using peptide nanoparticle technology, we have designed two novel vaccine constructs representing m2e in monomeric (mono - m2e) and tetrameric (tetra - m2e) forms. groups of specific pathogen free (spf) chickens were immunized intramuscularly with mono - m2e or tetra - m2e with and without an adjuvant. two weeks after the second boost, chickens were challenged with 107.2 eid50 of h5n2 low pathogenicity avian influenza (lpai) virus. m2e - specific antibody responses to each of the vaccine constructs were tested by elisa. vaccinated chickens exhibited increased m2e - specific igg responses for each of the constructs as compared to a non - vaccinated group. however, the vaccine construct tetra - m2e elicited a significantly higher antibody response when it was used with an adjuvant. on the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. the level of protection was evaluated using quantitative real time pcr at 4, 6, and 8 days post - challenge with h5n2 lpai by measuring virus shedding from trachea and cloaca. the tetra - m2e with adjuvant offered statistically significant (p < 0.05) protection against subtype h5n2 lpai by reduction of the ai virus shedding. the results suggest that the self - assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against ai.
drugs used in the perioperative period could have an effect on survival as recently pointed out by an international consensus conference on the reduction in mortality in cardiac anesthesia and intensive care. insulin infusion to achieve a strict glycemic control is the best example of how an ancillary (i.e. non - surgical) drug / technique / strategy might influence survival rates in patients undergoing cardiac surgery. presents her insights into the use of insulin in this setting and suggest that based on available evidence based medicine, insulin infusion, titrated to normoglycemia is a complex intervention, that not only requires the simple administration of a drug, the hormone insulin, but also needs tools and skills to accurately measure and control blood glucose to achieve normoglycemia while avoiding hypoglycemia and large glucose fluctuations. dr. landoni and colleagues are to be congratulated for their efforts to assess the existing evidence on all ancillary drugs / techniques / strategies that could potentially improve survival rates of patients undergoing cardiac surgery, and to draft a list of those that merit urgent further investigation. this list was the result of an international consensus conference that met last summer. the activities of this group to review the literature on these interventions and to weigh their potential impact using evidence - based methodology are vital to efficiently endorse effective strategies by practicing clinicians, and to systematically improve patient care. it will be interesting and important to see the results from further investigations as well as reports from the implementation process of these treatments into clinical practice. listed top, and annotated with the highest level of agreement, appears insulin infusion titrated to maintain normoglycemia in adult patients admitted to intensive care after cardiac surgery. this intervention has shown, in a large randomized controlled study (rct) of adult intensive care unit (icu) patients, among which 60% after cardiac surgery, to increase survival rate and to reduce morbidity. the publication of that study in 2001 was followed by another rct performed in infants and children, among which again 75% were patients after cardiac surgery, which confirmed the benefits. also in implementation studies, the intervention that was investigated comprises insulin infusion, which is titrated to normoglycemia. it is thus a complex intervention, that not only requires the simple administration of a drug, the hormone insulin, but also needs tools and skills to accurately measure and control blood glucose to achieve normoglycemia while avoiding hypoglycemia and large glucose fluctuations. the concept of infusing the hormone insulin in order to improve outcome of cardiac surgery patients was not new. sodi - pallares, who advocated the infusion of insulin together with glucose and potassium (gik) with the intention to provide the ischemic heart with an alternative metabolic substrate that requires less oxygen than fatty acids, and to prevent arrhythmia. however, the intervention to titrate insulin to normoglycemia in patients with critical illness was investigated for a different reason, namely to avoid complications that occur because of toxic effects of hyperglycemia during stress or illness. indeed, it has been shown in many large scale observational studies that there is a clear j - shaped association between adverse outcomes and blood glucose levels in all types of patients suffering from acute critical illnesses. this applies to the admission blood glucose values but also to the average blood glucose values during intensive care. the lowest risk is associated with age - adjusted normoglycemia, especially in patients without a history of diabetes mellitus. in patients with long - standing diabetes mellitus, this curve is flattened and somewhat shifted to the right, so slightly higher blood glucose levels could be associated with the lowest risk for diabetic as compared with non - diabetic patients. such an association could either suggest that hyperglycemia, any level higher than normoglycemia, is an adaptive response to illness, proportionately to the illness severity, or else that it contributes to adverse outcome as is the case in diabetes mellitus. in the rct published in 2001, the hypothesis was the latter : namely that toxic effects of hyperglycemia occur during stress and illness. hence, in the intervention group of this study of patients admitted to icu, targeting strict normoglycemia (80 - 110 mg / dl) with a continuous insulin infusion was compared with tolerating hyperglycemia up to 215 mg / dl in the control group. sixty percent of the patients had been admitted to intensive care after complex or complicated cardiac surgery with high euroscores. this study found that titrating insulin infusion to prevent blood glucose exceeding the upper normal limit, starting immediately after surgery and continuing throughout the entire stay in icu, substantially reduced morbidity and mortality, a benefit that was maintained at least up to 4 years after surgery. subsequently, a similar study was performed in critically ill infants and children, of which 75% was admitted to icu after surgery for complex congenital cardiac anomalies. titrating insulin to a blood glucose level that is equal to the age - normal fasting blood glucose level generated similar outcome benefits as in the adult population, with a striking protection of the heart, fewer infections, shortened icu stay and a reduced mortality. studies that documented implementation of a blood glucose lowering intervention to achieve normoglycemia in patients after lower risk cardiac surgery also confirmed the benefits [6, 12 ]. in depth analyses of the results suggested that the prevention of hyperglycemia, not the infusion of insulin per se, explained most of these benefits. studies in animal models of critical illness and in cell culture models further unraveled this important aspect of glucose toxicity, and pointed to direct damaging effects of hyperglycemia on cell integrity in liver, kidney, the endothelium, immune cells and the heart. a damaging effect on the mitochondria appears to play a crucial role in the adverse effects of hyperglycemia and anti - inflammatory effects could be involved as well. further post - hoc analyses of the available data from the rcts suggested a dose response : most effect on morbidity and on mortality was obtained when true normoglycemia was achieved. but to reduce mortality, 75% of the benefit may already have been present when blood glucose levels were effectively lowered to below 150 mg / dl. it remains hitherto unclear, however, from which level of blood glucose onward the toxic effects of hyperglycemia become clinically important, as rcts addressing this question have not been performed. the largest rct to date on the topic of blood glucose control in icu patients is the nice - sugar trial, that investigated 6100 patients in 42 centers predominantly in australia, new zealand and canada. multi - center repeat study of the leuven trials, it was not a repeat study as it studied a different intervention in a different patient population using different targets and tools for blood glucose control. second, it compared targeting strict normoglycemia with an intermediate target for blood glucose control (achieving mean blood glucose levels of around 150 mg / dl) instead of tolerating hyperglycemia to 215 mg / dl. third, the tools used to measure blood glucose levels (different types of hand - held meters) were highly inaccurate and therefore not suitable to target such a narrow range for blood glucose. fourth, the algorithm that was used did not allow stable control of the true blood glucose levels and resulted in a broad overlap between the measured blood glucose levels in both study arms. finally, the patients did not receive early parenteral nutrition to complete insufficient enteral nutrition, in contrast to the patients in the leuven trials. nice - sugar suggested that, with the tools and methods used, targeting an intermediate level of blood glucose is safer than attempting to reach strict normoglycemia. this is the correct conclusion from that trial, but all the above listed methodological differences make it difficult, if not impossible, to directly compare this trial with the proof - of - concept leuven trials. since cardiac surgery patients in addition, the outcome of nice - sugar underlines the importance of applying all aspects of a complex intervention when moving from a proof - of - concept study to a repeat study or when implementing the intervention in clinical practice. so where do we go from here ? based on high level evidence, insulin infusion titrated to normoglycemia in icu after cardiac surgery is ranked at the top of the list of ancillary drugs / techniques / strategies that could potentially improve survival rates of patients undergoing cardiac surgery by the international consensus committee. what should be done next before we can safely advise to implement the intervention in routine clinical practice ? in order to prove generalizability of the findings from the leuven studies, a large, multi - center repeat rct is required in the cardiac surgery patient population. this implies that the study should include cardiac surgery patients only and should investigate the full package of the intervention with all the details described above being copied. the intervention, titration insulin to achieve normoglycemia, should be compared against tolerating hyperglycemia, not against an intermediate target, as the latter will inevitably result in too broad overlap and thus insufficient separation of blood glucose levels in the groups to study an outcome difference. also, the study should be large enough to have enough statistical power. in view of the variable mortality rates for patients undergoing cardiac surgery, which can be as low as < 3% for isolated coronary bypass surgery up to 11% for complicated or combined valvular surgery, depending on the case - mix and types of surgery of the patients in the study, this could mean that a mega - trial is needed, with sample sizes that may range from 6.000 to 26.000 patients. in addition, if due to change in clinical practice, the control group would have an intermediate blood glucose target, even smaller benefits can be hypothesized and the number of patients required will further rise exponentially. in view of these complicating issues, it is highly unlikely that such a large trial will be performed in the very near future. in the absence of such results from generalizability studies, centers may consider the current evidence strong enough to implement the intervention in clinical practice. in that case, the clinical teams should be willing to implement all aspects of the package. this requires frequent blood glucose monitoring with accurate measurement tools by trained staff and nurses, validated guidelines for insulin treatment adjustment using accurate equipment for insulin administration, and all the educational steps that are required before embarking on such a complex intervention. it can be done, as shown elegantly by the report from a study on the transition to strict blood glucose control in a prestigious cardiac surgery center in aalst, belgium. but, in view of the complexity of the intervention, it will probably require the development of new accurate continuous sensors for blood glucose monitoring, ideally in combination with a closed - loop algorithm, before it will be adopted widely. such systems do not exist at this moment, although several companies are working on this development. finally, it is the responsibility of the icu teams to carefully document any impact on outcome of implementation of a novel intervention in clinical practice, in order to guarantee patient safety.	drugs used in the perioperative period could have an effect on survival as recently pointed out by an international consensus conference on the reduction in mortality in cardiac anesthesia and intensive care. insulin infusion to achieve a strict glycemic control is the best example of how an ancillary (i.e. non - surgical) drug / technique / strategy might influence survival rates in patients undergoing cardiac surgery. the author of this expert opinion presents her insights into the use of insulin in this setting and suggest that based on available evidence based medicine, insulin infusion, titrated to normoglycemia is a complex intervention, that not only requires the simple administration of a drug, the hormone insulin, but also needs tools and skills to accurately measure and control blood glucose to achieve normoglycemia while avoiding hypoglycemia and large glucose fluctuations.
the non - hodgkin 's lymphomas (nhl) are a group of b - cell and t - cell neoplasms that arise primarily in the lymph nodes. nhls of the oral cavity are rare and account for only 35% of the lymphomas reported. follicular lymphoma (fl) accounts for approximately 30% of adult nhl and is generally indolent in its clinical course with long survival rates, but it is currently incurable. a subset of fl cases transform into diffuse large b - cell lymphoma (dlbcl / dlbl) in 2560% of patients associated with a rapidly progressive clinical course, refractoriness to treatment and short survival. dlbl is the one of the common variants of nhls involving around two - third of the cases. primary lesion principally occurs in the areas of lymphoid tissue such as oropharynx, bone marrow, intestine and skin. dlbl is often associated with systemic symptoms such as night sweats, weight loss (10%) and fever. intraorally, lesions can occur as hard and diffuse tumors involving buccal vestibule, gingiva and the posterior region of the hard palate. in bone tissue, they may cause mild pain, paresthesia and swelling ; the radiographic examination often shows irregular radiolucid images. dlbl can occur in soft tissue as well as bone and sometimes it is difficult to determine its initial site of origin. dlbl corresponds to 68% of cases of nhl of the oral cavity and the average age of highest prevalence is 66 years. dlbcl is characterized by a diffuse proliferation of large neoplastic b cells having nuclear size equal to or exceeding normal macrophage nuclei or it can be more than twice the size of a normal lymphocyte. it is a heterogeneous neoplasm with marked biological heterogeneity and highly variable clinical course. here, we report a case of fl transforming into anaplastic dlbcl affecting the oral cavity of a 50-year - old man, along with clinical, histopathological and immunohistochemical features. a 50-year - old male patient reported to the dental hospital with a complaint of painless swelling in the lower left body of the mandible since 25 days following the extraction of left lower 1 and 2 molar teeth. the patient had a history of enlarged submandibular lymph node and fever since 15 days which was earlier thought to be due to infection after extraction. the swelling was insidious in onset and gradually increased in size causing facial asymmetry [figure 1 ]. examination revealed a nontender, solitary diffuse swelling measuring 3.5 cm 3.5 cm in the lower left body of the mandible. intraorally, the swelling extended from left premolar till left ascending ramus of mandible obliterating the buccal vestibule, with buccal cortical plate expansion [figure 2 ]. swelling on the left side of mandible causing facial asymmetry intraoral swelling involing left alveolar process of the mandible orthopantomogram of the patient was done to evaluate the invasion of the lesion and pathological fracture of lower left body of mandible [figure 3 ] was seen. orthopantomogram showing pathological fracture of the left lower jaw fine - needle aspiration cytology was done, but no aspirate was obtained. the hematological tests (complete blood count, fasting blood glucose, prothrombin time, partial thromboplastin time, erythrocyte sedimentation rate) were normal and the patient was hiv negative. correlating the history and clinical features, a provisional diagnosis of squamous cell carcinoma was made. histopathological picture showed the lesion to be composed of dysplastic parakeratinized stratified squamous epithelium overlying connective tissue stroma. basement membrane is indistinct at few places with infiltrating epithelial cells seen in the connective tissue. there are diffuse proliferations of large atypical lymphoid cells showing dysplastic features such as nuclear and cellular pleomorphism, nuclear hyperchromatism, increased number of nucleoli and abnormal mitotic figures. the above - mentioned findings favored the diagnosis of large cell lymphoma [figure 4a and b ]. (b) photomicrograph shows diffuse large lymphoblast in sheets showing nuclear and cellular pleomorphism with multiple nucleoli (black arrow) (h&e stain, x400) following this, immunohistochemical analysis was done for the expression of pan b - cell markers cd20, cd3, bcl-2, cd5, cd10 and ki67. cd20 and bcl-2 were positive in medium and large lymphoid cells in follicle and diffuse pattern. cd3 and cd5 were positive in few scattered lymphocytes. cd10 was positive in few focal areas and ki67 was positive in 30% area [figure 5a f ]. based on immunohistochemical report, the lesion was diagnosed as dlbcl anaplastic variant (70%) and fl, grade 3a (30%). (a) immunohistochemistry showing cd20 positivity (ihc stain, x40), (b) bcl-2 positivity in medium and large lymphoid cells in follicle and diffuse pattern (ihc stain, x40), (c) cd10 was positive in few focal areas (ihc stain, x40), (d) ki67 was positive in 30% areas (ihc stain, x40), (e) cd3 was positive in few scattered lymphocytes (ihc stain, x40), (f) cd5 were positive in few scattered lymphocytes (ihc stain, x40) based on histopathological examination and immunohistochemical study, the following lesions under differential diagnosis were ruled out squamous cell carcinoma, hodgkin 's lymphoma, large cell lymphoma of t - cell type, burkitt 's lymphoma, undifferentiated carcinoma and amelanotic melanomas. based on the final diagnosis, chemotherapy was advised by the oncologist, but the patient died within 1 month. malignant lymphomas involve a group of neoplastic proliferation process of the lymphocytes and their precursor cells. histologically, hodgkin 's lymphoma is characterized by the presence of multi - nucleated reed sternberg cells while all other neoplasms of lymphoid system named as nhl are derived predominantly from the cells of b - lymphocyte series. nhl arises primarily within the lymph nodes and up to 40% of cases present with extranodal presentation. nhl showing oral manifestations accounts for only 35% of cases and are rarely the initial manifestation of the disease. in the oral cavity, the most common locations are maxilla (11%), mandible (8%), palatal soft tissue (8%), vestibule and gingiva (7%). fl is one of the most common subtypes of nhl accounting for 22% of all cases worldwide. several analyses of genetic alterations that appear to affect the risk for fl transformation to dlbcl have been reported, including c - myc translocation, p53 mutation, deletions of the tumor suppressor genes p15 and p16 and chromosomal 6q23 - 26 and 17q aberrations. however, immunohistochemical analyses of fl shows it to be positive for the pan - b - cell marker cd20, cd10, bcl-2 and bcl-6. the who classifications of 2001 and 2008 recognized three grades - fl1 - 2, fl3a and fl3b [table 1 ], based on the number of centroblasts present per high power field ; the difference between fl3a and fl3b is the presence of a mixture of centrocytes and centroblasts in fl3a and the presence of follicles existing exclusively of centroblasts, immunoblasts or both in fl3b. the fact which supported the continuation of a grading system is that grades predict outcome, higher grades being associated with poor clinical outcome and more rapid progression to dlbcl. grading of follicular lymphoma (who 2008) dlbcl constitutes a heterogeneous group of lymphoid neoplasms with a diverse spectrum of features, response to therapy and survival. the normal architecture of lymph nodes is morphologically replaced by cells in a diffuse pattern. neoplastic cells are characterized by at least twice the size of normal lymphocytes with vesicular nuclei, prominent nucleoli and basophilic cytoplasm. the most common presenting symptoms of extranodal nhl in the head and neck region include pain, discomfort and local swelling in the region of involvement. clinical signs include localized swelling causing facial asymmetry, destruction of hard and soft tissue and ulceration. lymphomas can be diagnosed using hematoxylin and eosin - stained sections, but the most common technique currently is immunophenotyping. modern hematology relies on immunophenotyping to distinguish between benign and malignant diseases, as well as for a more detailed subtyping. the dlbcl can be subclassified depending on cytomorphology, gene expression profile and prognosis [table 2 ]. subclassification of diffuse large b - cell lymphoma affecting oral cavity dlbcl usually expresses pan b - cell markers lacking one or two such as cd19, cd20, cd75, cd79a, pax5 and cd22. in 5070% cases, surface and or cytoplasmic immunoglobulins (immunoglobulin m > immunoglobulin g > immunoglobulin a) dlbcl show homogeneously bright staining for cd20 showing differentiation from mature precursor b - cell until the preplasma cell. cd10 is a membrane metalloproteinase expressed in germinal centers (gcs) of secondary follicles. cd3 is expressed by t - cell lineage and transmits the activation signal to the cytoplasm. ki67 is a proliferation marker and it defines growth fraction of actively cycling cells. in dlbcl, the expression varies from 30 to 100% and tumors with ki 67 expression of > 80 % are termed as highly proliferative or aggressive tumors. bcl-2 protein functions as an anti - apoptotic protein associated with worse overall prognosis according to some studies and 3060% of dlbcl cases show its expression. their expression in dlbcls has been found in a majority of the cases ranging from 57% to 100%, but the biological significance of this expression is not clear. multiple myeloma oncogene 1 (mum1) is a lymphoid - specific member of the interferon regulatory factor family of transcription factors and is associated with worse overall survival rate. in dlbcls, mum1 is expressed in 5075% of cases and is seen both with and without bcl-6 expression. in normal b - cell differentiation, the b - cells go through the pre- and post - gc stages (non - gcb or activated b - cell - like). pre- germinal center b (gcb) cells are virgin b - cells while gc consists of small blast cells, centroblasts, centrocytes and occasionally plasma cells. the b - cells that leave the gc and enter the post - gc phase, differentiate either toward the memory cells or plasma cells. gcb lesions express genes normally expressed by gcb cells and the non - gcb dlbcl expresses genes normally induced during in vitro activation of peripheral blood b cells. more than 25% of dlbcl have a translocation t (14;18) and most of them show bcl-2 with or without a translocation. chromosomal rearrangements affecting the bcl-6 gene (regulator of germinal center formation) at 3q27 are seen in 30% of dlbcl extranodal tumors. the bcl-6 inhibits lymphocyte activation by inhibiting the expression of cd69 and cd44 and inhibits differentiation of gcb - cells toward plasma cells. current treatment of dlbcl usually begins with multiagent chemotherapy which includes typically cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone (chop). more than half the patients succumb to the disease in spite of initial response to therapy. early stage disease care involves either chemotherapy alone or a combination of chemotherapy and radiotherapy. the role of surgery is severely limited in the treatment of dlbcl. for advanced stage chemotherapy, other drugs which can be used in multiagent chemotherapy usually involve various combinations of methotrexate, bleomycin, doxorubicin, vincristine, dexamethasone and leucovorin, etoposide, mechlorethamine, procarbazine and cytarabine. this case report shows that diagnosis of these tumors is difficult considering the nonspecific nature of the presenting symptoms. an accurate morphologic and immunophenotypic diagnosis is required as early as possible so that to increase the life expectancy of the patient. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed.	follicular lymphoma (fl) is a common form of non - hodgkin 's lymphoma (nhl) with the ability to transform into a more aggressive disease, frequently to b cell - lymphoblastic lymphoma. diffuse large b - cell lymphoma (dlbcl) is a subtype of nhl, which is characterized by diffuse proliferation of large neoplastic b - lymphocytes. it accounts for 30% of all nhl and its occurrence in the mandible is very rare. it is often seen in young adults, but in the present case, a 50-year - old male patient presented with painless swelling in left lower jaw since 25 days following extraction of left lower molar teeth. there was a history of fever and submandibular lymph nodes were enlarged. on incisional biopsy, features of nhl - like lesion were observed and confirmed by immunohistochemistry using cd20, bcl-2, cd10, cd3, cd5, ki67 markers to be fl (3a) lymphoma transforming into dlbcl. this is a very uncommon presentation.
since 2003, valve implantation has evolved as a new therapy in patients with advanced emphysema.1,2 thereby, one - way valves that allow air to exit during expiration but block inspired air are implanted into the bronchi of the most emphysematous lobe, thus leading to lobar volume reduction. by this mechanism, valve therapy minimizes hyperinflation, which impairs functional capacity and influences mortality in patients with copd.3 the first randomized controlled trials (rcts) related to valve therapy, known as vent and euro - vent, demonstrated that particularly patients with complete inter - lobar fissure on high - resolution computed tomography (ct) and thus low interlobar collateral ventilation (cv) who had received a complete occlusion of the target lobe experienced relevant clinical improvement.4,5 based on this knowledge, only patients who fulfill these prerequisites were enrolled in the following recently published rct.6 in the stelvio trial, 25 patients with absent cv were treated with valves and compared with 33 patients of a control group.6 efficacy data demonstrated a statistically significant and clinically relevant outcome in the valve group. the adjusted patient selection in comparison to the vent influenced the spectrum of adverse events and led to a higher rate of complications of pneumothorax rate was reported at 4.2%, whereas pneumothorax rate increased up to 18% in stelvio. in further trials, the authors reported even higher pneumothorax rates of 20%25%.7,8 it is likely that a parenchymal rupture of the ipsilateral, untreated lobe due to a rapid expansion because of the volume reduction of the treated lobe is the reason for the occurrence of pneumothorax. although pneumothorax is a severe complication that may present a life - threatening situation and is associated with prolonged hospitalization, immobilization, and further invasive interventions, it does not appear to have a negative impact on patients outcome. one retrospective trial evaluated the clinical outcome of 25 patients who developed a pneumothorax following valve placement.9 despite the pneumothorax, patients achieved a substantial lobar volume reduction of 65%36%, which was associated in most cases with excellent clinical outcomes. however, as the number of patients is very low in this study population, it is not possible to make a general statement on the impact of pneumothorax on clinical outcomes. therefore, a further retrospective analysis evaluating the impact of pneumothorax on outcome measures following valve placement was performed in order to better assess the influence of this common complication. the database was queried for patients with severe emphysema, who were treated by endoscopic valve therapy between january 2009 and december 2013 at the thoraxklinik at the university of heidelberg. in this analysis, clinical outcome measures and radiological outcomes of patients who developed pneumothorax during a follow - up of up to the protocol of this trial was approved by the local ethics committee of the university of heidelberg (s-609/2012). the majority of the patients were treated within various prospective trials after written informed consent was obtained. as the data in this current analysis all patients with severe emphysema who experienced a pneumothorax following the placement of endobronchial valves (pulmonx, neuchatel, switzerland) and/or intrabronchial valves (olympus corporation, tokyo, japan) into the most emphysematous lobe were enrolled in the analysis. all patients treated with valves suffered from severe emphysema, which had been confirmed in baseline lung function tests demonstrating a significant forced expiratory volume in 1 second (fev1) reduction and severe hyperinflation. furthermore, each patient had undergone multi - detector ct, including software analysis (yet another ct analyzer, yacta) and perfusion scan in order to identify the most emphysematous lobe as the target lobe. the prevalence, onset, duration, and management of pneumothorax following valve treatment were assessed. in the case of pneumothorax, the subsequent procedure (chest tube insertion, valve removal, video - assisted thoracoscopy or thoracotomy) was dependent on the extent of the pneumothorax and/or clinical symptoms. the treatment algorithm for a post - interventional pneumothorax was derived from the expert statement published in 2014.10 in brief, the first step in symptomatic or large pneumothorax is the immediate insertion of a chest tube. in case of an ongoing air leak for > 7 days or in case of a lack of full lung re - expansion, the removal of one valve should be considered in order to inflate the target lobe again and thus re - establish pleural contact. if the air leak continues or no re - expansion is achieved, all valves should be removed. if these interventions remain unsuccessful, pleurodesis or surgical intervention needs to be considered. in summary, the pneumothorax management can vary depending on the clinical signs, the amount of air leak, and the existence of soft tissue emphysema. lung function parameters (vital capacity [vc ], fev1, residual volume [rv ], total lung capacity [tlc ]), exercise test (6-minute walk test [6-mwt ]), and dyspnea score (modified medical research council [mmrc ]) were collected from each patient prior to valve placement and 3 months after recovering from pneumothorax. for each patient, all chest x - rays and the patients were divided into two subgroups depending on whether a complete lobar atelectasis could be observed. the patients with complete lobar atelectasis as the maximum radiological result following valve placement belonged to the atelectasis group, whereas the other subgroup consisted of patients with partial atelectasis, dystelectasis or no change in volume. statistical analysis was performed using spss statistics (ibm spss statistics for windows, version 22.0 ; ibm corporation, armonk, ny, usa). data are presented as mean standard deviation, minimum and maximum, or n and % for frequency data. changes from baseline to follow - up in lung function, exercise capacity, and mmrc were expressed by descriptive statistics (mean, range, standard deviation). statistical comparison for the pre - interventional baseline examinations versus follow - up examinations was made using the paired two - sided t - test. missing data were imputed by an additional multivariate imputation on the basis of the patients baseline values. all patients with severe emphysema who experienced a pneumothorax following the placement of endobronchial valves (pulmonx, neuchatel, switzerland) and/or intrabronchial valves (olympus corporation, tokyo, japan) into the most emphysematous lobe were enrolled in the analysis. all patients treated with valves suffered from severe emphysema, which had been confirmed in baseline lung function tests demonstrating a significant forced expiratory volume in 1 second (fev1) reduction and severe hyperinflation. furthermore, each patient had undergone multi - detector ct, including software analysis (yet another ct analyzer, yacta) and perfusion scan in order to identify the most emphysematous lobe as the target lobe. the prevalence, onset, duration, and management of pneumothorax following valve treatment were assessed. in the case of pneumothorax, the subsequent procedure (chest tube insertion, valve removal, video - assisted thoracoscopy or thoracotomy) was dependent on the extent of the pneumothorax and/or clinical symptoms. the treatment algorithm for a post - interventional pneumothorax was derived from the expert statement published in 2014.10 in brief, the first step in symptomatic or large pneumothorax is the immediate insertion of a chest tube. in case of an ongoing air leak for > 7 days or in case of a lack of full lung re - expansion, the removal of one valve should be considered in order to inflate the target lobe again and thus re - establish pleural contact. if the air leak continues or no re - expansion is achieved, all valves should be removed. if these interventions remain unsuccessful, pleurodesis or surgical intervention needs to be considered. in summary, the pneumothorax management can vary depending on the clinical signs, the amount of air leak, and the existence of soft tissue emphysema. lung function parameters (vital capacity [vc ], fev1, residual volume [rv ], total lung capacity [tlc ]), exercise test (6-minute walk test [6-mwt ]), and dyspnea score (modified medical research council [mmrc ]) were collected from each patient prior to valve placement and 3 months after recovering from pneumothorax. for each patient, all chest x - rays and the patients were divided into two subgroups depending on whether a complete lobar atelectasis could be observed. the patients with complete lobar atelectasis as the maximum radiological result following valve placement belonged to the atelectasis group, whereas the other subgroup consisted of patients with partial atelectasis, dystelectasis or no change in volume. statistical analysis was performed using spss statistics (ibm spss statistics for windows, version 22.0 ; ibm corporation, armonk, ny, usa). data are presented as mean standard deviation, minimum and maximum, or n and % for frequency data. changes from baseline to follow - up in lung function, exercise capacity, and mmrc were expressed by descriptive statistics (mean, range, standard deviation). statistical comparison for the pre - interventional baseline examinations versus follow - up examinations missing data were imputed by an additional multivariate imputation on the basis of the patients baseline values. from january 2009 to december 2013, 381 patients with severe emphysema received endoscopic valve therapy. the mean age was 64 years (range : 4181 years), and 52% of them were males. the mean fev1 was 0.8 l and the mean rv was 262%21% predicted (table 1). overall, 18% (70/381) of the patients developed a pneumothorax as a complication following valve implantation (figure 1). in 51 out of 65 patients (79%), pneumothorax occurred within the first 3 days following intervention (median time to pneumothorax was 1 day [range : 0125 days ]). in the remaining five patients, pneumothorax onset and duration were unknown, as these patients developed pneumothorax outside the hospital and were referred to other centers. in 13% (9/70) of the patients, pneumothorax resolved under careful observation. in 87% (61/70) of the patients, chest tube insertion was necessary. in 51% (31/61) of these patients, chest drain insertion did not lead to the resolution of pneumothorax, so valve removal was required. despite chest tube drainage and valve removal, a persistent fistula was observed in 45% (14/31) of the patients, necessitating a talc slurry in one patient and additional surgical interventions in 13 patients (video - assisted thoracoscopy 77% [10/13 ], thoracotomy 15% [2/13 ] or both 8% [1/13 ]). during the first 3 months after valve explantation, 5 of the 31 patients in whom valve removal had been performed underwent reimplantation of the valve to achieve a lobar occlusion again. three months after recovering from pneumothorax, the patients experienced a modest improvement in lung function parameters, 6-mwt and mmrc (table 3). in 60% of the patients with pneumothorax (42/70), however, these patients did show a statistically significant improvement in rv (%) and tlc (% ; table 4). thirty - two percent (11/34) of these patients met the efficacy threshold of > 100 ml improvement in fev1, and 54% (14/26) of the patients experienced a 6-mwt improvement of > 262 m.11,12 fifteen percent (5/34) of the patients experienced an fev1 worsening of > 100 ml and 31% (8/26) developed a decrease in the 6-mwt of > 262 m (figure 2a and b). in 40% (28/70) of all pneumothorax patients, a lobar atelectasis was observed radiologically. in 64% (16/28) of the patients, atelectasis was confirmed after recovering from pneumothorax, and in 36% (12/28) of the patients, atelectasis was confirmed prior / during pneumothorax. three months following pneumothorax, valve therapy was associated with significant improvement in all lung function parameters except vc (table 5). forty - two percent (8/19) of the patients met the efficacy threshold of > 100 ml improvement in fev1, and 56% (9/16) of the patients experienced a 6-mwt improvement of > 262 m. in 5% (1/19) of the patients, an fev1 worsening of > 100 ml and in 25% (4/16) of the patients, a decrease in the 6-mwt of > 262 m could be observed (figure 2a and b). in 54% (15/28) of the patients, atelectasis was still confirmed 3 months after recovering from pneumothorax. regarding only these 15 patients with persistent lobar atelectasis 3 months after recovering from pneumothorax, great improvements were observed (vc : 299463 ml, vc : 11.7%15% predicted, fev1 : 122143 ml, fev1 : 5.1%5.4% predicted, rv : 995744 ml, rv : 43.8%33.9% predicted, tlc : 653573 ml, tlc : 10.7%9.1% predicted, 6-mwt : 22.8 m56.8 m, mmrc : 0.81.6 points). efficacy of endoscopic valve therapy in patients with severe emphysema and low cv was demonstrated in various different rcts.5,6,13 although valve placement is a minimally invasive therapeutic approach, it is associated with complications of which pneumothorax is the most common. in this analysis, the pneumothorax rate of 18% was comparable with the rates of 18%25% reported in other trials.68 regarding the management of pneumothorax, 87% of patients had to undergo chest tube insertion. a comparably high rate of chest drainage of 83% could also be seen in the recently published stelvio trial.6 the rates of necessary valve explantation of 44% in the current analysis and 50% in the stelvio were also similar. in 78% of the patients, the pneumothorax occurred during the first 3 days following valve placement, so that a hospital stay for 4872 hours for surveillance should be recommended routinely.10 in 22% of the patients, however, pneumothorax occurred after hospital discharge. therefore, it is of great importance to inform the patients of the symptoms of pneumothorax and explain to them to seek medical help quickly. one retrospective analysis published in 2014 based on the data of 25 patients with pneumothorax following valve implantation revealed that the patients will nevertheless experience a good outcome with a target lobe volume reduction of 65%36% following valve implantation.4,5,9,14 the current analysis of 70 patients with pneumothorax also confirms that a pneumothorax generally has no negative impact on the clinical status. thereby, mainly patients in whom lobar atelectasis occurred despite pneumothorax will experience a clinically relevant improvement in lung function parameters. the abscence of mean improvement of exercise capacity measured by the 6-mwt, despite the statistically significant improvement in lung function parameters, may result from the prolonged immobilization and the subsequent muscle wasting.15 the patients without a significant tlvr which represents the majority of all pneumothorax patients experienced only a slight mean improvement in lung function parameters. overall, 36% (19/53) and 55% (23/42) of all pneumothorax patients met the responder criteria for fev1 and the 6-mwt, respectively. in 11% (6/53) and 29% (12/42) of the patients, a clinically significant worsening of fev1 and 6-mwt overall, a great variability in the clinical outcome following valve therapy was observed in the total patient cohort. another retrospective analysis demonstrated that in patients who develop lobar atelectasis following valve therapy, the changes in lung function parameters, exercise capacity, and dyspnea score depend on different variables, such as the emphysema score of the treated lobe, 6-mwt, vc, and rv at baseline.16 it can be assumed that the outcomes following pneumothorax are also influenced by various baseline parameters, resulting in such a great variability. one explanation for the missing target lobe volume reduction in the majority of patients is the necessity for valve removal because of the pneumothorax. in 44% of all pneumothorax patients, valve removal was necessary due to persistent collapse of the lung despite chest tube insertion. these patients will not benefit significantly from valve therapy as only a complete occlusion of one lung lobe is associated with a good outcome.17 another explanation may be valve dislocation or dysfunction due to movement of lung parenchyma and bronchi in case of pneumothorax also resulting in a lack of target lobe volume reduction. efficacy data showed that pneumothorax is not associated with a clinically relevant improvement in the majority of patients, but it impairs the clinical status only in a minority.. nevertheless, the evaluation of pneumothorax predictors to find a balance between efficacy and safety and the evaluation of a prevention strategy of pneumothorax are crucial to minimize this anticipated adverse event of endoscopic lung volume reduction in emphysema patients. in one retrospective analysis, various ct parameters and clinical variables were found to be significant predictors of pneumothorax.18 these findings, however, will have to be confirmed in a prospective study. another retrospective trial evaluating a strategy to prevent pneumothorax found a reduction in risk by maintaining a 48-hour bed rest and the prescription of an antitussive therapy following valve placement. however, the number of treated patients in this analysis was too low to allow a general statement for or against this modified post - interventional strategy.7 in summary, pneumothorax is a frequent complication following endoscopic valve therapy in emphysema patients. although pneumothorax does not impair the clinical status in the majority of patients during follow - up, this complication needs to be considered a serious adverse event and an emergency in patients with advanced emphysema and limited respiratory reserve. adequate pneumothorax management, including chest tube insertion, valve removal, or surgical interventions, should be available in any institution performing endoscopic valve therapy. further research evaluating pneumothorax predictors and prevention strategies are crucial to balance the risk benefit ratio for patients undergoing endoscopic valve therapy.	introductionvalve implantation has evolved as a therapy for patients with advanced emphysema. although it is a minimally invasive treatment, it is associated with complications, the most common being pneumothorax. pneumothorax occurs due to the rapid target lobe volume reduction and may be a predictor of clinical benefit despite this complication.objectivethe objective of this study was to conduct an exploratory data analysis of patients who developed a pneumothorax following endoscopic valve therapy for emphysema.materials and methodsthis study performed a retrospective evaluation of pneumothorax management and the impact of pneumothorax on clinical outcomes in 70 patients following valve therapy in 381 consecutive patients.resultspneumothorax rate following valve therapy was 18%. pneumothorax management consisted of chest tube insertion, valve removal, and surgical intervention in 87% (61/70), 44% (31/70), and 19% (13/70) of the patients, respectively. despite pneumothorax, patients experienced modest but significant improvements in lung function parameters (forced expiratory volume in 1 second : 55148 ml, residual volume : 390964 ml, total lung capacity : 348876 ; all p<0.05). persistent lobar atelectasis 3 months after recovering from pneumothorax, which was associated with relevant clinical improvement, was observed in only 21% (15/70) of the patients.conclusionpneumothorax is a frequent severe complication following valve therapy that requires further intervention. nevertheless, the pneumothorax does not impair the clinical status in the majority of patients. patients with lobar atelectasis benefit after recovering from pneumothorax in terms of lung function parameters.
rheumatoid arthritis (ra) is a chronic systemic autoimmune disease characterized by nonspecific inflammation of peripheral joints, destruction of articular tissues, and deformities in the joints. as the disease progresses, there are enhanced chances of bone damage and destruction of cartilage causing substantial disability. the pathological conditions of ra are well known such as the leukocyte infiltration, a chronic inflammation, pannus formation, and extensive destruction of the articular cartilage and bone. the exact cause of ra is not yet known. in particular, it was reported that the inflammatory cytokines, such as tumor necrosis factor- (tnf-), interleukin- (il-) 1, and il-6, play key roles in the inflammation and joint damages during the development of ra. epidemiology of the arthritis in female to male is 3 : 1 and the prevalence is 1% of the world population. nonsteroidal anti - inflammatory drugs (nsaids) are widely used for the treatment of rheumatism diseases, such as rheumatoid arthritis and pain. the pharmacological effects of nsaids are due to inhibition of a membrane enzyme called cyclooxygenase (cox) which is involved in the prostaglandin biosynthesis. in spite of their extensive usage, nsaids are associated with many adverse effects like myocardial infarction : rofecoxib, gastric irritation (indomethacin), loose stool, nausea, vomiting, and dyspepsia (ketorolac). chronic usage of aspirin may also lead to gastric ulceration and liver damage [6, 7 ]. nonsteroidal anti - inflammatory drugs are widely used in the treatment of a number of inflammatory conditions, but gastrointestinal (gi) lesions have often limited their clinical utilization. topically applied nsaids rarely exhibit systemic side effects and most of the side effects are dermatological in nature - like rashes and/or pruritis. the fact of nsaids associated gastric damage is well explored for involvement of cox related pathway. the naturally occurring 1,8-dihydroxyanthraquinone derivatives (1,8 dad) were obtained from various families such as rhamnaceae (buckthorn, cascara), liliaceae (aloe), polygonaceae (rhubarbs), and caesalpiniaceae (senna). aloe emodin is an anthraquinone glycoside having antioxidant, neuroprotective, and in vitro anti - inflammatory activity due to inhibition of inducible nitric oxide (ino) and prostaglandin e2, via its action on murine macrophages. hence, taking into consideration reported activity of aloe emodin and molecular docking methodology, anthraquinone derivative was synthesized. the objective of forming this derivative was an attempt to reach an active anti - inflammatory agent with potent activity and selectivity toward cox-2. molecular docking studies were carried out on these compounds to identify the structured feature required for effective bind to cox-2 enzyme. hence, the present study was planned to explore possible modulation of pro - anti - inflammatory potential by modifying the aloe emodin into its derivative for evaluation of anti - inflammatory potential by considering the docking results, because no such study has been carried out in the past. (india), and complete freund 's adjuvant was obtained from sigma - aldrich (usa). the coordinates for the three - dimensional structure of cox-2 were obtained from protein data bank (http://www.rcsb.org/pdb/, entry code 4cox) [13, 14 ]. water molecules were removed and hydrogens were added to the structure of protein before docking. the structures of ligands (ae and aec) selected for the docking study were constructed using standard bond lengths and angles using advanced chemistry development (acd) chemsketch software. as the cocrystal structure of 4cox showed eight cavities, the selection of cavity for docking studies of the synthesized ligands the docking study of aloe emodin and its derivative was carried out using vlife mds docking software 3.0. the binding energy of ligands to the 4cox was calculated by using the following formula : gbinding = gcomplex (gligand + gprotein), gcomplex = g(ligand + protein), gprotein = the energy of the protein after optimization (uncomplexed), gligand = the energy of the ligand after optimization (uncomplexed), gbinding is binding energy required for a ligand to bind a protein. gbinding = gcomplex (gligand + gprotein), gcomplex = g(ligand + protein), gprotein = the energy of the protein after optimization (uncomplexed), gligand = the energy of the ligand after optimization (uncomplexed), gbinding is binding energy required for a ligand to bind a protein. synthesis of 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2 carboxylic acid from aloe emodin (see scheme 1) reaction. see scheme 2. procedure. in this procedure the oxidizing medium was prepared by dissolving 2.5 g of sodium nitrite in 12 ml of sulphuric acid ; the solution was heated to about 120c. one gram of aloe emodin was added in parts to this mixture over a period of 30 min. the reaction mixture was kept at this temperature for 3 h. at the end of 3 h the reaction mixture was poured into 700 ml distilled water at 2c to get orange brown precipitate (containing a mixture of 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2 carboxylic acid and the starting material aloe emodin). this was then dissolved in sodium carbonate solution ph below 9.5 and extracted with organic solvent. the precipitate is then filtered, washed, dried, and recrystallized from methanol to obtain pure compound with yield of 95%. albino wistar rats of either sex weighing 150250 g were used for present study. they were kept in polypropylene cages in an air - conditioned area at 22 3c in 1014 h light dark cycle. laboratory animal handling and experimental procedures were performed in accordance with cpcsea guidelines (approval number : 198/99). carrageenan induced paw edema in rats. anti - inflammatory activity of ae and aec was tested using the carrageenan induced rat paw edema model. experimental animals (wistar rats) were randomly divided into eight groups with six animals in each group. group ii (standard group) received diclofenac sodium at dose 10 mg / kg. groups iii v (ae) received aloe emodin at dose of 25, 50, and 75 mg / kg, respectively. groups vi viii (aec) received aloe emodin derivative at dose of 25, 50, and 75 mg / kg. the drugs were administered orally 1 h prior to the injection of 0.1 ml of freshly prepared suspension of carrageenan into the left hind paw of each rat. the paw volume was measured using a plethysmometer (ugo basile 7140, italy) at the time interval of 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, and 24 hr after administration of carrageenan. results were expressed as (1)edema volume = vtvc, where vt is paw volume in ml, at time t, after carrageenan administration. consider (2)inhibition rate % = ecetec100, where ec is edema volume of control group. adjuvant arthritis was induced as previously described by as modified by. on day 0, for the induction of arthritis, all the animals were anesthetized with intraperitoneal injections of 40 mg / kg thiopentone (0.3 ml/300 g rat) and arthritis was induced by the injection of 0.1 ml of complete freund 's adjuvant (cfa) containing 1.0 mg dry heat - killed mycobacterium tuberculosis per milliliter sterile paraffin oil into tibiotarsal joint of the left hind paw of female rats. the female wistar rats weighing 180230 g were divided into eight groups of six animals in each group as follows : group i : arthritic control / cfa (intraplantar injection of 0.1 ml cfa) ; group ii : standard treated with diclofenac sodium 10 mg / kg after intraplantar injection of 0.1 ml cfa, from 12th to 28th day ; groups iii v : treated with ae 25, 50, and 75 mg / kg, respectively, after intraplantar injection of 0.1 ml cfa, from 12th to 28th day ; groups vi viii : treated with aec 25, 50, and 75 mg / kg, respectively, after intraplantar injection of 0.1 ml cfa, from 12th to 28th day. group i : arthritic control / cfa (intraplantar injection of 0.1 ml cfa) ; group ii : standard treated with diclofenac sodium 10 mg / kg after intraplantar injection of 0.1 ml cfa, from 12th to 28th day ; groups iii v : treated with ae 25, 50, and 75 mg / kg, respectively, after intraplantar injection of 0.1 ml cfa, from 12th to 28th day ; groups vi viii : treated with aec 25, 50, and 75 mg / kg, respectively, after intraplantar injection of 0.1 ml cfa, from 12th to 28th day. the following parameters were measured. (a) paw volume evaluation (in ml). paw volume was measured on days 0, 4, 8, 14, 21, and 28 by using plethysmometer (ugo basile, 7140, italy). mean changes in injected and noninjected paw edema, with respect to initial paw volume, were calculated on respective day and % inhibition of paw edema with respect to untreated group was calculated using the following formula : (3)i=1v treatedv untreated100, where i is % inhibition of paw edema and v treated is mean changes in paw volume of treated rat. (b) visual arthritis scoring system. the visual arthritis scoring system described by [19, 20 ] was used to evaluate the severity of arthritis. in this scoring system each paw of animal was observed and separate score was given for each limb. the arthritis score ranged from 0 to 4, where 0 indicated the least but definite swelling and 4 represented the maximum swelling. the liver of each animal was removed rapidly and washed with ice cold tris buffer. liver of each animal was cut into small pieces and homogenized with homogenizer, so that clear homogenate is formed. homogenates were used for estimation of lpo (lipid hydroperoxide), gsh, sod, and catalase. reduced glutathione (gsh) in liver was estimated according to the method described by. the concentration of reduced glutathione was expressed as g of gsh / g of tissue. the sod activity (units / mg of tissue) was calculated by using the standard plot. the catalase (cat) activity was determined according to the method of [23, 24 ]. the lpo (lipid hydroperoxide) end product malondialdehyde (mda) measured in the homogenate was estimated by using method of. the concentration of lipid peroxidation was expressed as nmol / g of mda / g of tissue. the accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (no), was determined with a colorimetric assay with griess reagent (0.1% n-(1-naphthyl)ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid)., blood was withdrawn through retroorbital plexus puncture from all groups by under light ether anesthesia and the hematological parameters like hemoglobin content, total wbc count, esr, and rbc were analyzed using culter cb-9000, chariot., blood was withdrawn through retroorbital plexus of all groups and the biochemical parameters sgpt (serum glutamate pyruvic transaminase), sgot (serum glutamic oxaloacetic transaminase), and alp were analyzed using standard kits followed by fully automated analyzer. the results of docking studies of anthraquinone derivatives (dock score and net binding energy g) on cyclooxygenase protein (4cox) using vlife mds were as shown in table 1. the interactions between the active amino acid residues of the protein and ligand molecules are enlisted and diagrammatically represented. figures 1 and 2 represent the van der waals and hydrogen bond interaction, respectively, of ae with 4cox protein. the amino acid involved, type of interaction, and ligand atom involved in interaction are shown in table 2. the amino acid involved, type of interaction, and ligand atom involved in interaction are shown in table 3. from the docking study it was observed that the common amino acids which interact with the common ligand were glu 553a, lys557a, asn560a, and thr561a. the compound aec was most active while compound ae was least active as per docking. in the carrageenan induced rat paw edema model of anti - inflammatory activity, the ae and aec showed a significant inhibitory effect of the edema formation from the first hour to the sixth hour and after twenty - four hours. the highest inhibitory effect was found in late phase, that is, after the third hour (p < 0.01) at doses of 50 and 75 mg / kg when compared with control group (table 4). treatment with diclofenac sodium (10 mg / kg), ae (50 and 75 mg / kg), and aec (50 and 75 mg / kg) showed significant decrease in injected paw edema volume on the 14th, 21st, and 28th day (p < 0.001) as compared to arthritic control group. group treated with aec 25 showed significant decrease in paw volume on the 28th day (p < 0.05) as compared to arthritic control (figure 4). groups treated with diclofenac sodium, ae (75 mg / kg), and aec (50 and 75 mg / kg) showed significant decrease in the noninjected paw edema volume on the 14th, 21st, and 28th day (p < 0.01) as compared to arthritic control. ae 50 treated groups showed significant decrease in the noninjected paw edema volume on the 14th day (p < 0.05) also on the 21st and 28th day (p < 0.01) as compared to arthritic control. aec 25 treated groups showed significant decrease in the noninjected paw edema volume on the 28th day (p < 0.05) as compared to arthritic control (figure 5). arthritic control group treated with freund 's complete adjuvant showed increase in the arthritic index from the 4th day up to the 28th day, respectively, while diclofenac sodium treated group showed significant decrease in the arthritic index on the 14th, 21st, and 28th day (p < 0.01) as compared to arthritic control. groups treated with ae 50, ae 75, aec 50, and aec 75 showed significant decrease in the arthritic index, on the 21st and 28th (p < 0.001) day, respectively, while aec 25 treated groups showed significant decrease in the arthritic index on the 28th day (p < 0.05) as compared to arthritic control (figure 6). there was rise in wbc count and decrease in rbc and hb count in arthritic control group. diclofenac sodium treated group showed significant decrease in wbc count, rheumatoid factor (rf), and erythrocyte sedimentation rate (esr) (p < 0.01), while it showed significant increase in rbc and hb count (p < 0.01), as compared to arthritic control. groups treated with ae 50, ae 75, aec 50, and aec 75 showed significant decrease in wbc count, rf, and esr (p < 0.01), while they showed significant increase in rbc and hb count (p < 0.01), as compared to arthritic control. group treated with aec 25 showed significant decrease in wbc count and rf (p < 0.05) when compared with arthritic control (tables 5 and 6). there was an increase in lpo and no level and decrease in gsh, cat, and sod level in arthritic control group. diclofenac sodium treated group showed significant decrease in the lpo and no level (p < 0.01) as compared to arthritic control and significant increase in gsh, cat, and sod (p < 0.01) as compared to arthritic control. groups treated with ae 50, ae 75, aec 50, and aec 75 showed significant increase in gsh, cat, and sod (p < 0.01) as compared to arthritic control. group treated with aec 25 showed significant decrease in lpo and no level (p < 0.05), while it showed significant increase in gsh, cat, and sod (p < 0.05) as compared to arthritic control (table 7). diclofenac sodium treated group showed significant decrease in sgpt, sgot, and alp levels (p < 0.01) as compared to arthritic control. groups treated with ae 50, ae 75, aec 50, and aec 75 showed significant decrease in sgpt, sgot, and alp levels (p < 0.01) as compared to arthritic control. group treated with aec 25 showed significant decrease in sgot level (p < 0.05) as compared to arthritic control (table 7). rheumatoid arthritis (ra) is a symmetric polyarticular arthritis that primarily affects the small diarthrodial joints of the hands and feet. in addition to inflammation in the synovium, which is the joint lining, the aggressive front of tissue called pannus invades and destroys local articular structures. the synovium is normally a relatively a cellular structure with a delicate intimal lining. in ra, cd4 + t cells, b cells, and macrophages hyperplasia of the intimal lining results from a marked increase in macrophage - like and fibroblast - like synoviocytes. locally expressed degradative enzymes, including metalloproteinases, serine proteases, and aggrecanases, digest the extracellular matrix and destroy the articular structures. the structure of cox-2 was obtained from protein data bank (pdb code : 4cox) and used as target for docking studies. the docking study was carried out on aloe emodin (ae) and carboxylic acid derivative of aloe emodin (aec). the docking results of aec were compared with that of ae based on various parameters such as dock score, bond interactions, and binding free energy. the docking result showed that the aec have favorable interactions with active site residues and also have favorable dock score and binding free energy as compared with aloe emodin (ae), indicating that aec may be having a better anti - inflammatory activity than aloe emodin. in the present study, ae and aec exhibited significant anti - inflammatory and antiarthritic activity. the carrageenan is known for its classic biphasic effect ; the first phase is mediated by release of histamine and serotonin during the first hour and release of kinins up to 2.5 h, while the second phase is mediated by release of prostaglandins from 2.5 to 6 h. it has been reported that the second phase is found to be sensitive to most of the clinically effective anti - inflammatory drugs [16, 28 ]. this model is sensitive, conventional, and accepted for screening of newer anti - inflammatory agents. in the present study, ae and aec showed dose - dependent inhibition of second phase of carrageenan induced rat paw edema, suggesting the inhibition of prostaglandins release. in the present study, rats were selected to induce arthritis because rats develop a chronic swelling in multiple joints with influence of inflammatory cells, erosion of joint cartilage, and bone destruction. the pathogenesis or reasons for development of arthritis following injection of complete freund 's adjuvant preparations include reactivity to cartilage proteoglycans, heat shock proteins, and interactions with intestinal flora [30, 31 ]. the animals on exposure to cfa (or mycobacteria) in the early phases induce the release of cytokines such as tnf-, il-12, il-6, and ifn- and several chemokines. the determination of paw swelling is simple, sensitive, and quick procedure for evaluating and assessing the degree of inflammation and the therapeutic and curative effects of drugs. the swelling increases in the initial phase of inflammation and then becomes constant in two weeks. these changes in paw volume are associated with increase in granulocytes and monocytes. in chronic inflammation activation of macrophages results in the production of several cytokines including il-1, il-6, interferon-, and tnf- which have been implicated in immune arthritis [36, 37 ]. il-6 is considered to play a central role in chronic inflammation and is expressed in excess at sites of inflammation. it also elicits the development of specific cellular and humoral immune responses such as b cell differentiation and t cell activation. tnf- is mainly involved in the perpetuation of the inflammatory cascades in autoimmune diseases, which affect connective tissues where the connective tissues become hypercontracted due to inflammation. prostaglandins greatly potentiate exudates by inducing relaxation of arteriolar smooth muscle cells, increasing the blood supply to the tissue. in the present study, the standard drug diclofenac sodium and test drugs ae and aec significantly suppressed the paw edema swelling induced by the complete freund 's adjuvant (cfa), around tibiotarsal joint and paws. the appearance of secondary lesions, that is, cfa - noninjected paw swelling, is a manifestation of cell - mediated immunity. the appearance of secondary lesions is due to development of biochemical reactions into cfa - noninjected hind leg which results in swelling around tibiotarsal joint and paw. the suppression of such secondary lesions by a drug shows its immunosuppressive activity [41, 42 ]. this reveals potent suppression by ae and aec of cell - mediated immunity in arthritic rats. a selective reduction in the secondary lesions distinguishes the immunosuppressive effects of a drug from its anti - inflammatory effects. the significant reduction of the secondary lesions by ae and aec as observed in this study indicates a possible immunosuppressant effect. the two most common explanations are gastrointestinal blood loss due to arthritis medications and bone marrow changes in patients with inflammatory arthritis, which prevents the release of iron for incorporation into red blood cells [44, 45 ]. in cfa - induced arthritis model, arthritic control rats showed reduced rbc count, reduced hb count, and increased erythrocyte sedimentation rate (esr) and rf levels. it is proposed that the reduction in the hb count during arthritis results from reduced erythropoietin levels, a decreased response of the bone marrow erythropoietin, and premature destruction of red blood cells. similarly, an increase in the esr is attributed to the accelerated formation of endogenous proteins such as fibrinogen and / globulin, and such a rise in the esr indicates an active but obscure disease process. the acute phase proteins in esr share the property of showing elevations in the concentration in response to stress or inflammations like injection, injury, surgery, and tissue necrosis. prominent immunologic abnormalities that may be important in pathogenesis of ra include immune complexes that are found in joint fluid cells and in vasculitis. rf is the immunological expression of an individual 's immune system reaction to the presence of an immunoglobulin molecule that is recognized as nonself. this response to the nonself immunoglobulin results in the presence of immune complexes ; these in turn bind to the complement and may eventually lead to destruction of synovium, cartilage, and bone. the higher the levels of serum rf are, the higher the development of inflammation is. determination of serum rf levels in rheumatoid arthritis is essential to understand and measure the disease progression and to facilitate the development of novel treatments for rheumatoid arthritis. serum rf is a marker of systemic inflammation and antibody production against the injected adjuvant. in cfa - induced arthritic rats, activated cd4 + t cells stimulate b cells to produce immunoglobulins, which are associated with increase in the plasma levels of serum rf [50, 51 ]. the ae and aec treated groups showed a significant recovery from the induced anemia and serum rf level. this indicates that anemic conditions occurring during the inflammation in rheumatoid arthritis can be recovered by the treatment of ae and aec. in arthritic condition there is a mild to moderate rise in wbc count due to release of il - ib inflammatory response. il - ib increases the production of both granulocyte and macrophages colony stimulating factor [52, 53 ]. in the present study, the migration of leucocytes into the inflamed area was significantly suppressed by the standard drug, ae and aec. the body has effective antioxidant mechanism to prevent and neutralize the free radical induced damage. this is accomplished by a set of endogenous antioxidant enzymes, such as sod and cat. when the balance between ros (reactive oxygen species) production and antioxidant defense is lost, oxidative stress results, which through a series of events deregulates the cellular function leading to various pathological conditions. biological systems have evolved an array of enzymatic and nonenzymatic antioxidant defense mechanisms to combat the deleterious effects of oxidative free radicals (ofrs). sod is a metalloprotein while cat is a hemoprotein, localized in the peroxisomes or the microperoxisomes. both superoxide dismutase (sod) and catalase play an important role in the detoxification of superoxide anion and h2o2, respectively, thereby protecting the cells against oxidative free radicals induced damage. h2o2 may be reduced by enzymes glutathione peroxide but, alternatively, may react again with superoxide anion to form free hydroxyl radicals, which have a greater toxicity and a longer half - life than superoxide anion. although catalase is significantly increased in rheumatoid arthritis its concentration is very low to expect considerable protection against h2o2 [55, 56 ]. in arthritis the lowered levels of sod activity may be due to the inhibition of the enzyme by hydrogen peroxide, which might be an indicator of high degree of superoxide anion production. the reduced cat level in ra is due to its inactivation by h2o2 and suggests that these enzymes may play an important role in the rheumatic process and increased oxidative stress. the gsh is a predominant low molecular weight thiol in the cytoplasm, which protects the tissue against in vivo toxicity of sulfhydryl binding toxicants [58, 59 ]. the level of gsh appears to be reflux mechanism to protect against extracellular free radicals in chronic arthritis. glutathione is endogenously synthesized in the liver and is the first line of defence against peroxidation. the reduced gsh, in turn, keeps up the cellular level of the active form of vit - c. lipid peroxidation (lpo) is an important marker of oxidative stress and is analyzed by malonaldehyde. increased ros levels in ra may result in a prooxidation environment, which in turn could result in increased mda levels. as a result, lpo may have a role in the pathogenesis of the ra. in the present study, ae and aec significantly decreased the lpo level in cfa - induced arthritis rats probably indicating the prevention of the cell damage by reducing oxidative stress. in present study ae and aec significantly increased the levels of sod, cat, and gsh possibly by preventing the inactivation of these enzymes by h2o2 or by reducing the oxidative stress. studies have revealed increased nitric oxide (no) levels in the serum and synovial fluids of arthritic patients owing to the upregulation of inducible nitric oxide synthase (inos), indicating thereby a role of no in arthritis. in experimental models of arthritis, selective inhibitors of inos no levels were found to be drastically increased in disease controls indicating oxidative stress due to inflammation. the ae and aec treatment have significantly prevented the rise in no ; the tentative mechanism maybe like treatment had prevented the formation of ros or helped to boost the natural antioxidant system of body by preventing the disturbance in normal function. assessment of liver injury is generally done by ascertaining the levels of biomarkers such as sgot, sgpt, and alp. elevated levels of these enzymes in serum suggest injury to the architecture of hepatic cells resulting in leaching of these enzymes into the circulation. the present study in which significant rise in the level of aminotransferase was observed in animals treated with freund 's complete adjuvant suggests that it might be released from the damaged cells of the liver. in the present study, ae and aec treated group showed a significant improvement in serum sgot, serum sgpt, and alp levels, thus indicating anti - inflammatory activity which may be due to the prevention of cell damage via restoration of natural antioxidants of body.	aloe emodin is isolated compound of aloe vera which is used traditionally as an anti - inflammatory agent. in vitro pharmacokinetic data suggest that glucuronosyl or sulfated forms of aloe emodin may provide some limitations in its absorption capacity. aloe emodin was reported to have in vitro anti - inflammatory activity due to inhibition of inducible nitric oxide (ino) and prostaglandin e2, via its action on murine macrophages. however, present work evidenced that molecular docking of aloe emodin modulates the anti - inflammatory activity, as well as expression of cox-2 (cyclooxygenase-2) in rodent. the aec (4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2 carboxylic acid) was synthesized using aloe emodin as starting material. the study was planned for evaluation of possible anti - inflammatory and antiarthritic activity in carrageenan rat induced paw oedema and complete freund 's adjuvant induced arthritis in rats. the ae (aloe emodin) and aec significantly (p < 0.001) reduced carrageenan induced paw edema at 50 and 75 mg / kg. complete freund 's adjuvant induced arthritis model showed significant (p < 0.001) decrease in injected and noninjected paw volume, arthritic score. ae and aec showed significant effect on various biochemical, antioxidant, and hematological parameters. diclofenac sodium 10 mg / kg showed significant (p < 0.001) inhibition in inflammation and arthritis.
the most critical difficulty with the concept of mci is that it is an arbitrary label on a continuum of cognitive changes that occur in people as they age. as a result the arbitrary nature of the label can be seen most explicitly in the neuropsychological criteria, which may specify the threshold for applying the terms (one or one and a half standard deviations less than age - matched controls), the composition of the battery, and the norms. the criteria concerning preserved or relatively preserved activities of daily living also permit considerable variability as to where the line is drawn by different clinicians. how complex must an impaired instrumental activity of daily living be before the label mci is applied ? for that matter, how simple should the task be before the affected person is said to convert to alzheimer 's disease (ad) ? differences in an individual 's performance of life 's tasks create both patient and clinician variability in perceptions as well as cross - cultural challenges in multinational studies (gaines a, whitehouse pj, unpublished data). the existence of a continuum of cognitive changes is illustrated by mci being bounded on one side by ad and on the other by labels such as age - associated memory impairment (aami) or age - related cognitive decline. the emergence of aami was also closely linked to attempts to develop medicines to treat this condition. the criteria for applying this label included demonstrating test performance one standard deviation below younger - age controls, thus creating a large number of older individuals who could be labeled with aami. yet whether mci is normal or not is at the heart of the conceptual and practical ambiguities associated with this concept. clinicians know logically that there is a time in the life course of a patient, who will eventually be diagnosed as having ad, when the symptoms are present, but not sufficiently severe to warrant the label dementia. any progressive medical condition must have a phase in which the symptoms are emerging, but not of sufficient intensity to warrant a disease label. in medicine, increasing attention is being paid to so - called preclinical states, such as in hypertension, depression, and parkinson 's disease. thus, it is not at all surprising that different variants of mci have been identified, including amnestic mci, mci with symptoms in several different cognitive domains, and mci with focal symptoms in an intellectual area other than memory. the mci associated with frontal lobe dementia and vascular dementia would more likely be predicted to be nonamnestic. the symptoms in mci are mild and perhaps more variable than in dementia ; therefore, it is not surprising that the outcomes of longitudinal follow - up studies and drug studies might also be more variable. the logically complete set of outcomes for a patient with mci includes no change over time, further deterioration or even improvement. all these outcomes can be demonstrated in epidemiological studies when patients are followed for several years. there are, however, some people who improve, at least for a period of time, suggesting either a benign course to their medical condition or a mislabeling of the individual in the first place, perhaps due to a bad testing day or mild depression. if one accepts the 15% annual conversion rate, one also has to ask what happens over a more extended period of time, such as that usually associated with epidemiological studies. at 15% a year, most people would have been expected to convert to ad within 10 years. this point returns us to the issue of mci as an arbitrary label on the continuum of cognitive aging and raises the unresolved question of whether all human beings would develop ad if they lived long enough. most studies of those in their 80s, 90s, and beyond suggest that the incidence of ad continues to increase with age. thus, the major conceptual challenge to the further development of drugs to treat mci is the ambiguity around definition and the relationship to normal aging. other challenges also exist in the development of trial designs to demonstrate the effectiveness and safety of drugs. most studies are classified as either trials to demonstrate symptomatic benefit or trials to demonstrate disease modification. most of the interest in mci surfaced because of the desire to develop medications to prevent ad. in order to conduct a primary prevention protocol, one needs to enter into the study individuals who do not currently suffer from dementia. one of the easiest ways to enrich the sample in a prevention study is to include people who already suffer from minor degrees of cognitive difficulties, as they are more likely to proceed to full dementia. of course, this begs the question as to whether this is primary prevention, or really secondary prevention in which the enrolled individuals were already suffering from a dementia at an early stage and the observed deterioration was the further progression of the already existing disease condition. the problem with conducting either primary or secondary prevention studies is that there are no agreedupon designs. survival analysis as promoted by the national institute of aging 's alzheimer 's disease cooperative study in their studies of vitamin e, for example, can not clearly differentiate a prolonged symptomatic benefit from a disease - modifying or neuroprotective effect. the staggered - start, staggered - stop design, elaborated most clearly by leber, has been used in a few studies. however, it has been difficult for regulators to interpret the complex slope changes necessary to make the claim that a drug is disease - modifying. although considerable effort has been placed in developing biological markers, particularly neuroimaging, no test can currently replace a clinical diagnostic process for mci. regulatory bodies will most likely not consider surrogate markers such as hippocampal atrophy unless they are clearly linked to clinical improvement. thus, from a regulatory perspective, we are puzzled to know what designs to use to demonstrate a disease - modifying process that prevents the conversion of mci to ad. attempts have also been made to demonstrate that medications provide symptomatic benefit for people with mci. such studies have been designed in a fashion parallel to those in ad, using outcome measures tailored to persons with less cognitive impairment. here, the conceptual challenges are less evident in developing the trial designs, but the practical implications of such studies are perhaps less clear. even if such studies show positive effects, what are the functional benefits to individuals and the pharmacoeconomic impacts on societies ? another area of conceptual and practical confusion that permeates the study of people with mild degrees of cognitive impairment is the overlap with the emerging field of cognitive enhancement. at what point on the continuum of cognitive aging is a drug not treating a disease, but rather enhancing a person 's normal ability to function intellectually. our pilot study of the use of donepezil in 53-year - old pilots flying in flight simulators suggests that cholinergic drugs may benefit individuals in their middle years who are performing complex cognitive tasks in society. studies of the biology of brain aging, particularly changes in neurotransmitter systems, support the idea that persons with ad and mci lie on the continuum of neuronal alterations that begin perhaps quite early in life. a related conceptual and practical problem in developing drugs to treat mci is the overlap between western scientific allopathic medicine and so - called complementary and alternative medicine (whitehouse pj, juengst e, unpublished data). many individuals take herbal and nutraceutical products to try to improve their memories or slow the progression of age - related cognitive deterioration. such therapies to treat brain aging overlap with those designed to slow the aging process itself. practically, this means that decisions must be made about whether to enroll individuals in studies who are taking such products (and at what doses). conceptually, and from a regulatory perspective, it raises issues of what products are to be regulated by the food and drug administration (fda) and other drug regulatory bodies or monitored through other means or by no means at all. in both the usa and europe, there is increasing concern about the lack of oversight of such complementary and alternative medicines. yet, as we have seen vitamin e and ginkgo are examples of biological products that have been sold as essentially unregulated products, but that are also being studied scientifically. a final major area of challenge to the development of more effective drugs from mci is ethics. given current science and resources, what would a drug profile look like that cured or prevented ad ? what are the implications of such a term for the individual labeled with it and for their partner and potential caregiver (corner l, bond j, unpublished data) ? if i have a label of mci, does that mean that i do not have ad, that i have a mild form of ad or another dementia, or that i may or will eventually get dementia ? moreover, we already noted that some persons with the label mci improve. the implications of the term mci for an individual patient and clinician are closely linked to the fear of ad itself. perhaps in our enthusiasm for creating new medications, we have also intensified the terror that people feel about the possibility of suffering from dementia. perhaps the greatest ethical issue facing the development of drugs for cognitive impairment has to do with conflict of interest between researchers, physicians, and the drug industry. one of the lessons of the introduction of drugs to treat erectile dysfunction is that the line between disease and normality is thin. moreover, the ability to enhance cognition already motivates many people to take complementary and alternative medical products. the concern about the use of serotonin reuptake blockers to treat depression in childhood is but one example. a major challenge to biological psychiatry, but also to neurology, is maintaining the trust of our research participants and patients. one important issue that surfaced around the treatment of depression is the suppression of negative trials. we need to ensure that trials in dementia are entered into an international database and that the trial results made available to the scientific community or that research subjects are appropriately compensated. academic experts for hire as authors of papers in which their contributions are limited is another example of a major problem. the pharmaceutical industry is amazingly effective at not only selling their drugs, but also at influencing the very way we think about health. the amount of money put into drug treatments limits our incentive to think about alternative ways of addressing social problems due to various age - related cognitive challenges. the fda held a one - day hearing in march 2001 to address some of the conceptual issues surrounding the development of drugs for mci. although no official statement was made concerning the acceptability of mci as a therapeutic target, many experts interpreted the fda 's position that mci is very early dementia, most likely ad. further elaboration of this issue will likely require submission by industry of drugs for mci to the fda for consideration for approval. the regulatory process in the usa is relatively open. whether or not experts believe that a diagnostic entity is an appropriate target for drug development influences the regulators in the evaluation of protocols. the european medicines evaluation agency (emea) has not held open hearings about the concept of mci. such presentations suggest that the attitude in europe is similar to that in the usa, ie, the regulators will wait for more development in the field and for the submission of actual trials. in the fall of 2004, a group of investigators in canada will meet to examine the draft academic guidelines that were issued several years ago. regulators will be involved in the meeting, and the topic of mci and related conditions will be discussed. no regulatory bodies in asia have taken a stance on mci as a target condition. under the auspices of the international working group for the harmonization of dementia drug guidelines, organized for the first time 8 years ago by the author and currently by jean - marc orgogozo, three asian regional meetings have been held. from the first in singapore in 2001, to the second in china, and now this year in thailand, there has been growing interest in the concept of mci among academic opinion leaders in asia. of course, this is largely influenced by the western experts expressing their enthusiasm for the concept. attitudes toward the elderly and to age - related cognitive changes are different in asia. the back - translation into english of the term, mci, by a leading opinion leader in china is labeling millions of chinese with this term has some interesting social implications and potentially profound effects on attitudes toward the elderly in china. the international working group for the harmonization of dementia drug guidelines has had regulatory issues at the heart of its mission to promote global discussion about designs to treat mci, ad, and other conditions like vascular dementia. currently under the direction of lon schneider with input from other experts, including the author, the international psychogeriatric association is also planning a meeting for this winter (2004) on mci, which will involve discussions about regulatory issues. the fda held a one - day hearing in march 2001 to address some of the conceptual issues surrounding the development of drugs for mci. although no official statement was made concerning the acceptability of mci as a therapeutic target, many experts interpreted the fda 's position that mci is very early dementia, most likely ad. further elaboration of this issue will likely require submission by industry of drugs for mci to the fda for consideration for approval. the regulatory process in the usa is relatively open. whether or not experts believe that a diagnostic entity is an appropriate target for drug development influences the regulators in the evaluation of protocols the european medicines evaluation agency (emea) has not held open hearings about the concept of mci. such presentations suggest that the attitude in europe is similar to that in the usa, ie, the regulators will wait for more development in the field and for the submission of actual trials. in the fall of 2004, a group of investigators in canada will meet to examine the draft academic guidelines that were issued several years ago. regulators will be involved in the meeting, and the topic of mci and related conditions will be discussed. no regulatory bodies in asia have taken a stance on mci as a target condition. under the auspices of the international working group for the harmonization of dementia drug guidelines, organized for the first time 8 years ago by the author and currently by jean - marc orgogozo, three asian regional meetings have been held. from the first in singapore in 2001, to the second in china, and now this year in thailand, there has been growing interest in the concept of mci among academic opinion leaders in asia. of course, this is largely influenced by the western experts expressing their enthusiasm for the concept. attitudes toward the elderly and to age - related cognitive changes are different in asia. the back - translation into english of the term, mci, by a leading opinion leader in china is labeling millions of chinese with this term has some interesting social implications and potentially profound effects on attitudes toward the elderly in china. the international working group for the harmonization of dementia drug guidelines has had regulatory issues at the heart of its mission to promote global discussion about designs to treat mci, ad, and other conditions like vascular dementia. currently under the direction of lon schneider with input from other experts, including the author, the international psychogeriatric association is also planning a meeting for this winter (2004) on mci, which will involve discussions about regulatory issues. he doubts that more conferences alone will lead to consensus, since there have been many such conferences and the differences of opinion remain. at the 9th conference on alzheimer 's disease and related disorders in philadelphia, pa, in july 2004, this author received the impression of a growing split between the usa and europe. in fact, within the usa, the original mayo clinic concept of the mci (perhaps to be renamed petersen 's syndrome) is still meeting resistance. the main issue that remains is the need to address more seriously the continuum of aging. of course, such a reconsideration of the categories of age - related cognitive impairment would have implications for ad as well. despite the work in genetics and neuroimaging, we are having a harder time differentiating the various types of dementia from each other. this is most likely explained by the fact that the process of brain aging affects individuals in many different ways and our attempts to assign dementias into discrete categories are failing because of the overlap in biologies at work in our brain. neuronal loss occurs in multiple different systems to different degrees creating a wide spectrum of cognitive, affective, and motor symptoms. as i have suggested previously, it may be possible to treat cognitive impairment as a nonspecific symptom rather than a feature of different discrete conditions. the biological substrate for such a claim is that overall loss occurs continuously in various brain nuclei as we age. for example, loss of cells in the cholinergic basal forebrain occurs in a variety of dementias and with normal aging. cholinesterase inhibitors appear to work, albeit in modest ways, in a variety of dementias characterized by cholinergic pathology, including not only ad, but also parkinson 's disease, vascular dementia, and other overlapping conditions. psychosis, which may be considered a discrete category, exists in a variety of conditions ; agitation occurs on a continuum. drugs to treat agitation are being developed and submitted for approval based on finding positive effects in three or more conditions, like dementia and mental retardation. could we consider submitting a portfolio of results for a drug to treat cognitive impairment in several conditions and get a label for a general indication ? such an approach would not only avoid the problems of nosology in the field of dementia, which are only increasing, but allow the use of these medications to treat mild degrees of symptoms as well as more profound cognitive deficits. such an approach would greatly expand the market for potential therapies. the boundaries between what is a disease and what is normality would grow even more unclear with an approach that labels cognitive impairment on a continuum. the costs of drugs to our health care system would likely increase. as an advocate for the importance of pharmacoeconomic studies, especially studies of quality of life, i would urge that we stress the importance of such cost - utility approaches even in the current regulatory and reimbursement environment, and even if that would increase the size of the potential market. a focus on drug treatment for cognitive impairment first, we are constantly focusing on what is wrong with our cognition as we age. more emphasis on cognitive vitality and the potential for older people to further develop cognitively and gain wisdom would be helpful in society. moreover, a focus on drugs makes us think that the only answers to the challenges of cognitive aging lie in medicine and biology. clearly, there are many ways to prevent the deterioration that can occur in cognitive abilities as we age, besides waiting for a magic bullet. developing a sense of purpose, engaging in civic activities, and taking responsibility for one 's personal legacy are all activities that can contribute to a sense of cognitive vitality, even in persons who suffer from mci and ad.	the development of the concept of mild cognitive impairment (mci) and its practical application have been intimately tied to attempts to produce therapeutic agents. understanding the regulatory environment and determining the way in which it can be influenced are critical to the development of drugs and their eventual approval. in this article, we review some of the current challenges surrounding the concept of mci relevant to drug development, summarize activities in various regions of the world, and conclude with some suggested next steps and an alternative framing for approving drugs for mci and related conditions.
cv is a technique that measures current as a function of applied potential. this technique uses an electrochemical cell with three electrodes : working, reference, and counter. different working electrodes (e.g., pd, gc, or cp) are studied as the anode. this laboratory involves students in the use and interpretation of cyclic voltammograms and determination of electro - active surface area for different electrodes to evaluate the anode performance of the cell. this experience was divided into a prelaboratory and three experimental parts that gradually increase the level of reasoning. it has been implemented four times in the undergraduate instrumental analysis laboratory course. a total of 180 undergraduate students have been impacted so far. in each laboratory section, the students were divided in groups of 2 members for a total of 12 groups. the activity was done in two laboratory periods of 3 h each. in the first period, the following parts were carried out : discussion of the prelaboratory, preparation of the aqueous solutions and construction of the home - built cp electrode, including the measurement and calculation of the geometric area of the three different electrodes. in the second period, they worked on the pdnp ink preparation, modification and electrochemical testing of the electrodes, and data analysis for ethanol oxidation. the experience served to provide connections between the different components of fuel cells, nanotechnology, fundamental electrochemical concepts, surface area, and the use of the cv technique. as a prelaboratory activity, students performed a literature search to predict and select the best materials to construct a fuel cell anode. in addition, they determined the resistance to corrosion and chose the most appropriate electrode based on conductivity, resistance to corrosion, and cost. table 1 summarizes some of the properties that students considered to select the best material for the construction of the fuel cell anode. this activity was done by the students as homework and discussed in the classroom. standard reduction potentials are referred to aqueous solutions at 25 c, 1 atm. versus ag / agcl (3 m nacl). the undergraduate students constructed a home - built cp electrode in the laboratory using mineral oil, graphite, and a copper wire (see supporting information). once the electrode was constructed, the students measured the diameter of the cp surface using a ruler and calculated the geometric area. they then repeated the same procedure for the different commercial electrodes : pd and gc. in this session, students were exposed to the concepts of nanotechnology and surface area. the pdnp were synthesized via a simple chemical reduction using sodium borohydride (see supporting information). the students prepared a pdnp ink paste by mixing 0.50 mg of pdnp with 2-propanol and nafion solution in a vial and placing it in an ultrasonic bath for 30 min. then, 10 l of the pdnp ink paste was placed on the gc and cp surface and allowed to air - dry at room temperature. once dried, the working electrodes (gc and cp) with pdnp were placed in the electrochemical cell with 0.10 m h2so4 solution. students were required to generate their own cyclic voltammograms using an applied potential from 0.00 to 1.30 v vs ag / agcl at a scan rate of 100 mv / s with the pdnp modified electrodes. then, they interpreted the generated cyclic voltamograms for each electrode and compared their results with the literature. in this part, the surface areas of different electrodes were calculated using data obtained from voltammograms in 1.0 m koh solution. the catalytic activity for ethanol oxidation in 1.0 m koh solution was tested using the cp and gc electrodes modified with pdnp. the working electrode was placed in the electrochemical cell, and the applied potential was cycled between 0.90 and 0.40 v vs ag / agcl at a scan rate of 100 mv / s. once the cyclic voltammograms were obtained, the students determined the peak current density for ethanol oxidation using the electrode geometric and surface area and compared the results for each working electrode tested. students must wear a lab coat, goggles and gloves at all times during the lab. concentrated sulfuric acid is dangerous to the skin and eyes ; they must be used in a fume hood. in the prelaboratory activity, students analyzed the requirements for an effective anode using the information in table 1 and their own literature search. then, they selected the materials to be tested as an anode. in part i, they measured each working electrode surface diameter and calculated the geometric area. with this data collection, students were able to see a difference in the diameters for the different electrodes. in part ii then, the presence of pdnp on the gc and cp electrodes was confirmed using the cv technique in 0.10 m h2so4 solution and a potentiostat. figure 2 shows the cyclic voltammograms for bare gc and gc modified with pdnp in 0.10 m h2so4 solution.. however, gc modified with pdnp showed current peaks corresponding to the palladium oxide (pdo) formation at positive potential and pdo reduction at negative potential. the students were guided to make a detailed interpretation of each cyclic voltammogram, explaining the characteristic peaks and its corresponding redox reaction. cyclic voltammogram of gc electrode modified with pdnp (solid line) and bare gc electrode (dash line) in 0.10 m h2so4 solution. in part iii, the student calculated the active surface area of pd using the voltammograms obtained from in 1.0 m koh for each electrode (see supporting information). in addition, the students tested the catalytic activity for ethanol oxidation using the different electrodes modified with the pdnp. the obtained results were compared with the voltammogram of the commercial pd electrode. the current density was calculated using the ethanol oxidation peak current divided by both the electrode geometric and surface areas for each electrode, and the results were compared. the potential, current, and current density values were written in table 2. figure 3 shows cyclic voltammograms of the ethanol oxidation using an alkaline medium, students observed two peaks : the first peak appears in the potential region from 0.50 to 0.00 v vs ag / agcl during the forward scan (positive direction). at positive potentials, the formation of pdo blocks the catalytic activity for ethanol oxidation. in the reverse scan (negative direction), pdo is reduced to metallic palladium starting at 0.30 v, returning the ethanol oxidation catalysis, and showing an anodic peak again. students observed this second peak in the region between 0.30 and 0.60 v vs ag / agcl in 1.0 m ethanol and 1.0 m koh solution. this voltammetry experiment showed that both peaks are anodic, a typical ethanol oxidation behavior in alkaline conditions. cyclic voltammogram of cp / pdnp (solid line), gc / pdnp (dashed line), and pd (dotted line) electrodes in 1.0 m ethanol and 1.0 m koh solution. finally, the students choose the best cost - effective anode electrode for an alkaline fuel cell through the literature search, experimental method, and analysis of data summarized in table 1 and the results are presented in table 2. results shown in table 2 will allow students to learn about the effect of the electrode active surface area on the performance of the electrode. it is expected that they understand that electrodes with equal area surface (1 cm) for each catalyst will have high catalytic surface area and will produce a higher peak current density. a new laboratory experience that introduces students to applications of nanotechnology and fuel cells was successfully implemented in the instrumental analysis laboratory course. during this experience, students developed skills related to literature search, and interpretation and analysis of electrochemistry data related to fuel cell. in the prelaboratory, they were encouraged to convert resistivity values to conductivity, resistance to corrosion, and to consider the economical aspect in the selection of materials for anode electrodes. in the experimental process, they were exposed to fundamental electrochemical concepts, and tested and analyzed how different parameters such as material and surface area affect the catalytic activity for ethanol oxidation in alkaline media. the principal limitations confronted in the experience were related to students previous knowledge of fuel cells components and the use of the cv technique. in spite of the wide use of cv in academic and research, this technique was not well understood by the students in comparison to other instrumental techniques such as chromatography and spectroscopy. at the beginning of the experience, the students demonstrated poor understanding of the concepts involving the cv technique such as the peculiar shape of the voltammograms. therefore, a brief description of the cv technique and operation of fuel cells components presented in most textbooks is insufficient to attain a comprehensive understanding. it was necessary to explain the technique in detail to the students with an emphasis on the analysis of the voltammograms to characterize the function of the fuel cell components. the most challenging part for the students was the interpretation of the cyclic voltammograms obtained for ethanol oxidation. since in most textbooks cv is explained using voltammograms where oxidation and reduction peaks are observed, the ethanol oxidation voltammogram was confusing for the students, because the reduction peak was absent. this behavior was explained in detail to the students with the use of the literature.	nanotechnology allows the synthesis of nanoscale catalysts, which offer an efficient alternative for fuel cell applications. in this laboratory experiment, the student selects a cost - effective anode for fuel cells by comparing three different working electrodes. these are commercially available palladium (pd) and glassy carbon (gc) electrodes, and a carbon paste (cp) electrode that is prepared by the students in the laboratory. the gc and cp were modified with palladium nanoparticles (pdnp) suspensions. the electrodes efficiencies were studied for ethanol oxidation in alkaline solution using cyclic voltammetry techniques. the ethanol oxidation currents obtained were used to determine the current density using the geometric and surface area of each electrode. finally, students were able to choose the best electrode and relate catalytic activity to surface area for ethanol oxidation in alkaline solution by completing a critical analysis of the cyclic voltammetry results. with this activity, fundamental electrochemical concepts were reinforced.
millions of patients underwent lasik in their 20s and 30s when this technology emerged in the early 1990s. these patients have already expressed their wish to be spectacle independent and made the decision to undergo refractive surgery. it seems to be logical to assume that these patients would continue to be interested in remaining spectacle - free, and therefore the probability is high that they would opt for a presbyopia - correcting intraocular lens (pciol) if given the option. unfortunately, there are limited data available regarding successful visual restoration with pciol implantation in patients having undergone previous refractive laser surgery. the few available published studies have been mainly performed by the same research group with the same lens models [2, 3, 4, 5 ]. to our best knowledge, nothing has been published on the use of accommodating, enhanced depth of focus and extended range of vision (erv) intraocular lenses (iols). we present a case with successful implantation of the erv iol tecnis symfony (abbott medical optics, santa ana, calif. a 46-year - old myopic male patient underwent multifocal ablation refractive laser surgery (lvc) in 2003 when he was 46 years old. the treatment was performed with the patented visx (abbott medical optics) multifocal ablation pattern that was created using the wavescan aberrometer during a feasibility study. this system was customized to ablate a central, steepened zone for near correction and a peripheral zone targeted for distance. he presented at our clinic in late 2014 and was interested in a presbyopia - correcting solution as his reading vision had dropped recently. his optical scatter index was elevated to 2.8 (od) and 2.3 (os) indicating changes in lens nuclear density. the patient was informed in detail about the challenges of iol calculation after lvc and the potential limitations of pciols and agreed to undergo surgery in may 2015. his preoperative refraction was + 0.50/0.50 165 in the right eye and + 0.50/0.50 005 in the left eye. for the iol calculation we targeted emmetropia in his dominant left eye and 0.50 d in his right eye. one day postoperatively the patient 's refraction was 1.00 (od) and 0.75/0.25 165 (os) but binocularly his distance visual acuity was 20/25. with distance correction in place, his visual acuity was still 20/20 at near, intermediate and distance. after 1 week, his uncorrected binocular visual acuity was 20/25 at distance, 20/30 at intermediate and 20/20 at near. his refraction was 0.25 d/1.25 d 110 (od) and 0.50 d/0.50 d 180 (os). although the patient was slightly myopic in both eyes, he was very satisfied with the quality of his vision. thirteen months after surgery, the patient 's binocular visual acuity was 20/25 at distance, 20/20 at intermediate and 20/16 at near. when his mild myopia was corrected to plano in both eyes, his visual acuity increased to 20/20 at distance, 20/20 at intermediate and still 20/20 at near. the patient is very satisfied with the outcome and not bothered by any night vision issues or other photic phenomena. this case represents successful implantation of an erv iol 12 years after multifocal ablation in a patient with the strong desire for spectacle independence. his uncorrected visual acuity across all distances was excellent at the last follow - up, which was 13 months after surgery. multifocal ablations for treatment of presbyopia with the excimer laser can be performed either by steepening the peripheral cornea for near vision and using the central cornea for distance vision [6, 7, 8 ] or by customized ablation patterns for a central, steepened zone for near correction and a peripheral zone targeted for distance [9, 10, 11 ]. to our best knowledge, no publications exist on pciol implantation after multifocal laser ablation. a few studies investigated the outcome of multifocal iol (miol) implantation after myopic and hyperopic lasik. alfonso. compared visual acuity under photopic and mesopic conditions with and without glare, as well as higher - order aberrations (hoas) between patients after myopic lasik who were implanted either with the diffractive acri.lisa 366d iol (acri.tec gmbh, germany) or with the apodized refractive - diffractive acrysof restor sn60d3 iol (alcon laboratories, fort worth, tex. mean photopic corrected visual acuity (cdva) after 6 months was 20/25 or better in all groups. under glare or low - contrast situations, the acri.lisa patients performed better than the restor patients. all eyes in both iol groups achieved a distance - corrected near visual acuity (dcnva) of 20/32 or better. the same authors evaluated further parameters in a second publication on the same patient cohort and concluded that the optical and visual performance of the acri. lisa eyes was comparable to eyes in the control group, but that the group with this lens performed better than the restor group. the same research group investigated the outcome after implantation of a spherical diffractive iol (acrysof restor sn60d3) in eyes with previous hyperopic lasik. after 6 months, mean cdva was 20/25 or better in 58.54% of the patients. mean udva and cdva were 20/26 and 20/22, and the patients achieved a mean unva and dcnva of 20/21, respectively. in a second evaluation of further parameters, the authors reported a comparable visual performance of the restor eyes and a control group with phakic eyes after hyperopic lasik under photopic conditions, but a worse outcome of the iol group under mesopic conditions. the results after myopic and hyperopic lasik suggest that the implantation of diffractive multifocal lenses is a viable option for presbyopia correction. however, the patients should be informed about the potential risk of a refractive surprise and the possibility of poorer visual performance under low light conditions, beyond what they may have already experienced after lasik. our case is only partly comparable to the outcomes of the discussed studies because our patient had a multifocal ablation before iol implementation, and the implanted iol has a different optical principle. this erv optical technology of the tecnis symfony iol provides a full range of vision with reduced incidence of halos and glare and contrast sensitivity close to that typically associated with monofocal iols. the basis of this technology is an achromatic diffractive echelette design that corrects the corneal chromatic aberration for enhanced contrast sensitivity and generates an erv. correcting chromatic aberration in combination with the correction of spherical aberration has been shown to improve retinal image quality with no impact on the depth of focus. therefore, the correction of corneal chromatic aberration with an iol should result in an improved focus of light. it would be interesting to study the interaction of a multifocal ablation profile with the asphericity of an iol. in our case, clinical data with the tecnis symfony iol do not show the typical behavior of miols as this lens has an elongated focal area instead of multiple focal points. in addition to the improved intermediate vision, an important advantage is the low occurrence of photic phenomena and visual symptoms, which are typically seen with miols as one image is in focus while the out - of - focus image is suppressed. visual symptoms, such as halos, are caused by this out - of - focus image. in conclusion, our case demonstrates that excellent distance, intermediate and near visual acuity results can be achieved with an erv iol after multifocal excimer laser ablation. we would like to encourage surgeons to feel comfortable utilizing this type of iol in the increasing number of post - lasik patients who seek spectacle independence. controlled studies are needed to create evidence for the suitability of pciols in patients with a history of myopic, hyperopic and multifocal excimer laser ablation.	as our baby boomer population is aging and developing cataracts, so are our post - lasik patients. these patients underwent lasik surgery as they wished to be spectacle - free and are hoping to remain so after intraocular lens (iol) surgery. unfortunately, very little information is available regarding the suitability of presbyopia correcting iols for post - lasik patients. this case represents successful implantation of an extended range of vision iol in a 59-year - old patient who underwent multifocal ablation excimer laser surgery 12 years before. emmetropia was targeted for the dominant eye and 0.5 d for the fellow eye. the 13 month follow - up after bilateral implantation of the tecnis symfony iol revealed an uncorrected visual acuity of 20/25 for distance, 20/20 for intermediate and 20/16 for near. the patient is very happy and did not report any visual symptoms when asked. this successful case should encourage surgeons to consider implanting an extended range of vision iols in post - lasik patients.
pharmacopuncture does not pass through the digestive system, so it works faster and is more effective compared to medicines that are administered orally. the constituents of the saeng maek san (sms) are three herbs, panax ginseng, ophiopogon japonicas, and schisandra chinensis. in traditional chinese medicine (tcm), sms is used as a remedy or clinical prescription to treat symptoms related to cardiovascular diseases. in previous studies, sms was found to inhibit inflammatory cytokines, such as tumor necrosis factor- and interleukin-8, and to reduce the systemic inflammatory reaction. protective effects against oxidative damage in mitochondria, cells, and tissues, as well as amyloid--induced cytotoxicity in pc12 cells, were also verified [5 - 8 ]. additionally, sms is known to enhance humoral immunity and to inhibit cellular immunity after a cardiopulmonary bypass. in a previous single - volume toxicity study (biotoxtech study no : b12877), 0.1, 0.5 and 1.0 ml of sms were administered to the experimental groups and 1.0 ml of saline to the control group. in all four groups, the administration of 1.0 ml / animal of sms did not cause any significant changes or any incidence of mortality. therefore, sms administration up to this volume was determined to be a safe option for treatment. however, signs of hematuria were noted in the animals that received sms doses of 0.5 and 1.0 ml / animal. therefore, in this study, 0.3 ml / animal was set as the high dosage and 0.2 and 0.1 ml / animal as the medium and the low dosages, respectively. all experiments were performed at biotoxtech (chungwon, korea), an institute certified to perform non - clinical studies under the regulations of good laboratory practice (glp). the sms consisted of panax ginseng, ophiopogon japonicas, and schisandra chinensis in the ratio 2 : 1 : 1, respectively (total : 5,500 g). the sms pharmacopuncture was prepared in a sterile room at the korean pharmacopuncture institute (k - gmp). sms was extracted by decocting the dried herbs in distilled water for 2 hours (total extracts : 12 l), and the ph was controlled to between 7.0 and 7.5 by adding naoh to make a 0.9% isotonic solution. 5-week - old male and female sprague - dawley (sd) rats with weights in the ranges of 119.3 137.0 g and 101.9 118.2 g, respectively, were provided by orient bio inc. sd rats have been widely used in drug - safety tests, so the use of sd rats in this study allowed the data obtained to be easily compared to similar data available in numerous existing databases. the animals were housed in stainless - steel wire - mesh cages (260 mm (w)350 mm (d)210 mm (h)) at a constant temperature of 21.8 23.5 under a relative humidity of 48.7% 68.1% with 10 15 air changes per hour. the room was provided with artificial lighting (150 300 lux) from 07:00 to 19:00. the animals were allowed free access to tap water and commercial rodent chow (teklad certified irradiated global 18% protein rodent diet 2918c, harlan laboratories, inc. this study was conducted with the approval of the institutional animal ethics committee (no. forty male and forty female sd rats were used as the subjects of this test after a week of adaptation. rats of each gender were randomly distributed, based on average weights, into four groups, with 10 rats per group (table 1). at the first injection, the 6-week - old male and female sd rats had weights in the ranges of 189.6 212.5 g and 149.0 175.1 g, respectively. according to a previous single - volume toxicity study (biotoxtech study no : b12877), 0.1, 0.5 and 1.0 ml of sms were administered to the experimental groups and 1.0 ml of saline to the control group. in all four groups, no deaths occurred, but hematuria was noted in the animals that received sms in doses of 0.5 and 1.0 ml / animal. therefore, in this study, 0.3 ml / animal was set as the high dosage and 0.2 and 0.1 ml / animal as the medium and the low dosages, respectively. all animals were observed daily for clinical signs for 4 weeks from the first injection day. the body weight and food consumption of each rat were measured at the initiation of treatment and once a week during the treatment period. the amounts of food and water intake were averaged every week during the treatment period. ophthalmological examinations and urinalyses of five rats in each group were carried out at the end of the recovery period. in the ophthalmological examinations, after the use of a mydriatic (lot no. : 12k21b, isopto atropine eye drops 1%, alcon, korea), and the anterior segment, lenses, vitreous body and fundus were examined by using an opthalmoscope (all pupil, keeler, u.k.). urinalyses were conducted on fresh urine to assess specific volume, protein, bilirubin, and occult blood ; a combur10test m stick (roche, germany) system (miditron junior ii, roche, germany) was used. hematological analyses were performed before autopsy ; all animals were anesthetized by using isoflurane after fasting for more than 18 hours, and blood was collected from the abdominal aorta. the blood samples, about 1 ml, were collected into tubes with ethylene diamine tetraacetic acid (edta) and were analyzed using a blood counting analyzer (advia 2120i, siemens, germany). for the blood coagulation analyses, about 2 ml of blood were collected into tubes with 3.2% sodium citrate, centrifuged at 3,000 rpm for 10 minute, after which measurements were taken using an automated coagulation analyzer (coapresta 2000, sekisui, japan). the serum biochemistry analyses were performed using an auto - analyzer (7180, hitachi, tokyo, japan). biochemical tests were performed by using an automatic analyzer (7180, hitachi, japan) and an electrolyte analyzer (ilyte, instrumentation laboratory, usa). weight, food intake, hematology and blood biochemistry data were analyzed using the statistical analysis system (sas) software (versions 9.3, sas institute inc. the bartlett test (p < 0.05) was conducted to evaluate the homogeneity of the variance and the significance. if the test had equal variance, the data were analyzed by using the one - way analysis of variance (anova) (p < 0.05) and multiple range tests for dunnett s t - test for a post - hoc analysis (p < 0.05, p < 0.01). if the test did not have equal variance, the data were analyzed by using the kruskal - wallis test (p < 0.05) and multiple range tests for the steel test for a post - hoc analysis (p < 0.05, p < 0.01). compared to the control group, the male and the female rats in groups 3 and 4 (0.2 and 0.3 ml / animal / day) showed hemoglobinuria, but the low - dosage group (g2, 0.1 ml / animal / day) showed no significant changes in the clinical signs test (table 2). in addition, no significant differences in food consumption were observed (table 4). in the ophthalmological tests, no abnormalities were detected in any group (table 5). in the urinalysis, occult blood of male rats in groups 3 and 4 (0.2 and 0.3 the female rats in groups 3 and 4 (0.2 and 0.3 ml / animal / day) exhibited significantly increased protein. in addition, groups 2, 3 and 4 (0.1, 0.2 and 0.3 ml / animal / day) showed increased bilirubin, and occult blood (table 6). no remarkable changes were observed in the hematology (table 7). finally, no changes were observed in the blood chemistry, the necropsy, or the histopathology. sms, a traditional medicine, is a mixture of panax ginseng, ophiopogon japonicas, and schisandra chinensis. in a recent study, sms was used to treat symptoms of cardiovascular diseases, such as heart failure and stroke, as well as neuronal damage [10 - 12 ]. even though sms is widely used in clinics, further research is needed to assess the safety of the medication by using tox - icity tests. toxicity tests are mostly used to examine the toxicity of a specific sample and to calculate the no - observed adverse - effect level (noael) volume. in this study, the toxicity test was performed at biotoxtech (chungwon, korea), an institute certified to perform non - clinical studies under the regulations of glp. during the observation period, the medium- and the high - dosage groups (g3 and g4, 0.2 and 0.3 ml / animal / day, respectively) showed signs of hemoglobinuria while the low - dosage group (g2, 0.1 ml / animal / day) showed no significant signs of hemoglobinuria. in the medium - dosage group (0.2 ml / animal/ day), from the 4 day, hemoglobinuria was observed in 8 male rats, and from the 9 day, it was observed in 10 female rats. in the high - dosage group (0.3 ml / animal / day) compared to the control group, male rats in groups 3 and 4 (0.2 and 0.3 ml / animal / day) exhibited increased occult blood, and female rats in groups 3 and 4 (0.2 and 0.3 ml / animal / day) exhibited significantly increased protein. also, groups 2, 3 and 4 (0.1, 0.2 and 0.3 ml / animal / day) showed increased bilirubin and occult blood. in the medium - dosage group (0.2 ml / animal/ day) and the high - dosage group (0.3 ml / animal / day), one and two cases, respectively, of amber - colored urine were observed in female rats. no remarkable changes were observed in the hematological examination. finally, no changes were observed in the blood chemistry, necropsy, or histopathological examinations. in conclusion, the present study corroborated that administration of 0.1 ml / animal / day of sms did not cause any significant changes in body weight, food consumptions or the results of hematological, blood biochemistry, and necropsy examinations. also, no mortality was observed in any group, which indicates that sms pharmacopuncture can be used as a safe treatment. noa, no observable abnormality. significantly different from control by dunnett s t - test : p < 0.01. significantly different from control by dunnett s t - test : p < 0.05, p < 0.01. rbc, red blood cell ; hgb, hemoglobin ; hct, hematocrit ; mcv, mean corpuscular cell volume ; mch, mean corpuscular hemoglobin ; mchc, mean corpuscular cell hemoglobin concentration ; plt, platelet ; reti, reticulocytes ; wbc, white blood cell ; neu, neutrophils ; lym, lymphocytes ; mono, monocytes ; eos, eosinophils ; baso, basophils ; pt, prothrombin time ; aptt, active partial thromboplastin time.	objectives : this study used repeated intravenous injections of saeng maek san (sms) injection in sprague - dawley (sd) rats to assess the toxicity and the stability of smsmethods : six - week - old male and female sd rats reared by orient bio inc were chosen for this pilot study. they were randomly split into four groups : group 1 (g1), the control group (0.3 ml of normal saline solution / day / animal), and groups 2, 3 and 4 (g2, g3 and g4), the experimental groups (0.1, 0.2 and 0.3 ml / day / animal of sms), respectively. each animal received an intravenous injection of sms once a day for four weeks. clinical signs, body weight changes, and food consumption were monitored during the observation period, and urinalysis and hematology were conducted after four weeks of sms or saline administration.results:no deaths occurred in any of the four groups during the observation period. compared to the control group, male and female rats in groups 3 and 4 (0.2 and 0.3 ml / animal / day) showed hemoglobinuria, but the low - dosage group (g2, 0.1 ml / animal / day) showed no significant changes in the clinical signs test. no significant changes due to sms were observed in the experimental groups regarding body weight changes, food consumption urinalysis, or hematology.conclusion:during this study, no mortalities were observed in any of the experimental groups and no hemoglobinuria was observed in the low dosage group (0.1 ml / animal / day) while it was intermittently observed in groups 3 and 4 (0.2 and 0.3 ml / animal / day). thus, we suggest that the no - observed adverse - effect level (noael) is 0.1 ml / animal / day in male and female sd rats.
according to the patient survey of 2005 carried out by the ministry of health, labour and welfare, japan1 of 326,000 patients hospitalized due to mental and behavioral disorders, approximately 43.5%, i.e., approximately 142,000 patients, were elderly persons aged 65 years or older. moreover, according to the patient survey of 20112 of 282,000 hospitalized patients, approximately 48.2%, i.e., approximately 136,000 patients, were 65 years of age or older. while the patients in psychiatric hospitals are aging, a tendency has been observed for the duration of hospitalization to be as long as 296 days limiting the opportunities for patients to act of their own accord. recently, the rate of falling is high for hospitalized patients in psychiatric hospitals with falling being the most frequently occurring issue when it comes to managing medical safety3. regarding the relationship between falling and drugs in nursing home residents, while it has been reported that the risk of falling increases as the psychotropic dosage increases4, or that the risk of falling for patients taking tricyclic antidepressant drugs is approximately three times higher than that for those not taking psychotropic drugs5, some negative reports exists as well6, and the debate on this is still controversial. however, there are few reports that showed relationship between falling and physical function in patients with schizophrenia. regarding the relationship between falling and physical function, physical factors in addition to environmental factors are noted in elderly persons without mental disorders ; specifically, a decrease in muscle strength of the lower limbs, walking ability, and balance ability are seen as problematic7. after falling, it is noted that activities of daily living (adl) deteriorate due to bone fractures, inducing limited activity caused by post - fall syndrome, losing confidence, developing symptoms of depression or anxiety, and decreasing the sphere of action due to the fear of falling8, 9. since the daily life activities of patients are further limited after falling, it is important to prevent mental disorder patients from falling during hospitalization. in the present study, we evaluated patients with schizophrenia hospitalized in psychiatric hospitals with the objective of analyzing the factors causing falling. the study subjects included 19 patients with schizophrenia (male : 8 patients, female : 11 patients, mean se age : 63.3 6.1 years) hospitalized in a psychiatric hospital. the exclusion criteria were patients using a wheelchair, those with mental disorders too severe to allow them to understand the study explanation, and those whose adl was decreasing due to orthopedic disorders such as spinal cord injuries. consent to participate in the study was obtained from the subjects upon providing a verbal explanation about the study purport and study content. the study was carried out with the approval of the research ethics committee of kansai university of welfare sciences (approval no. assessment items measured included muscle strength (grip strength, and 30-second chair stand test10), balance ability (one leg standing time with eyes open / closed, functional reach test [frt11 ], and tugt12), flexibility (long sitting position toe touching distance13), walking ability (10 m maximum walking speed14), sleeping condition (athens insomnia scale15), global assessment of functioning scale (gaf)16, the antipsychotic drug intake, drug induced extra - pyramidal symptoms scale (diepss)17, and number of falls. grip strength of the dominant hand was measured using grip - d (takei scientific instruments co., ltd.). the one leg standing time with eyes open / closed was measured for the pivotal foot, standing bare foot with both hands at the waist and elevating one foot, with 60 seconds set as the maximum. frt was measured with both hands in accordance with the method reported by tsushima.18, using a reach measuring instrument (og wellness technologies co., ltd.). long sitting position toe touching distance was measured using ekj09 (evernew inc.), in accordance with the measuring methods of the new physical fitness tests by the ministry of education, japan culture, sports, science and technology. grip strength, the 30-second chair stand test, one leg standing time with eyes open / closed, frt, tugt, long sitting position toe touching distance, and 10 m maximum walking speed were each measured twice, with the maximum and fastest values adopted as representative values. regarding the antipsychotic drug intake, the average dosage from one month before measurement day was extracted using the chlorpromazine - equivalent dosage. a fall was defined as when body parts other than the sole of the foot touched the floor / ground, against ones will based on the definition by gibson19, with patients having experienced a fall at least once during the past 1 year assigned to the fall group, while those not having experienced a fall were assigned to the non - fall group. in addition, we investigated the fall situation using the incident and accident reports. regarding the statistical analyses, a mann - whitney u - test and test were used to evaluate the differences in gender, age, bmi, complication, muscle strength, balance ability, flexibility, walking ability, sleeping condition, gaf, the antipsychotic drug intake, and diepss between the fall group and the non - fall group. a total of 12 patients (male : 4 patients, female : 8 patients, mean se age : 59.1 15.5 years) were included in the fall group while 7 patients (male : 4 patients, female : 3 patients, mean se age : 61.3 7.1 years) were included in the non - fall group. the results of a univariate analysis are provided in table 1table 1.the comparison between factors in fall group and non - fall groupitemfall group (n=12)non - fall group (n=7)gender (male / female)5/74/3age (yrs)64.5 5.4 (5372)61.3 7.1 (5474)bmi (kg / m)22.4 2.723.2 3.7complicationdiabetes mellitus (normal/ abnormal) (n)(10/2)(7/0)hyperlipidemia (normal/ abnormal) (n)(9/3)(7/0)hypertension (normal/ abnormal) (n)(9/3)(7/0)grip strength (kg)18.0 6.626.8 10.930-s chair stand test (times)12.2 2.716.4 7.5one leg standing time with eyes open (seconds)4.7 6.219.9 21.5one leg standing time with eyes close (seconds)1.8 2.83.2 2.7functional reach test (cm)20.2 10.622.3 9.5time up and go test (seconds)8.7 1.66.6 1.0long sitting position toe touching distance (cm) 26.0 7.526.1 16.910 m maximal walking speed (m / min)7.4 2.35.9 1.4athens insomnia scale (scores)4.6 5.85.0 2.8gaf (scores)30.6 9.037.0 10.5antipsychotic drug doses (mg)545.4 491.3481.4 697.4diepss : gait2.1 0,81.4 1.0bradykinesia1.8 0.91.4 0.8sialorrhea1.1 0.90.1 0.4muscle rigidity1.0 1.00.7 0.8tremor1.0 1.00.4 0.8akathisia0.6 0.70.4 0.8dystonia0.5 1.00 0dyskinesia0.8 0.60.1 0.4overall severity1.8 0.91.4 0.8p<0.05 : significant difference between the groups ; athens insomnia scale : 024 scores ; gaf : 0100 scores ; diepss : 04 rates (0 : normal, 1 : minimal, 2 : mild, 3 : moderate, 4 : severe). significant differences were observed for one leg standing time with eyes opened, tugt, and diepss sialorrhea between the fall group and the non - fall group (p<0.05). p<0.05 : significant difference between the groups ; athens insomnia scale : 024 scores ; gaf : 0100 scores ; diepss : 04 rates (0 : normal, 1 : minimal, 2 : mild, 3 : moderate, 4 : severe) in the present study, the presence or absence of having experienced a fall in patients with schizophrenia along with their muscle strength, balance ability, flexibility, walking ability, sleeping condition, gaf, and the antipsychotic drug intake were evaluated. regarding the physical function factors affecting falling, they reported that lower limbs muscle weakness20,21,22 and decreased balance ability20, 21 are the factors involved in the elderly without mental disorders and stroke patients. in addition, ikai.22 pointed out that there is a relationship between falling and dynamic balance ability, so it is useful to evaluate the dynamic balance ability in order to predict falling. furthermore, it is reported that the balance exercise was useful in fall prevention23. based thereon, it is inferred that in subjects without mental disorders, falling is strongly associated with the physical functions of lower limb muscle strength and balance ability. as a result of the present study, since the static and dynamic balance ability in the fall group was lower than the non - fall group, it was suggested that a decrease in balance ability has a strong effect as a factor inducing falling in mental disorder patients as well. regarding the relationship between drugs and falling, drugs are involved in falling, and for psychotropic drugs in particular, which act on the central nerve system, it has been reported that psychotropic drugs increase the risk of falling / tumbling24. moreover, it has been indicated that the risk of falling / tumbling further increases in patients taking multiple psychotropic drugs4. at the same time, reported that while they evaluated the relationship between the antipsychotic drug dosage and falls / tumbles based on the results that the number of falls / tumbles was significantly higher when antipsychotic drugs were administered, there was no obvious relationship observed between them25. in the present study, the antipsychotic drug dosage did not demonstrate any significant differences between the fall group and the non - fall group. the reason for the low involvement of antipsychotic drug dosage was suspected to be due to the fact that antipsychotic drugs are prescribed by physicians only after assessing adl and physical function / ability, in addition to mental function, assuming there is no negative impact on the assessed items. this was a cross - sectional study and we believe that the effect of decreasing the number of falls by improving balance ability can be determined by follow - up or intervention studies, which will require further evaluation.	[purpose ] the purpose of this study is to investigate the factors causing falling among patients with schizophrenia hospitalized in psychiatric hospitals. [subjects and methods ] the study subjects were divided into either those having experienced a fall within the past one year (fall group, 12 patients) and those not having experienced a fall (non - fall group, 7 patients), and we examined differences between the two groups. assessment items measured included muscle strength, balance ability, flexibility, body composition assessment, global assessment of functioning scale (gaf), the antipsychotic drug intake, and drug induced extra - pyramidal symptoms scale (diepss). [results ] as a result, significant differences were observed in regard to one leg standing time with eyes open, time up and go test (tugt), and diepss sialorrhea between the fall group and the non - fall group. [conclusion ] these results suggest that a decrease in balance ability was significantly correlated with falling in schizophrenia patients.
to a k channel, the alkaline earth metal ba is like a divalent k ion. barium 's size allows it to fit into the selectivity filter, but its charge apparently causes it to bind too tightly. when it is bound in the filter, ba prevents the rapid flow of k. the k channel inhibitory properties of ba have been analyzed in detail (armstrong and taylor 1980 ; eaton and brodwick 1980 ; armstrong. 1982 ; vergara and latorre 1983 ; benham. 1985 ; miller 1987, neyton and miller 1988a, neyton and miller 1988b ; zhou. 1996 neyton and miller 1988a, neyton and miller 1988b showed through single channel studies of ca - activated k channels that ba inhibition is sensitive to the presence of k ions in the pore. they observed that the mean blocking dwell time of ba depended on the k concentration on both sides of the membrane. low external (extracellular side of the membrane) k ion concentrations (0.01 mm) tended to prevent ba from exiting to the external side, while higher concentrations (500 mm) actually caused ba to dissociate more rapidly to the internal side (intracellular side of the membrane) as if it could be pushed through. these phenomena were attributed to the presence of two k ion sites located between the blocking ba ion and the external solution. the presence of k at the first of these sites, termed the external lock - in site, would obstruct barium 's outward movement. occupancy of both sites by k (the site closest to ba was termed the enhancement site) would destabilize ba and speed its exit to the inside. by evaluating a series of different ions, the lock - in and enhancement sites were shown to be highly selective for alkali metal ions that permeate k channels such as k and rb, but not na. an additional monovalent ion site, termed the internal lock - in site, was hypothesized to lie between the position of ba and the internal entryway ; this site became occupied with an affinity constant around 10 mm. in contrast to the sites lying external to ba, the internal lock - in site was not very selective for k when compared with na. the membrane voltage dependence of lock - in and enhancement effects exerted by k on ba provided an estimate of the electrical distance that k must travel through the channel to reach a specific site. ions in the pore presumably move in a somewhat concerted fashion, making the electrical distance a complicated quantity without a simple structural interpretation. the external lock - in and enhancement sites appeared to reside 15 and 50%, respectively, of the way across the membrane electric potential difference relative to the outside, and the internal lock - in site appeared to reside 70% of the way (i.e., 30% of the electrical distance from the internal solution). these experiments supported the picture in fig. 1, right. following the neyton and miller 1988a, neyton and miller 1988b analysis of ca - activated k channels, similar results were obtained by vergara. x - ray structure determination of the kcsa k channel showed that it contains multiple permeant ions in its pore (doyle. 1998). given the high degree of amino acid sequence conservation in the pore - forming regions of the ca - activated k channel, the shaker k channel, and the kcsa k channel, we expect their three - dimensional structures to be conserved. in this study, we determined the position of ba in the pore of the kcsa k channel. the results allow a comparison of the ionic configuration deduced by structural (x - ray crystallographic) and functional (electrical analysis of single channels) methods. the ba - containing crystals were prepared by first washing crystals in a solution of 50% peg400, 200 mm cacl2, 150 mm nacl, 5.0 mm ldao, 100 mm hepes, ph 7.5, and then soaking them in an otherwise identical solution containing 100 mm cacl2 and 100 mm bacl2 for 1 h. crystals were frozen in a cold nitrogen stream (100k) and stored in liquid nitrogen cooled propane. data were collected at the cornell high energy synchrotron source, a1 station, and processed with denzo and scalepack (otwinowski 1993 ; otwinowski and minor 1997). the electron density of ba in the channel was calculated from the fourier transformation of (fbarium fnative) e, where fbarium and fnative are the observed structure factors for the ba - containing and native crystals, respectively, and is the experimental (mir, solvent - flattened, averaged) phase. due to crystal damage during the soaking procedure, ionic strength was kept constant by replacing cacl2 present in the crystal growth solution with bacl2. at the same time, 150 mm kcl was replaced by an equal amount of nacl. the location of ba in the pore is well defined in a difference fourier map (fbarium fnative) calculated at 5.0- resolution (fig. 1, left and center, green mesh). electron density for ba is compared with that for rb ions determined in a separate experiment (red mesh). the rb - difference fourier map, calculated at 3.8- resolution, shows two ions in the selectivity filter and one in the cavity at the membrane center, below the selectivity filter (doyle. the outer ion is near the extracellular entryway and the inner ion is closer to the cavity and can be at either of two positions. the ba ion very clearly binds at the location of the inner ion closest to the central cavity. the consistency of structural data on the kcsa k channel with functional data on ca - activated k channels is quite remarkable. the external lock - in effect implied the existence of a first ion binding site external to ba this site had to be highly selective for permeant alkali metal cations and very near the extracellular solution, because external lock - in is a weakly voltage - dependent process. the external enhancement effect implied a second site, also external to ba and highly selective for permeant ions. the stronger voltage dependence of its apparent occupancy suggested that the enhancement site is deeper in, closer to the ba ion. the effective kd for achieving the enhancement configuration of two k ions external to ba is nearly 500 mm, indicating that it is a relatively unstable configuration. in the structure, the outermost peak of the inner ion is too close to ba to be the precise site of the enhancement ion. however, the structure of the selectivity filter provides several potential ion binding sites external to ba. the internal lock - in effect required a site internal to ba that, in contrast to the external sites, exhibits little ion selectivity. the cavity ion in the crystal structure, being fully hydrated and not in direct contact with protein functional groups, fulfills the requirements of the internal lock - in site completely. even the relatively weak voltage dependence of internal lock - in is consistent with the idea that a large fraction of the membrane voltage falls across the narrow selectivity filter (doyle. 1998). given the high affinities of the external and internal lock - in sites, it seems likely that they would contain a k most of the time at physiological ion concentrations. therefore, the picture of the pore with three ions (two in the selectivity filter and one in the cavity), derived from the crystal structure, is probably not very different from a snapshot of what the conducting channel looks like in the membrane. the external enhancement effect is interesting because it suggests that a fourth ion enters the pore (that is, a third ion in the selectivity filter) to push the queue along (and ba out). the affinity for the enhancement site is low, and corresponds reasonably well to the concentration range over which the conductance increases as k concentration is raised (0.31.0 m, depending on the k channel). this enhancement configuration would not have been abundant in the crystal bathed in only 150 mm kcl (or rbcl). the idea that k channels have multiple ions in their pore is as old as the ion channel field itself (hodgkin and keynes 1955). the mind 's eye picture of multiple ions in a queue earned k channels the name long - pore channels because it seemed that multiple ions with a large separation between them would require a long pore. when mutational analysis of k channels identified a short pore - region sequence, and even shorter signature sequence forming the selectivity filter how could so few amino acids form a long enough pore to accommodate the queue of ions ? ironically, in the answer, we see that the long pore is not very long at all : three ions are compressed into a distance of roughly 15 about half the membrane thickness. the close spacing is probably not happenstance ; it allows for strong interactions between ions in the pore. ionic interactions, mediated by electrostatics and perhaps by the structure of the selectivity filter itself, offer a solution to the apparent paradox of simultaneous high turnover and high selectivity in k channels.	x - ray diffraction data were collected from frozen crystals (100k) of the kcsa k+ channel equilibrated with solutions containing barium chloride. difference electron density maps (fbarium fnative, 5.0 resolution) show that ba2 + resides at a single location within the selectivity filter. the ba2 + blocking site corresponds to the internal aspect (adjacent to the central cavity) of the inner ion position where an alkali metal cation is found in the absence of the blocking ba2 + ion. the location of ba2 + with respect to rb+ ions in the pore is in good agreement with the findings on the functional interaction of ba2 + with k+ (and rb+) in ca2 + -activated k+ channels (neyton, j., and c. miller. 1988. j. gen. physiol. 92:549567). taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.
dental health - care workers are at high risk of exposure to cross infection with bloodborne pathogens such as hepatitis b virus (hbv) and hepatitis c virus (hcv), human immunodeficiency virus (hiv) and mycobacterium tuberculosis, streptococci and other viruses and bacteria that colonize the oral cavity and the respiratory tract. during dental procedures, transmission of infections could occur through direct contact with blood, saliva or contaminated treatment water from dental units, injury with an anesthetic needle or splash exposure of the mucous membranes, droplets and aerosols as well as indirect contact with contaminated instruments and surfaces. accidental exposure to infections in dental settings can be avoided by using safety precautions at work and implementing infection control guidelines. however, since some exposures can not be prevented, vaccination and proper post - exposure management are the main forms of protection. direct involvement in patient treatment as part of their clinical training puts dental students at risk of exposure to pathogens. since the majority of carriers of infectious diseases can not be identified, implementation of standard universal precautions in dental schools is the most effective way to control cross infection. dental schools should provide their students with current guidelines and training in infection control and facilitate appropriate immunization. implementation can be achieved at all levels and is the first step toward changing the attitudes and habits of oral health - care professionals. in the college of dentistry at university of sharjah infection control procedures are applied in dental laboratories in the 2 and 3 year. in the 4 and 5 years of study, the personal protective equipment (ppe) at dental clinics at university of sharjah includes disposable caps, gowns, gloves and protective eyewear. students are to refrain from wearing jewelry in the dental clinics and must have short fingernails. students are required to have taken the hb vaccination before entering the clinical years. in year 4 they deal mainly with simple periodontal procedures and operative dentistry as well as pedodontics and single rooted endodontic procedures. as they progress into year 5, they further develop and consolidate their skills pertaining to patient management in complex conservative dentistry, pediatric dentistry, periodontics and endodontics. the purpose of this study was to investigate awareness, knowledge and compliance with recommended infection control procedures among year 4 and year 5 senior clinical dental students at the college of dentistry, university of sharjah, united arab emirates. in the united arab emirates, there are five undergraduate private dental schools, graduating approximately 250 students yearly. this study was conducted in one of these schools, at the college of dentistry, university of sharjah in sharjah city during the spring semester of 2011. the study group comprised of 4 and 5 year dental students (n = 119). our sample of students in university of sharjah would be representative of the other private dental schools in the country. the questionnaire used was adapted and modified from the article published by de souza. on his study conducted in rio the questionnaire consisted of 25 open and closed - ended questions related to barrier techniques, vaccination status, percutaneous and mucous membrane exposures in addition to the dental treatment of infected patients, infection control practices and awareness. it was distributed to all senior dental students. after obtaining permission from the deanship of the college, three of the co - authors approached to the students at the beginning of the students clinical sessions and invited them to participate in the study. the authors used the class lists obtained from the dean 's office to verify that the questionnaires were distributed to all of the senior students. students, who agreed to participate in the study, signed the consent form prior to answering the questionnaire. it took 2 days to access all clinical students because of different clinical groups. to ensure confidentiality of responses all analyses were conducted using the statistical package for social sciences (spss11.5) by ibm. the questionnaire was distributed to 223 dental students from year 4 to year 5, of which only 119 of them responded (response rate = 53%). distribution of the students according to gender and the year of study in dental college the average age was 24 years, ranging from 21 to 27 years. vaccinations for hbv were 94.6% and 96.8% for year 4 and year 5 students respectively. percentages of students distributed according to the doses of hbv immunization taken are shown in table 2. there was a significant difference between 4 and 5 year students (p 0.05). frequency and percentage of hepatitis b vaccination doses taken by senior dental students p 0.05). frequency and percentage of hepatitis b vaccination doses taken by senior dental students p < 0.001 the uses of protective barrier techniques reported by dental students are shown in table 3. nearly, all of the year 4 and year 5 students mentioned wearing gloves and masks at all times, with the exception of 3.6% of year 4 students reportedly wearing masks only some of the time. only 26.8% of year 4 students and 28.6% of year 5 students always used protective eyewear, while 64.3% of year 4 and 65% of year 5 students used it only occasionally. there were a reported 8.9% of year 4 and a 6.3% of year 5 students who claimed to have never used protective eyewear. both 4 and 5 year students also reported similar percentages pertaining to the use of caps, ranging from 30% to 41%. there was no significant difference in the usage of gowns or scrubs between 4 and 5 year students. use of protective barrier techniques reported by senior dental students nearly, 20.2% of students reported not removing their gloves upon leaving the immediate area of patient care. there was a statistically significant difference between year 4 and year 5 students regarding the changing of gloves after each procedure with the same patient. 75% of the 4 year students compared with 63.5% of the 5 year students changed their gloves after each procedure (p < 0.05). in general, only 47.9% of students reported washing hands after changing gloves. nearly, 95.8% reported sterilizing instruments after each dental procedure. 30.7% of students mentioned not removing their jewelry while working in the clinic and 33.3% reported keeping long fingernails. nearly, 87.1% thought that dental clinics were more prone to infectious contamination than other medical clinics. statistically significant differences were observed between year 4 and year 5 students regarding the attitude and the number of patients treated with infectious diseases. compared with 66% of the 4 year students, only 44% of the 5 year students did not mind treating patients with infectious diseases (p < 0.05). however, 20.6% of the 5 year students treated at least 1 patient with an infectious disease compared with none among 4 year students (p < 0.05). among 5 year students, 9 of them reported to have treated patients with hbv, while 6 students reported they had treated patients with hcv. only 1 student mentioned having treated patients with hbv and hcv and 4 students reported they had treated patients with herpes labialis infection. the number of students who had occupational exposure to blood and other fluids while treating patients is shown in table 4. the maximum number of reported exposures was related to the use of local anesthesia needles. there was a considerable difference between 4 and 5 year students (p < 0.05) who suffered from non - sterile percutaneous and mucous membrane exposures, with higher percentages reported among 5 year students (61.9%) compared with the 4 year students (44.6%). number of students who had percutaneous and mucous membrane exposures during dental treatment and type of exposures around 93.3% of the participants in this study were keen to follow the same infection control procedures in their own clinics, upon graduation. the results of our study regarding hbv immunization of senior dental students, stood at 95.8%. de souza. reported that 90.8% of all senior students received vaccinations in 6 dental schools in rio de janeiro, brazil. mccarthy and britton 's study showed 100% immunization among the final year undergraduate dental, medical and nursing students at the university of western ontario, canada. on the other hand, singh. stated that 61.2% of undergraduate students in a dental school in central india were not vaccinated for hbv even though it was mandatory. he concluded a positive attitude but poor compliance of infection control practices among dental students. among the vaccinated students in our study only 60.7% in year 4 and 68.2% in year 5 completed the minimum required dosage (three doses) needed to obtain adequate immunity. these numbers are quite below the percentage of students who have completed the required doses reported by de souza.. the lower rate of complete hbv vaccination in our sample emphasizes the need to further encourage vaccination and serological testing to reduce the risk of acquiring hbv, in the light of recent infection control guidelines. in our survey, 50.4% of students who were immunized tested for post immunization serology. in comparing previous studies, although 83.3% took the required doses for immunity in de souza 's survey, only 27.5% of participants reported post hbv immunization serology. in mccarthy and britton 's study, a significant proportion failed to confirm the adequacy of post immunization anti - hb titer. there is a concern that since hbv immunization does not always produce a sufficient response, the immunization may give students a false feeling of safety even if they did not have an adequate response. the use of gloves among the dental students in our study was 99.2% and the use of masks was 98.3%. these results are comparable with previous studies assessing the use of ppe among dental students. compliance with the use of protective eyewear was quite low, only 30%, when compared with 59.7%, 93.5% and 84.2% use of protective eyewear reported in other studies. students should be reminded that neglecting the use of protective eyewear puts them at risk of transmission of infectious diseases through exposed membranes. there was a 40% compliance with the use of caps by dental students at the university of sharjah, which is lower than the previous studies. personal protective clothing such as gowns or scrubs are worn as a barrier to prevent transmission of microorganisms between patients and dental health - care workers. research has shown that aerosol and splatter containing pathogens can contaminate clinical wear, targeting the chest and forearms and remain alive for several days. the majority of subjects in our study mentioned changing their scrubs when it was visibly contaminated. it has been recommended that dental uniforms be worn only in dental clinics, changed daily and immediately after a blood splatter to prevent cross contamination. furthermore, leivers. and qureshi. suggested that the uniforms should be washed separately and stressed the importance of using disposable gowns. (2010) reported only 45% compliance of hand hygiene among the graduation students. the low compliance with regular hand washing necessitates stricter measures to remind the students of the importance of hand washing. hand washing signs can be placed near each basin in dental clinics since hand washing is a significant element in infection control. in our college, a newly appointed infection control officer, other than the scheduled instructors of the clinics, makes rounds in every clinical session to ascertain implementation of infection control in students clinics. the significant difference between the 4 and 5 year students who suffered from accidental injuries may be due to the fact that fifth year students had longer clinical exposure when compared with the 4 year students. mccarthy and britton reported 82% accidental injuries whereas de souza. reported 31% accidental injuries. in our survey non - sterile occupational injuries may pose a risk of transmission of bloodborne pathogens especially hbv, c and hiv. at university of sharjah, there is a post exposure management program for non - sterile occupational injuries during the students clinical training. recapping the needles with both hands was found to be the most common cause of percutaneous injuries. when multiple injections of local anesthetic are needed for one patient, dentists may recap the needles even when it is not recommended. at university of sharjah, college of dentistry of recent, every local anesthetic syringe is paired with artery forceps in a pouch to enable the removal of needles after usage, without direct hand contact. the limitation of our study was that responses were based on students self - assessments rather than under the supervision by investigators of the study in a clinical environment. therefore, the responses may not accurately reflect the actual infection control practices of dental students. the results of our study regarding hbv immunization of senior dental students, stood at 95.8%. de souza. reported that 90.8% of all senior students received vaccinations in 6 dental schools in rio de janeiro, brazil. mccarthy and britton 's study showed 100% immunization among the final year undergraduate dental, medical and nursing students at the university of western ontario, canada. on the other hand, singh. stated that 61.2% of undergraduate students in a dental school in central india were not vaccinated for hbv even though it was mandatory. he concluded a positive attitude but poor compliance of infection control practices among dental students. among the vaccinated students in our study only 60.7% in year 4 and 68.2% in year 5 completed the minimum required dosage (three doses) needed to obtain adequate immunity. these numbers are quite below the percentage of students who have completed the required doses reported by de souza.. the lower rate of complete hbv vaccination in our sample emphasizes the need to further encourage vaccination and serological testing to reduce the risk of acquiring hbv, in the light of recent infection control guidelines. in our survey, 50.4% of students who were immunized tested for post immunization serology. in comparing previous studies, although 83.3% took the required doses for immunity in de souza 's survey, only 27.5% of participants reported post hbv immunization serology. in mccarthy and britton 's study, a significant proportion failed to confirm the adequacy of post immunization anti - hb titer. there is a concern that since hbv immunization does not always produce a sufficient response, the immunization may give students a false feeling of safety even if they did not have an adequate response. the use of gloves among the dental students in our study was 99.2% and the use of masks was 98.3%. these results are comparable with previous studies assessing the use of ppe among dental students. compliance with the use of protective eyewear was quite low, only 30%, when compared with 59.7%, 93.5% and 84.2% use of protective eyewear reported in other studies. students should be reminded that neglecting the use of protective eyewear puts them at risk of transmission of infectious diseases through exposed membranes. there was a 40% compliance with the use of caps by dental students at the university of sharjah, which is lower than the previous studies. personal protective clothing such as gowns or scrubs are worn as a barrier to prevent transmission of microorganisms between patients and dental health - care workers. research has shown that aerosol and splatter containing pathogens can contaminate clinical wear, targeting the chest and forearms and remain alive for several days. the majority of subjects in our study mentioned changing their scrubs when it was visibly contaminated. it has been recommended that dental uniforms be worn only in dental clinics, changed daily and immediately after a blood splatter to prevent cross contamination. furthermore, leivers. and qureshi. suggested that the uniforms should be washed separately and stressed the importance of using disposable gowns. the low compliance with regular hand washing necessitates stricter measures to remind the students of the importance of hand washing. hand washing signs can be placed near each basin in dental clinics since hand washing is a significant element in infection control. in our college, a newly appointed infection control officer, other than the scheduled instructors of the clinics, makes rounds in every clinical session to ascertain implementation of infection control in students clinics. the significant difference between the 4 and 5 year students who suffered from accidental injuries may be due to the fact that fifth year students had longer clinical exposure when compared with the 4 year students. mccarthy and britton reported 82% accidental injuries whereas de souza. reported 31% accidental injuries. in our survey non - sterile occupational injuries may pose a risk of transmission of bloodborne pathogens especially hbv, c and hiv. at university of sharjah, there is a post exposure management program for non - sterile occupational injuries during the students clinical training. recapping the needles with both hands was found to be the most common cause of percutaneous injuries. when multiple injections of local anesthetic are needed for one patient, dentists may recap the needles even when it is not recommended. at university of sharjah, college of dentistry of recent, every local anesthetic syringe is paired with artery forceps in a pouch to enable the removal of needles after usage, without direct hand contact. the limitation of our study was that responses were based on students self - assessments rather than under the supervision by investigators of the study in a clinical environment. therefore, the responses may not accurately reflect the actual infection control practices of dental students. it is necessary to effectively communicate to students the associated risks and importance of transmission of infectious diseases and exposures during dental treatments. efforts are needed to improve attitudes, to implement information and motivate students in the correct and routine use of infection control measures. with all infection control protocols already implemented in dental schools,	objective : the purpose of this study was to investigate compliance, awareness and practices of infection control procedures among senior dental students at the college of dentistry, university of sharjah, united arab emirates.materials and methods : the study comprised of 119 subjects of 4th and 5th year dental students. a questionnaire was developed with 25 open and closed - ended questions related to barrier techniques, vaccination status, infection control practices and awareness. this was distributed among the senior dental students and completed upon signing the consent form. each questionnaire was coded to ensure the confidentiality of responses.results:the questionnaire was distributed among 223 senior dental students at the university of sharjah of which only 119 students (53%) responded. compliance with the use of protective barriers was high with the exception of protective eye wear, utilized by a mere 27% of students. there was a significant difference between 4th and 5th year dental students attitudes (p < 0.05) regarding the treatment of patients with infectious diseases. compared with 44.4% of the 5th year students, 68.5% of the 4th year students did not mind treating patients with infectious diseases. owing to this, 61.9% of the 5th year students suffered from non - sterile percutaneous and mucous membrane exposures compared with 44.6% of the 4th year students (p < 0.05).conclusions : efforts are needed to improve attitudes, implement information and motivate students in the correct and routine use of infection control measures. with all infection control protocols already implemented in dental schools, the challenge remains on improving compliance with infection control recommendations.
mycobacterium tuberculosis was isolated from the cerebrospinal fluid (csf) of a patient diagnosed with tuberculosis at a local hospital. the sample was cultured in bbl mgit mycobacterial growth indicator tube supplemented with bbl mgit oadc enrichment and bbl mgit panta antibiotic mixture (becton - dickinson, oxford, united kingdom). since the isolate was found to be resistant to rifampicin and isoniazid, it was labeled as multidrug resistant m. tuberculosis strain pr10 (mdr - tb pr10). genomic dna was extracted and sequenced using miseq (illumina, ca, usa), generating a total of 10,595,456 reads in a 300-cycle run. raw reads were trimmed and assembled de novo using clcbio (clc genomics workbench version 7.0.3) (clcbio, aarhus, denmark), producing 163 contigs with an average coverage of 334. annotation was performed using the bacterial annotation system (basys) and rapid annotation using subsystem technology (rast) online services, and the pathogenicity and virulence genes were determined. the genes identified were validated using the following external gene annotation databases : tuberculist (http://tuberculist.epfl.ch), uniprotkb (http://www.ebi.ac.uk/uniprot), virulence factor database (vfdb) (http://www.mgc.ac.cn), and tbdatabase (tbdb) (http://www.tbdb.org). the size of the draft genome mdr - tb pr10 is 4,347,484 bp with a g + c content of 65.6%. basys annotation software predicted 4637 genes and 50 rna - encoding elements. using rast, a total of 400 subsystems and 4286 protein - coding genes were annotated in the mdr - tb pr10 genome (fig. a gene that encodes dna - directed rna polymerase beta ' subunit (rpob) (ec 2.7.7.6) was annotated in mdr - tb pr10. the gene was reported to be involved in the invasion and intracellular resistance subsystem that may play a role in resistance towards certain drugs such as rifampicin. the rpob gene is responsible for the transcription of dna into rna using the four ribonucleoside triphosphates as substrates catalyzed by dna - dependent rna polymerase. mutations that occur in the rpob gene may cause the changes in the protein translation of mycobacterium that may confer resistance to rifampin,. comparison of genome sequences using rast showed that the strains closest to pr10 are m. tuberculosis ncgm2209 (score of 521), followed by m. tuberculosis na - a0008 (score of 459) and m. tuberculosis um 1072388579 (score of 400). the whole genome shotgun project has been deposited at ddbj / embl / genbank under the accession number cp010968. the authors declare that there is no conflict of interests with respect to the work published in this paper.	here, we report the draft genome sequence and annotation of a multidrug resistant mycobacterium tuberculosis strain pr10 (mdr - tb pr10) isolated from a patient diagnosed with tuberculosis. the size of the draft genome mdr - tb pr10 is 4.34 mbp with 65.6% of g + c content and consists of 4637 predicted genes. the determinants were categorized by rast into 400 subsystems with 4286 coding sequences and 50 rnas. the whole genome shotgun project has been deposited at ddbj / embl / genbank under the accession number cp010968.
the us healthcare costs are assuming an increasing level of importance. medicare expenditures for inpatient care are expected to increase from us$129.1 billion in 2008 to us$234.9 billion in 2019. approximately 19.6% of medicare recipients are rehospitalized within 30 days following discharge from an acute care setting. in 2010, the patient protection and affordable care act (ppaca ; p.l.-11 - 148) was signed into law in the united states. hospital readmission is a complex problem with multiple etiologies, and there are no simple strategies to reduce their incidence. despite the complexity, readmission is seen as an important performance and accountability measure for hospitals. hospital readmission following hip fracture is a frequent and serious sentinel event that may be avoidable and may indicate a gap in care. hip fracture is the leading orthopedic discharge diagnosis associated with 30-day readmission in terms of numbers. because readmission to the hospital following a hip fracture is so common, it adds considerably to the costs on an already overburdened health care system. readmission rates following hip fracture have increased slightly from 14.3% in 2004 to 14.5% in 2009. hip fracture has been shown to be the third most costly diagnosis in medicare recipients aged 65 and older accounting for 4.6% share of total spending. most hip fractures occur in patients aged 65 years and older, which is the most rapidly growing segment of the population in the united states. the number of hip fractures is predicted to increase by 51% by 2025. with increasing number of patients treated and discharged, the associated economic impact of hospital readmission is also growing. this manuscript will analyze the costs of the initial inpatient admission, readmission, and 30-day hospital readmission rates of 1081 patients with a native nonpathologic, low - energy hip fracture treated at a single level 3 trauma center over a 65-month period. the causes of readmission and the outcomes of the readmitted patients will also be examined. the objective of this study is to evaluate the costs, frequencies, and reasons for readmission after hip fracture. a secondary purpose of the study would be to compare the costs of readmission to the costs of the original admission. all patients aged 65 and older admitted to a 261-bed university - affiliated level 3 trauma center between april 30, 2005, and september 30, 2010, with a unilateral, native, nonpathologic low - energy proximal femur fracture were identified from a fracture registry and included for analysis. patients with periprosthetic fractures, pathologic fractures, bilateral injuries, and high - energy mechanisms were excluded. all patients had retrospective chart reviews completed by a member of the research team as part of a hospital quality management initiative. data were collected by a study nurse from patients directly and from their medical records and included demographic information, comorbidities, surgical management, in - hospital complications as well as any readmission within 30 days of original discharge. readmissions within the original health care system, which includes 2 hospitals, were confirmed with the hospital s admission tracking computer system. these patients were contacted by telephone for information regarding their readmission. because data on costs of care could not be obtained, these patients were excluded from financial analysis. we also analyzed charges for care rather than actual costs because charges are typically reported by governmental reports. statistical analyses were performed on spss v20 software with statistical significance being reached on the 2-tailed student t test when p 851.581.02 - 2.26.02 male1.491.00 - 2.24.05assisted living1.520.82 - 2.59.12skilled nursing1.240.84 -1.85.29time to surgery > 24 hours1.461.00 - 2.15.05parker mobility score medium (5 - 8)1.810.98 - 3.35.06 low (0 - 4)1.500.85 - 2.64.16activities of daily living partial or complete disability1.511.03 - 2.25.03 charlson score medium (2 - 3)1.530.97 - 2.55.11 high (4 or more)1.651.00 - 2.74.05in - hospital complications (initial hospitalization for index fracture) delirium1.661.14 - 2.41.01 hematoma7.510.47 - 121.16 urinary tract infection1.840.39 - 8.84.44 preoperative arrhythmia1.621.09 - 2.39.02 past medical history pacemaker1.751.11 - 2.76.02 gerd1.440.99 - 2.10.05 diabetes1.911.22 - 2.99.005 dementia1.611.12 - 2.22.01 cardiac disease1.020.66 - 1.59.92 alcoholism1.120.46 - 2.68.81 tobacco use0.990.56 - 1.73.54abbreviation : gerd, gastroesophageal reflux disease. the parker mobility score is a functional assessment that rates the patient s ability to get about the house, to get out of the house, and to go shopping, with no difficulty (3), with an aid (2), with help from another person (1), and not at all (0). denotes statistical significance (p <.05) hospital charges were available for 123 of the 129 readmitted patients. these patients accumulated an average hospital charge of us$16 308 us$6400 during their initial hospitalization for their native hip fractures. the average charges accumulated during their readmission within 30 days was us$$14 191 us$25 035 (p =.36). us$4200.81congestive heart failureus$14 526.71 us$3921.52myocardial infarctionus$15 131.50 us$1720.39 clostridium difficile infectionus$16 016.40 us$2193.88obstipationus$15 693.00 us$4922.85gastrointestinal bleedus$15 418.00 us$6153.86small bowel obstructionus$18 322.50 us$5438.36ileusus$17 017.67 us$2570.99thromboembolic eventus$10 508.33 us$10 453.86decubitus ulcerus$16 343.00 us$6398.96deep infectionus$15 842.75 us$3233.37superficial infectionus$10 029.50 us$1207.03hip dislocationus$22 262.00 us$1711.20failure of fixationus$11 976.00 us$3710.90second fractureus$18 676.29 us$4840.90strokeus$14 836.00 us$5456.59deliriumus$11 314.33 us$1049.60seizureus$15 439.50 us$5479.37copd exacerbationus$20 908.67 us$5972.60pneumoniaus$16 145.20 us$4765.17acute kidney injuryus$13 119.67 us$8624.22fluid / electrolyte disturbanceus$16 003.67 us$4703.08urinary tract infectionus$16 796.00 us$11 724.00abbreviation : copd, chronic obstructive pulmonary disease ; sd, standard deviation. average age (number of patients)1 - 7 days8 - 14 days15 - 21 days22 - 30 dayspulmonary pneumonia89 5 (n = 27)13932 respiratory failure88 5 (n = 6)4101 chronic obstructive disease82 7 (n = 2)0101gastrointestinal gastrointestinal bleed87 6 (n = 5)1301 small bowel obstruction90 6 (n = 3)2100 fecal impaction92 13 (n = 3)0102 clostridium difficile infection93 2.5 (n = 6)2301 illeus72 8 (n = 2)2000 failure to thrive88 4 (n = 2)2000neurologic stroke83 11 (n = 5)2201 delirium86 6 (n = 2)1001 seizure77 8 (n = 2)1010 intracranial hemorrhage87 (n = 1)0010cardiovascular congestive heart failure92 8 (n = 7)1015 atrial fibrillation85 6 (n = 7)5002 myocardial infarction94 1(n = 2)1001musculoskeletal refracture78 9 (n = 3)2001 failure of fixation92 3 (n = 3)1110 new site fracture87 4 (n = 7)4021 deep wound infection85 10 (n = 3)0300 superficial wound infection79 6 (n = 2)0110 dislocation of joint74 16 (n = 2)0020 pressure ulcer89 6 (n = 3)0012 hematoma89 (n = 1)1000genitourinary urinary infection90 7 (n = 5)1220 urosepsis87 5 (n = 2)1100 urinary retention98 (n = 1)1000 acute renal failure83 10 (n = 3)1101 electrolyte abnormality96 1 (n = 2)1100hematologic anemia82.5 1 (n = 2)2000 pulmonary emboli or deep vein thrombosis78 6 (n = 3)2010other 90 3 (n = 5)2201 number of patients readmitted in each time period. this is the first study looking at patient - level clinical and financial data on patients with hip fracture from the united states. readmission is often associated with serious medical and surgical complications of the original hospitalization, and this was true in this study.. however, here we identified some reasons for readmissions that are indicators or poor quality and are potentially preventable (table 2). in this study, these preventable causes can serve as targets for future quality improvement efforts. under ppaca, the centers for medicare and medicaid services (cms) will begin to hold hospitals accountable for their medical readmission rates starting with 4 specific diagnoses. this will be accomplished with public reporting of individual hospital readmission rates and decreased hospital reimbursement from cms. it is clear that government policy is capable of altering practice habits of clinicians through financial incentives or penalties. reducing payments and ultimately reducing monetary resource allocation toward the most costly medical conditions does not necessarily result in similar outcomes. patients with hip fracture may increase financial burden on the health care system before they have sustained a fracture. kilgore recently studied 60 354 medicare patients with hip fractures and found 88% of increased health care expenditure is directly associated with the fracture. furthermore, in the months leading up to their fracture, these patients consumed significantly more health care resources than matched controls. expenditures on every body system studied (ie, cardiovascular, pulmonary, endocrine, neurologic, genitourinary, etc) increased significantly after their hip fracture. this suggests that the patient who sustains a hip fracture is experiencing a general decline in health prior to his or her fracture. more critical research into this topic may yield models that could potentially predict a patient s risk of hip fracture, which may allow for preventive measures to be developed. one study of 606 patients for 180 days after hip fracture found an readmission rate of 8.3%. the rate varied by discharge destination with inpatient rehabilitation (4.5%) and home (5.1%) having the lowest rates. multivariate analysis in this study further supported that inpatient rehabilitation decreased readmission rates, while patients with longer loss had higher odds of readmission. buecking reviewed 402 patients with hip fracture (80% living at home alone or with family) and found a 12% readmission rate. the majority (79%) were not related to their fracture, with respiratory failure (25%), cardiovascular morbidity (15%), and infectious disease (10%) being the most common reasons for readmission, all similar to our data in this study. multivariate analysis suggested that males and specifically femoral neck fractures had an increased risk of readmission. discharge to inpatient rehabilitation, especially those with more comorbidities, may be a potential route for improving readmission rates. french described a 30-day readmission rate of 18.3% using claims data from 41 331 us veterans aged 65 years with a hip fracture. the readmitted patients in that study had a 1-year mortality rate of 48.5% compared with a 24.9% mortality rate in veterans who were not readmitted. bookvar described a prospective analysis of 562 patients with hip fracture aged 50 years. of these readmissions, 11% were readmitted for surgical causes and 89% were readmitted for medical reasons. readmitted patients in their series were found to have an increased risk of mortality, impaired gait, and placement in a nursing home 6 months following fracture. jencks published a 30-day readmission rate of 17.9% after major hip or femur surgery and cited pneumonia and chf as being the 2 most frequent causes of readmission. the recently published dartmouth atlas report on readmissions highlights the considerable variation seen in readmission rates seen among both community and academic medical centers. the specific causes for this variation are not clear. there has been no improvement in readmission rates over the past decade, 14.3% in 2004 and 14.5% in 2009. for new york state hospitals, many causes for readmission have been described including communication issues, problems with medication reconciliation, lack of satisfactory follow - up care, and defects in the original inpatient care. other causes for readmissions may include shorter loss, increased age of the patients, and increased burden of comorbidity carried by these patients. typically, the inpatient care team only addresses the inpatient care phase with no interventions extending past the inpatient stay. the authors believe that some of our readmissions are likely of a preventable nature including some cases of constipation, cutout of implants, and congestive failure. there are likely some cases that could be avoided with improved communication with receiving providers at the time of the discharge handoff. there have been several successful methods published for reduction in readmissions following medical hospitalization including early follow - up care with the primary care physician, the coleman discharge coaching model, and the naylor model. additional efforts at improving the discharge process, communication, and postdischarge follow - up may improve the readmission rates this is a single - center study conducted in a hip fracture program with a strong history for quality improvement, comanaged care, and utilizing standardized protocols. the sample size of 1081 with 129 readmissions is certainly not large enough to generalize these results. larger, multicenter studies may be useful to determine whether these results can be generalized to other centers. another limitation is the retrospective nature of the data collection which may not fully capture all readmissions or adverse events. as a countermeasure, we have tried to capture all the 30-day readmissions by reviewing medical records and calling the patients, families, or caregivers following discharge. the patients included in this study may not accurately represent the populations seen at many centers. half of our patient population were admitted from a nursing home or assisted (residential care) living home, whereas most published studies describe 80% to 90% of patients with hip fracture admitted from a home living setting. we were also unable to access a detailed data sample for 6 of the patients readmitted to regional hospitals. this is a limitation inherent to the us health care system where medical and economic data are typically not shared between regional hospitals. this is a single - center study conducted in a hip fracture program with a strong history for quality improvement, comanaged care, and utilizing standardized protocols. the sample size of 1081 with 129 readmissions is certainly not large enough to generalize these results. larger, multicenter studies may be useful to determine whether these results can be generalized to other centers. another limitation is the retrospective nature of the data collection which may not fully capture all readmissions or adverse events. as a countermeasure, we have tried to capture all the 30-day readmissions by reviewing medical records and calling the patients, families, or caregivers following discharge. the patients included in this study may not accurately represent the populations seen at many centers. half of our patient population were admitted from a nursing home or assisted (residential care) living home, whereas most published studies describe 80% to 90% of patients with hip fracture admitted from a home living setting. we were also unable to access a detailed data sample for 6 of the patients readmitted to regional hospitals. this is a limitation inherent to the us health care system where medical and economic data are typically not shared between regional hospitals. the most common reasons for readmission include pneumonia, chf, new fractures, intestinal obstructions, and infections. of the patients, readmitted patients generated similar average hospital charges during readmission (us$14 191) compared to their initial hospitalization (us$16 308).	introduction : hip fracture is the leading orthopedic discharge diagnosis associated with 30-day readmission in terms of numbers. because readmission to the hospital following a hip fracture is so common, it adds considerably to the costs on an already overburdened health care system.methods:patients aged 65 and older admitted to a 261-bed university - affiliated level 3 trauma center between april 30, 2005, and september 30, 2010, with a unilateral, native, nonpathologic low - energy proximal femur fracture were identified from a fracture registry and included for analysis. readmissions within 30 days of hospital discharge, costs, and outcomes were collected and studied.results:of 1081 patients, 129 (11.9%) were readmitted within 30 days. the average hospital length of stay for readmissions was 8.7 18.8 days, which was significantly longer than the initial stay (4.6 2.3 days) (p =.03). nineteen percent (24 patients 19%) died during readmission versus 2.8% during the index admission. these patients accumulated an average hospital charge of us$16 308 us$6400 during their initial hospitalization for compared with charges for their readmissions of us$14 191 us$25 035 (p =.36).discussion : readmission was usually associated with serious medical or surgical complications of the original hospitalization.conclusions:readmission after hip fracture is costly and harmful. charges were similar between the original fracture admission and the readmission. patients were readmitted most frequently for medical diagnoses following their original hospital stay. some of these readmissions may have been avoidable.
	more than a million workers are at risk for methylene chloride exposure. aerosol sprays and paint stripping may also cause significant nonoccupational exposures. after methylene chloride inhalation, significant amounts of carbon monoxide are formed in vivo as a metabolic by - product. poisoning predominantly affects the central nervous system and results from both carboxyhemoglobin formation and direct solvent - related narcosis. in this report, we describe a case of methylene chloride intoxication probably complicated by exogenous carbon monoxide exposure. the worker 's presentation of intermittent headaches was consistent with both methylene chloride intoxication and carbon monoxide poisoning. the exposures and symptoms were corroborated by elevated carboxyhemoglobin saturations and a workplace inspection that documented significant exposures to both methylene chloride and carbon monoxide. when both carbon monoxide and methylene chloride are inhaled, additional carboxyhemoglobin formation is expected. preventive efforts should include education, air monitoring, and periodic carboxyhemoglobin determinations. methylene chloride should never be used in enclosed or poorly ventilated areas because of the well - documented dangers of loss of consciousness and death.imagesfigure 1
during the last 5 years, advances in laparoscopic equipment and technique, including the addition of the da vinci surgical system (intuitive surgical, mountain view, california), have resulted in significant progress in the development of minimally invasive surgery for localized prostate cancer. the combination of less postoperative morbidity, improved cosmesis, shorter convalescence, and the possibility of comparable outcomes has lured patient demand away from the conventional open retropubic prostatectomy technique. robot - assisted prostatectomy has been widely popularized with over 1000 cases already reported in the literature by a single surgeon. at our institution, we developed a total extraperitoneal laparoscopic and robotic approach and found this procedure to be technically feasible and reproducible. robot - assisted prostatectomy has been performed at multiple centers in both the united states and europe, with early oncological and functional results still being reported. objective data using validated questionnaires on postrobot - assisted nerve - sparing radical prostatectomy are sparse. we objectively assessed erectile function after robot - assisted extraperitoneal prostatectomy by using the 5-item version of the international index of erectile function (iief-5), also known as the sexual health inventory for men (shim), validated questionnaire. analysis of our first 150 patients undergoing extraperitoneal robotic prostatectomy by the same surgeon (jvj) was performed. we prospectively collected baseline demographic data, such as race, body mass index, serum psa, prostate volume, gleason grade / sum, clinical stage, and associated co - morbidities. perioperative, intraoperative, and postoperative data along with early functional and short - term oncological results were also prospectively enrolled, recorded, and analyzed. the indications for laparoscopic prostatectomy were identical to those of the conventional open radical retropubic prostatectomy. a nerve - sparing procedure was applied to all patients where oncological control was not jeopardized, regardless of preoperative sexual activity. bilateral and unilateral nerve - sparing procedures were performed in 93 and 24 patients, respectively. table 1 reviews the clinical features of the first 150 patients undergoing extraperitoneal robot - assisted nerve - sparing radical prostatectomy at our institution. clinical parameters and intraoperative data of 150 patients undergoing extraperitoneal robot - assisted nerve - sparing radical prostatectomy at our institution our method for development of extraperitoneal pneumoperitoneum and development of the space of retzius is based on our preliminary experience with laparoscopic radical prostatectomy and has been described else - wherere. briefly, the procedure is performed in an antegrade fashion with initial transection of the bladder neck and exposure of the seminal vesicles. the nerve - sparing operation commences after the seminal vesicles are freed, and the lateral prostatic pedicles are dissected with the reflection of the lateral pelvic fascia off the prostate. the vessels entering the prostate base are selectively coagulated by using bipolar forceps before their transection with da vinci articulating scissors. the shim validated questionnaire was used to assess potency status at the 6-month and yearly follow - up visit or during a separate phone interview by a third party. all patients receive a prescription for oral pharmacological assistance postprocedure and are instructed on its use. the degree of erectile function was classified into 4 grades : < 11=impotent ; 11 to 15=moderate erectile dysfunction (ed) ; 16 to 21=mild erectile dysfunction ; 22 to 25=potent. analysis of our first 150 patients undergoing extraperitoneal robotic prostatectomy by the same surgeon (jvj) was performed. we prospectively collected baseline demographic data, such as race, body mass index, serum psa, prostate volume, gleason grade / sum, clinical stage, and associated co - morbidities. perioperative, intraoperative, and postoperative data along with early functional and short - term oncological results were also prospectively enrolled, recorded, and analyzed. the indications for laparoscopic prostatectomy were identical to those of the conventional open radical retropubic prostatectomy. a nerve - sparing procedure was applied to all patients where oncological control was not jeopardized, regardless of preoperative sexual activity. bilateral and unilateral nerve - sparing procedures were performed in 93 and 24 patients, respectively. table 1 reviews the clinical features of the first 150 patients undergoing extraperitoneal robot - assisted nerve - sparing radical prostatectomy at our institution. clinical parameters and intraoperative data of 150 patients undergoing extraperitoneal robot - assisted nerve - sparing radical prostatectomy at our institution our method for development of extraperitoneal pneumoperitoneum and development of the space of retzius is based on our preliminary experience with laparoscopic radical prostatectomy and has been described else - wherere. briefly, the procedure is performed in an antegrade fashion with initial transection of the bladder neck and exposure of the seminal vesicles. the nerve - sparing operation commences after the seminal vesicles are freed, and the lateral prostatic pedicles are dissected with the reflection of the lateral pelvic fascia off the prostate. the vessels entering the prostate base are selectively coagulated by using bipolar forceps before their transection with da vinci articulating scissors. the shim validated questionnaire was used to assess potency status at the 6-month and yearly follow - up visit or during a separate phone interview by a third party. all patients receive a prescription for oral pharmacological assistance postprocedure and are instructed on its use. the degree of erectile function was classified into 4 grades : < 11=impotent ; 11 to 15=moderate erectile dysfunction (ed) ; 16 to 21=mild erectile dysfunction ; 22 to 25=potent. a nerve - sparing procedure was performed in 117 of the first 150 patients, 24 unilateral and 93 bilateral. the additional 33 patients had preplanned nonnerve - sparing robotic - assisted prostatectomies to avoid compromising cancer control. the high - risk features used to exclude patients from a nerve - sparing approach were gleason sum / grade, psa, number of positive biopsies, and clinical stage. the iief-5 questionnaire was used to assess postoperative potency in 67 patients who were at least 6 months postsurgery. erectile function was classified as impotent (< 11) ; moderate dysfunction (11 to 15) ; mild dysfunction (16 to 21) ; and potent (22 to 25). twelve patients were excluded who abstained from all sexual activity after surgery for emotional or social reasons. from the remaining 55 patients evaluated, 4 had nonnerve - sparing, 6 had unilateral nerve - sparing, and 45 had bilateral nerve - sparing. of the 55 patients, 22 (40%) were impotent, 3 (5.5%) had moderate ed, 12 (21.8%) had mild ed, and 18 (32.7%) were fully potent. table 2 compares iief-5 scores with nerve - sparing status. of patients who had bilateral nerve sparing, 28/45 (62.2%) had mild or no ed within 6 to 12 months postsurgery, and all expressed satisfaction with their current sexual function or rate of improvement after robotic prostatectomy (table 2). when stratified by age, men under 60 more frequently had an erection sufficient for sexual intercourse than those who were older (mean iief-5 scores, 17.6 versus 10.8 ; p<0.05). at 6-month follow - up, 20/30 patients (67%) who were 60 years of age reported mild ed or were fully potent, compared with 10/25 (40%) in patients older than 60. ability to reach an orgasm was asked as a separate question after the set of validated questions. independent of whether the patients were potent or had erectile dysfunction, greater than 80% of the men remarked that they were able to attain an orgasm or did not have a change in their ability to attain an orgasm. a nerve - sparing procedure was performed in 117 of the first 150 patients, 24 unilateral and 93 bilateral. the additional 33 patients had preplanned nonnerve - sparing robotic - assisted prostatectomies to avoid compromising cancer control. the high - risk features used to exclude patients from a nerve - sparing approach were gleason sum / grade, psa, number of positive biopsies, and clinical stage. the iief-5 questionnaire was used to assess postoperative potency in 67 patients who were at least 6 months postsurgery. erectile function was classified as impotent (< 11) ; moderate dysfunction (11 to 15) ; mild dysfunction (16 to 21) ; and potent (22 to 25). twelve patients were excluded who abstained from all sexual activity after surgery for emotional or social reasons. from the remaining 55 patients evaluated, 4 had nonnerve - sparing, 6 had unilateral nerve - sparing, and 45 had bilateral nerve - sparing. of the 55 patients, 22 (40%) were impotent, 3 (5.5%) had moderate ed, 12 (21.8%) had mild ed, and 18 (32.7%) were fully potent. table 2 compares iief-5 scores with nerve - sparing status. of patients who had bilateral nerve sparing, 28/45 (62.2%) had mild or no ed within 6 to 12 months postsurgery, and all expressed satisfaction with their current sexual function or rate of improvement after robotic prostatectomy (table 2). when stratified by age, men under 60 more frequently had an erection sufficient for sexual intercourse than those who were older (mean iief-5 scores, 17.6 versus 10.8 ; p<0.05). at 6-month follow - up, 20/30 patients (67%) who were 60 years of age reported mild ed or were fully potent, compared with 10/25 (40%) in patients older than 60. ability to reach an orgasm was asked as a separate question after the set of validated questions. independent of whether the patients were potent or had erectile dysfunction, greater than 80% of the men remarked that they were able to attain an orgasm or did not have a change in their ability to attain an orgasm. the introduction of the anatomical nerve - sparing prostatectomy resulted in improved sexual function without compromising cancer control. in the psa era, as patients are being increasingly diagnosed with localized prostate cancer, a main focus of treatment has become prevention of significant nonlife - threatening morbidity (such as incontinence and erectile dysfunction) while maintaining cancer control. the multiple advantages afforded by the robot (increased precision and dexterity with wristed instrumentation, ergonomic manipulation, and 3-dimensional visualization with 10x magnification) have encouraged both laparoscopically nave and trained surgeons to embrace this technique. current reports have already shown superior return of urinary control when compared with that of open surgery. in addition, early series have demonstrated comparable early tumor control and oncological outcomes. as opposed to tumor control and incontinence, where there are objective tests that can accurately determine functional and oncological outcomes, the process of achieving a successful erection for intercourse is dependent on multiple patient parameters including age, co - morbid medical conditions like diabetes and peripheral vascular disease, psychological, behavioral, and social factors. in addition, varying surgical technique and operative modality as well as differences in surgeons ' patient selection has further compromised our understanding of postoperative potency status. finally, the different definitions of postoperative potency and erectile dysfunction used as well as the multiple validated questionnaires in existence have made it even harder to assess. there has been a wide range of postnerve - sparing prostatectomy potency rates in the world literature. walsh reported on 64 patients who underwent conventional open prostatectomy with bilateral neurovascular nerve preservation and found 73% of the patients were potent and sexually active. in a series by catalona, 68% recovery of erections after bilateral, and 41% after unilateral nerve - sparing surgery was reported. when stratified by age, they found a 75% return of erection in patients < 60 who had bilateral nerve - sparing surgery. gralnek demonstrated a 39% potency rate in 46 men after unilateral nerve preservation, and in a more recent series, rabbani reported on 314 men undergoing open radical prostatectomy and found unilateral nerve preservation to be associated with a 25% rate of erectile function, while bilateral nerve sparing was associated with a 47% rate in patients older than 65 years of age and 76% in patients who were younger. others have reported a large range of potency rates varying between 11% to 86% (table 3). reported potency rates and method of prostatectomy denotes unilateral resection and unilateral / bilateral damage, respectively. denotes inclusion of unilateral preservation with the contralateral nerve being partially excised or widely excised. denotes a subset of the entire group analyzed. a large series from johns hopkins university demonstrated age to be a predictor of recovery of potency. they showed that in patients < 50 years of age, regardless of the extent of nerve sparing, there was no difference in potency rates. in patients between 50 and 60 years of age, not only was the overall potency rate decreased, but recovery of erectile function was strongly dependent on whether one or both neurovascular bundles were preserved. reports by catalona and rabbani also demonstrated that age and extent of nerve preservation were strong predictors of the return of erectile function. reports on erectile function after laparoscopic and robotic nerve - sparing radical prostatectomy have been scant. of 44 patients having either unilateral or bilateral nerve preservation, they found 18 patients having spontaneous intercourse and 8 needing pharmacological assistance. rassweiler reported on 100 patients undergoing laparoscopic radical prostatectomy, whereby 10 patients underwent unilateral nerve - sparing surgery with only 4 of these men having intercourse with the aid of pharmaco - therapy. six of 10 patients undergoing laparoscopic bilateral nerve - sparing prostatectomy had successful intercourse in a series by bollens, and 9 of 20 (preoperative potent patients) undergoing laparoscopic bilateral nerve - sparing prostatectomy had successful intercourse in guillonneau and vallancien 's early series. subsequently, both teams reported potency rates of 65% and 66%, respectively, in their larger series. using sexual function questionnaires, katz reported on 143 patients undergoing laparoscopic nerve - sparing radical prostatectomy. fifty - eight percent of patients who had erections preoperatively maintained their erections after surgery. there have been even fewer reports of potency outcome after robotic - assisted nerve - sparing surgery compared with laparoscopic and open surgery. menon reported 82% of preoperatively potent patients younger than 60 having a return of some sexual activity using the expanded prostate cancer index composite, and 64% having sexual intercourse at 6-month follow - up. of patients over 60 years of age, 75% had sexual activity and 38% had sexual intercourse. using shim, ahlering reported a lower 43% potency rate after cautery - free robotic - assisted prostatectomy when including patients up to the age of 65. recent reports by menon describe the outcomes after robotic - assisted prostatectomy using both conventional and prostatic fascia - sparing techniques. at 12-month follow - up, 74% of patients having undergone conventional nerve - sparing robotic - assisted prostatectomy, and 97% of those having undergone the fascia - sparing procedure achieved erections sufficient for intercourse. in patients who did not use medications, however, shim scores demonstrated that the percentage achieving normal erections in the conventional and fascia - sparing groups was only 17% and 51%, respectively. our data are based on the 55 eligible patients able to fulfill the abovementioned criteria and undergo objective questioning using the iief-5 validated questionnaire. currently, the iief questionnaire published by rosen in 1997 is a worldwide accepted and validated tool in assessing all aspects of sexual function and dysfunction. of the 55 patients we evaluated, 22 (40%) were impotent, 3 (5.5%) had moderate ed, 12 (21.8%) had mild ed, and 18 (32.7%) were fully potent. of patients who had bilateral nerve sparing, 28/45 (62.2%) had mild or no ed within 6 to 12 months postsurgery, and all expressed satisfaction with their current sexual function or rate of improvement after robotic prostatectomy. the overall rate of sexual function at 1 year of 62.2% is comparable to the results in open and early laparoscopic and robotic surgery. as can be expected, the majority of these patients had undergone bilateral nerve - sparing techniques. all of our patients receive a prescription for oral pharmacologic assistance and take the medication routinely after surgery and discontinue it when they feel they can do without it. when the patients are stratified to younger or older than 60, men under 60 years of age had a statistically significant increase in the rate of erection sufficient for sexual intercourse ; however, those who were older did not (mean iief-5 scores of 17 versus 10 ; p<0.05). at 6-month follow - up, 20/30 patients (67%) who were 60 reported mild ed or were fully potent, compared with 10/25 (40%) in those patients one limitation of our study is that preoperative potency scores were not attained, which alters the interpretation of our postoperative results, as a patient who was impotent preoperatively will not become potent after surgery thereby lowering our results. this would render our potency rate to be artificially low due to inclusion of impotent patients or patients with poor baseline erectile status. despite this, our results are encouraging, which also adds a functional outcome to the benefit of robotic - assisted radical prostatectomy. follow - up times after surgery have been variable with many studies showing that recovery of erection is a function of time and may occur more than 1 year after surgery. walsh reported an improvement in potency of 72% to 86% between 12 and 18 months postoperatively. in the series by rabbani, the benefits of the robot allow for better visualization and subsequent meticulous dissection and preservation of the neurovascular bundles. our preliminary results provide comparable objective outcomes to open surgery with regards to erectile function. assessment of the ultimate maximal erectile function will require continued analysis, as this is likely to further improve beyond 6 to 12 months.	background and objectives : erectile function after prostate surgery is an important criterion for patients when they are choosing a treatment modality for prostate cancer. improved visualization, dexterity, and precision afforded by the da vinci robot allow a precise dissection of the neurovascular bundles. we objectively assessed erectile function after robot - assisted extraperitoneal prostatectomy by using the shim (iief-5) validated questionnaire.methods:between july 2003 and september 2004, 150 consecutive men underwent da vinci robot - assisted extraperitoneal radical prostatectomy for clinically localized prostate cancer. the iief-5 questionnaire was used to assess postoperative potency in 67 patients who were at least 6 months postsurgery. erectile function was classified as impotent (< 11), moderate dysfunction (11 to 15), mild dysfunction (16 to 21), and potent (22 to 25). all patients used oral pharmacological assistance postprocedure.results:sixty-seven patients were available to complete the iief-5 questionnaire 6 months to 1 year postprostatectomy. twelve patients were excluded from the study who abstained from all sexual activity after surgery for emotional or social reasons. of the 55 patients evaluated, 22 (40%) were impotent, 3 (5.5%) had moderate ed, 12 (21.8%) had mild ed, and 18 (32.7%) were fully potent. the table compares iief-5 scores with nerve - sparing status. of patients who had bilateral nerve sparing, 28/45 (62.2%) had mild or no ed within 6 to 12 months postsurgery, and all expressed satisfaction with their current sexual function or rate of improvement after robotic prostatectomy.conclusion:robot-assisted extraperitoneal prostatectomy provides comparable outcomes to those of open surgery with regards to erectile function. assessment of the ultimate maximal erectile function will require continued analysis, as this is likely to further improve beyond 6 to 12 months.
hearing loss is caused by genetic factors in more than 60% of the patients. until date, more than 300 genetic loci have been found to be related with nonsyndromic hearing loss. the strategy used to identify causes of hereditary hearing impairment (hhi) is continuously improving. traditional linkage analysis and candidate gene sequencing have been proven to be useful in identifying the genes that are responsible for autosomal dominant nonsyndromic hhi (adnshhi). however, this method is not effective to detect all of the cases of adnshhi. mapping the genes that are responsible for deafness recently, next generation high - throughput sequencing (ngs) has been verified to be a valuable molecular diagnostic tool for hhi patients. tjp2, also known as zo2, encodes the zonula occludens 2, which belongs to the family of the membrane - associated guanylate kinase homolog with three pdz domains, one sh3 module, and a guk domain. it is predominantly expressed at the apical region between the hair cells and supporting cells in the organ of corti. its expression decreases with age from embryonic development to adulthood, aiding the maintenance of the barrier between the endolymph and perilymph. tjp2 overexpression is associated with the induction of apoptosis, which was the reason of adnshhi in a family of jewish ancestry. a missense mutation or a more complex gain - of - function mutation of tjp2 may be a viable and identifiable cause of adnshhi in human patients. however, complete loss of tjp2 leads to embryonic lethality and hence, tjp2 knockout mice are not viable. our research group has identified a chinese genealogy family (family 686) with adnshhi in five generations. through a series study conducted over the last 10 years, we deciphered the genetic code of an unconditional chinese family with adnshhi, while simultaneously demonstrating the numerous strategies for understanding the pathogenesis of hhi. this study was approved by the committee of medical ethics of chinese people 's liberation army (pla) general hospital. we obtained written informed consent from all the participants in this study. written informed consents were obtained from the next of kin on the behalf of the minors / children participants involved in this study. a five - generation pedigree with 99 members showing adnshhi was ascertained from the department of otolaryngology, head and neck surgery, at the institute of otolaryngology of pla, chinese pla general hospital 10 years ago [figure 1a ]. personal and familial medical history, including hearing loss, tinnitus, vestibular symptoms, use of aminoglycosides, and other clinical abnormalities was taken by a team of experienced doctors and audiologists, audiometric evaluations included pure tone audiometry, auditory brainstem responses, and distortion product otoacoustic emissions. high - resolution computed tomography (hrct) was also performed on some subjects to verify whether the family members had other complications other than hearing disorders. (a) pedigrees of family 686. filled symbols for males (squares) and female (circles) represent affected individuals, and empty represent unaffected individuals. a total of four recombination events were observed in family 686, including two recombination events in iii7 between d9s259 and d9s169, d9s1874 and d9s1777 respectively, one recombination event in iv4 in which the exchange was between d9s169 and d9s1853, and one significant recombination in v5 in which the exchange occurred at d9s205. figure shows the cytogenetic chromosome bands and regional distribution from the 9p13.2 to the 9p13.3 region. black mark column on the right of the chromosomal region is the positioning region of 686 lines, locating between 9p13.2 and 9p13.3. we performed a genome - wide linkage scan, fine - mapping, and haplotype analysis on family 686. genomic dna from 21 participants have been used for genetic analysis (family members iii2, iii4, iii5, iii6, iii7, iii8, iii10, iv1, iv3, iv4, iv8, iv13, iv15, iv17, iv20, iv21, iv22, iv23, iv26, v5, and v10). the polymorphic loci single nucleotide polymorphism (snp) microsatellite markers came from the prism linkage mapping sets - md10 version 2.0 kit (abi, usa). a genome - wide screening was carried out using 386 microsatellite markers, distributed with an average spacing of 10 centimorgan on 22 chromosomes, excluding x and y (abi prism linkage mapping set 2, applied biosystems, foster city, ca, usa). we followed the procedures using a pe9600 thermocycler made by applied biosystems. pcr products were loaded onto a 6% denaturing polyacrylamide gel (7 m urea) and visualized on an abi prism 3700 sequencer. further screening within the linked region on chromosome 9 was performed with markers from the marshfield 9 map (http://research.marshfieldclinic.org/genetics) for fine mapping. linkage analysis between the disease locus and the markers was performed using the mlink program of the linkage version 5.1 software package (http://linkage.rockefeller.edu/pub/). the two - point log odds (lod) score was calculated under a 90% penetrance autosomal dominant mode of inheritance, considering the genetic heterogeneity of hereditary hearing loss, and setting the disease allele frequency of 0.0001. haplotype analysis was constructed using cyrillic software, version 2.1 (cyrillic software, wallingford, uk). in accordance with the linkage analysis results, the aqp3 gene was chosen as the candidate gene and sanger sequencing was first performed in one affected family member and one unaffected member. design primers were used to amplify the six exons of aqp3 [supplementary table 1 ]. bi - directional sequencing was carried out using both the forward and reverse primers and was performed using the abi prism big dye dna - sequencer (applied biosystems, usa). primer sequences of aqp3 a customized capture array (nimblegen, roche) was performed on the proband, iv26 who had a different phenotype from other affected family members, was then added to sequence. the array can capture exons, splicing sites and immediate flanking intron sequences of 307 human kayo genes responsible for either human or mouse deafness and all the mitochondrial genes [supplementary table 2 ]. the targeted region captured by array was constructed a library and then sequenced on illumina hiseq2000 to generate paired end reads (90 bps at each end). raw image files were processed by illumina pipeline (version 1.3.4) for base - calling with default parameters. snps and indels (inserts and deletions) were detected using gatk (http://www.broadinstitute.org/gatk/index.php). targeted captured genes list abr : auditory brainstem responses. after filtering against common snps reported by public databases, sanger sequencing was used in all available members from family 686 to determine whether the potential mutation c. 2081g > a in tjp2 co - segregated with the disease phenotype in these individuals or not. direct pcr products were sequenced using bigdye terminator version 3.1 cycle sequencing kits (applied biosystems. a total of 100 dna samples from a panel of unaffected individuals from a chinese background made up the control genomic dna samples. a five - generation pedigree with 99 members showing adnshhi was ascertained from the department of otolaryngology, head and neck surgery, at the institute of otolaryngology of pla, chinese pla general hospital 10 years ago [figure 1a ]. twenty - one members of the family chosen participated in our study. personal and familial medical history, including hearing loss, tinnitus, vestibular symptoms, use of aminoglycosides, and other clinical abnormalities was taken by a team of experienced doctors and audiologists, audiometric evaluations included pure tone audiometry, auditory brainstem responses, and distortion product otoacoustic emissions. high - resolution computed tomography (hrct) was also performed on some subjects to verify whether the family members had other complications other than hearing disorders. (a) pedigrees of family 686. filled symbols for males (squares) and female (circles) represent affected individuals, and empty represent unaffected individuals. a total of four recombination events were observed in family 686, including two recombination events in iii7 between d9s259 and d9s169, d9s1874 and d9s1777 respectively, one recombination event in iv4 in which the exchange was between d9s169 and d9s1853, and one significant recombination in v5 in which the exchange occurred at d9s205. figure shows the cytogenetic chromosome bands and regional distribution from the 9p13.2 to the 9p13.3 region. black mark column on the right of the chromosomal region is the positioning region of 686 lines, locating between 9p13.2 and 9p13.3. we performed a genome - wide linkage scan, fine - mapping, and haplotype analysis on family 686. genomic dna from 21 participants have been used for genetic analysis (family members iii2, iii4, iii5, iii6, iii7, iii8, iii10, iv1, iv3, iv4, iv8, iv13, iv15, iv17, iv20, iv21, iv22, iv23, iv26, v5, and v10). the polymorphic loci single nucleotide polymorphism (snp) microsatellite markers came from the prism linkage mapping sets - md10 version 2.0 kit (abi, usa). a genome - wide screening was carried out using 386 microsatellite markers, distributed with an average spacing of 10 centimorgan on 22 chromosomes, excluding x and y (abi prism linkage mapping set 2, applied biosystems, foster city, ca, usa). we followed the procedures using a pe9600 thermocycler made by applied biosystems. pcr products were loaded onto a 6% denaturing polyacrylamide gel (7 m urea) and visualized on an abi prism 3700 sequencer. further screening within the linked region on chromosome 9 was performed with markers from the marshfield 9 map (http://research.marshfieldclinic.org/genetics) for fine mapping. linkage analysis between the disease locus and the markers was performed using the mlink program of the linkage version 5.1 software package (http://linkage.rockefeller.edu/pub/). the two - point log odds (lod) score was calculated under a 90% penetrance autosomal dominant mode of inheritance, considering the genetic heterogeneity of hereditary hearing loss, and setting the disease allele frequency of 0.0001. haplotype analysis was constructed using cyrillic software, version 2.1 (cyrillic software, wallingford, uk). in accordance with the linkage analysis results, the aqp3 gene was chosen as the candidate gene and sanger sequencing was first performed in one affected family member and one unaffected member. design primers were used to amplify the six exons of aqp3 [supplementary table 1 ]. according to standard conditions bi - directional sequencing was carried out using both the forward and reverse primers and was performed using the abi prism big dye dna - sequencer (applied biosystems, usa). a customized capture array (nimblegen, roche) was performed on the proband, iv26 who had a different phenotype from other affected family members, was then added to sequence. the array can capture exons, splicing sites and immediate flanking intron sequences of 307 human kayo genes responsible for either human or mouse deafness and all the mitochondrial genes [supplementary table 2 ]. the targeted region captured by array was constructed a library and then sequenced on illumina hiseq2000 to generate paired end reads (90 bps at each end). raw image files were processed by illumina pipeline (version 1.3.4) for base - calling with default parameters. snps and indels (inserts and deletions) were detected using gatk (http://www.broadinstitute.org/gatk/index.php). after filtering against common snps reported by public databases, sanger sequencing was used in all available members from family 686 to determine whether the potential mutation c. 2081g > a in tjp2 co - segregated with the disease phenotype in these individuals or not. direct pcr products were sequenced using bigdye terminator version 3.1 cycle sequencing kits (applied biosystems. a total of 100 dna samples from a panel of unaffected individuals from a chinese background made up the control genomic dna samples. a total of 21 members in the family 686, composed of 8 clinically affected and 13 unaffected individuals, were recruited in this study. most of the patients had relatively consistent audiograms, except for iii7 and iv26, initially presenting at 2 khz, 4 khz, and 8 khz. the hearing loss of these three frequencies declined rapidly in the first decade, with the thresholds worse than 4050 db hl. the audiograms were the downslope type. the hearing impairments then implicated the 1 khz and the 0.25 khz frequencies, and finally 0.5 khz. the subsequent progression of hearing loss to a severe level at all frequencies at a later age was gradual in most cases. all patients had associated lofty - tone tinnitus, but no vestibular symptoms or signs were reported [table 1 and figure 2 ]. hrct results of the temporal bone in some of the patients showed normal middle ears structure, including a normal vestibular aqueduct and internal auditory canal. none of the affected members had a history of exposure to aminoglycosides, noise, or other causes that may account for the hearing impairment. summary of clinical data for hearing impaired members in family 686 pta : pure - tone air - conduction averages (0.5, 1, 2 and 4 khz) for the better - hearing ear of affected subjects in family 727 ; diagnosed at the time of test. the severity of hearing impairment was defined as mild (2640 db hl), moderate (4155 db hl), moderately severe (5670 db hl), severe (7190 db hl) and profound (> 90 db hl). o and x denote air conduction pure - tone thresholds at different frequencies in the right and left ear, respectively. the genome - wide linkage study located the hearing impairment gene on the short arm of chromosome 9 (9p13.2-p13.3) and excluded the remainder of the genome. the maximum two - point lod score of 3.25 (= 0) was observed at marker d9s1817, between d9s171 and d9s175. fine mapping of the gene with dense microsatellite markers from the initially targeted area refined the position of the section to d9s165 and d9s1874, between which the genetic distance is 4.12 centimorgan (cm). this candidate region showed no overlap with the critical intervals identified in the previously studied families [table 2 ]. two - point lod scores between 9q microsatellite markers for family 686 lod : log odds score, the lod scores were computed under an autosomal dominant mode of inheritance. the gene was located between the d9s165 and d9s1874 interval, which is about 4.12 cm (the genetic distance was calculated from http://www.ensemble.org/per1) [figure 1b and 1c ]. twenty - one family members were screened for aqp3, including 8 affected members and 13 unaffected members. two polymorphisms in aqp3 were found : 390c > t/390c > t and 394g > a / wt. all the members in this family have 390c > t/390c > t and only four members have 394g > a / wt, including three patients and one unaffected member, who was 32 years old. the latter mutation can change the 132 amino acid from asp to asn, possibly leading to changes in both the functionality and spatial structure of the resulting protein. it is reasonable to assume that the unaffected member with this functional polymorphism might show the disease at an older age. however, not all of the patients in this family had this variant. approximately, 1.44 mbp of the exons and adjacent intronic regions of the 307 genes were captured and sequenced. the average sequencing depth for the target region was approximately 468x, and the average coverage for the targeted region with > 20x was 97.51%, satisfying the requirements for identifying snps and indels. for the proband of family 686, through function and frequency filtration, the missense variant c. 2081g > a (p.g694e) in exon14 of tjp2 (nm_201629.3) was identified and no other possibly pathogenic variants were found. in iv26, who was an affected member without tjp2 c. 2081g > a [supplementary figure 1 ], two previously reported missense mutations, c. 408c > a (p.y136) and c. 134g > a (p.g45e) in gjb2, were identified [supplementary figure 2 ]. dna sequence chromatograms showing the heterozygous missense mutation c.2081g > a of tjp2 gene in affected individuals iii2, iii4, iii5, iv4, iv13, iv22. iv8 was the unaffected control in this family. iii7 and iv26 were affected members without the tjp2 c.2081g > a mutation. dna sequence chromatograms showing the two heterozygous missense mutations c.134c > a and c.408g > a of gjb2 gene in affected individuals (lower panel) compared with the wild type controls (upper panel). results showed that all the patients carried the variant except for iii7 and iv26, while none of the unaffected members of the family carried the variant [supplementary figure 1 ]. sanger sequencing confirmed the co - segregation of p.g694e with the disease phenotype in family 686, with the exception of iii7 and iv26. the tjp2 mutation occurred at highly conserved amino acids, and the sift, polyphen2, lrt, mutation taster, gerp++, and phylop programs all predicted it to be deleterious. the amino acid sequence encoded by tjp2 was compared between different species, including mouse, rat, dog, cattle, and human, and it was found that c. 2081g > a (p.g694e) is located in a highly conserved region. no tjp2 c. 2081g > a variation was identified in 100 control genomic dna samples from a panel of unaffected individuals [supplementary figure 3 ]. based on these results and the phenotypes of family 686, we concluded that the mutations in tjp2 and gjb2 are responsible for hearing loss in this family. a total of 21 members in the family 686, composed of 8 clinically affected and 13 unaffected individuals, were recruited in this study. most of the patients had relatively consistent audiograms, except for iii7 and iv26, initially presenting at 2 khz, 4 khz, and 8 khz. the hearing loss of these three frequencies declined rapidly in the first decade, with the thresholds worse than 4050 db hl. the audiograms were the downslope type. the hearing impairments then implicated the 1 khz and the 0.25 khz frequencies, and finally 0.5 khz. the subsequent progression of hearing loss to a severe level at all frequencies at a later age was gradual in most cases. all patients had associated lofty - tone tinnitus, but no vestibular symptoms or signs were reported [table 1 and figure 2 ]. hrct results of the temporal bone in some of the patients showed normal middle ears structure, including a normal vestibular aqueduct and internal auditory canal. none of the affected members had a history of exposure to aminoglycosides, noise, or other causes that may account for the hearing impairment. summary of clinical data for hearing impaired members in family 686 pta : pure - tone air - conduction averages (0.5, 1, 2 and 4 khz) for the better - hearing ear of affected subjects in family 727 ; diagnosed at the time of test. the severity of hearing impairment was defined as mild (2640 db hl), moderate (4155 db hl), moderately severe (5670 db hl), severe (7190 db hl) and profound (> 90 db hl). o and x denote air conduction pure - tone thresholds at different frequencies in the right and left ear, respectively. the genome - wide linkage study located the hearing impairment gene on the short arm of chromosome 9 (9p13.2-p13.3) and excluded the remainder of the genome. the maximum two - point lod score of 3.25 (= 0) was observed at marker d9s1817, between d9s171 and d9s175. fine mapping of the gene with dense microsatellite markers from the initially targeted area refined the position of the section to d9s165 and d9s1874, between which the genetic distance is 4.12 centimorgan (cm). this candidate region showed no overlap with the critical intervals identified in the previously studied families [table 2 ]. two - point lod scores between 9q microsatellite markers for family 686 lod : log odds score, the lod scores were computed under an autosomal dominant mode of inheritance. the gene was located between the d9s165 and d9s1874 interval, which is about 4.12 cm (the genetic distance was calculated from http://www.ensemble.org/per1) [figure 1b and 1c ]. twenty - one family members were screened for aqp3, including 8 affected members and 13 unaffected members. two polymorphisms in aqp3 were found : 390c > t/390c > t and 394g > a / wt. all the members in this family have 390c > t/390c > t and only four members have 394g > a / wt, including three patients and one unaffected member, who was 32 years old. the latter mutation can change the 132 amino acid from asp to asn, possibly leading to changes in both the functionality and spatial structure of the resulting protein. it is reasonable to assume that the unaffected member with this functional polymorphism might show the disease at an older age. approximately, 1.44 mbp of the exons and adjacent intronic regions of the 307 genes were captured and sequenced. the average sequencing depth for the target region was approximately 468x, and the average coverage for the targeted region with > 20x was 97.51%, satisfying the requirements for identifying snps and indels. for the proband of family 686, through function and frequency filtration, the missense variant c. 2081g > a (p.g694e) in exon14 of tjp2 (nm_201629.3) was identified and no other possibly pathogenic variants were found. in iv26, who was an affected member without tjp2 c. 2081g > a [supplementary figure 1 ], two previously reported missense mutations, c. 408c > a (p.y136) and c. 134g > a (p.g45e) in gjb2, were identified [supplementary figure 2 ]. dna sequence chromatograms showing the heterozygous missense mutation c.2081g > a of tjp2 gene in affected individuals iii2, iii4, iii5, iv4, iv13, iv22. dna sequence chromatograms showing the two heterozygous missense mutations c.134c > a and c.408g > a of gjb2 gene in affected individuals (lower panel) compared with the wild type controls (upper panel). results showed that all the patients carried the variant except for iii7 and iv26, while none of the unaffected members of the family carried the variant [supplementary figure 1 ]. sanger sequencing confirmed the co - segregation of p.g694e with the disease phenotype in family 686, with the exception of iii7 and iv26. the tjp2 mutation occurred at highly conserved amino acids, and the sift, polyphen2, lrt, mutation taster, gerp++, and phylop programs all predicted it to be deleterious. the amino acid sequence encoded by tjp2 was compared between different species, including mouse, rat, dog, cattle, and human, and it was found that c. 2081g > a (p.g694e) is located in a highly conserved region. no tjp2 c. 2081g > a variation was identified in 100 control genomic dna samples from a panel of unaffected individuals [supplementary figure 3 ]. based on these results and the phenotypes of family 686, we concluded that the mutations in tjp2 and gjb2 are responsible for hearing loss in this family. to the best of our knowledge, this study is the third report on disease - causing missense mutations in tjp2 in patients with hhi. the c. 2081g > a mutation that was predicted to be pathogenic was detected in six patients from the family 686, and was absent in unaffected members. this mutation caused a transition from a guanine to adenine at nucleotide position 2081, causing a glycine to glutamic acid substitution at amino acid position 694 (p.g694e). however, the frequency of this mutation in dbsnp and 1k genome database was 0.0000 and 0.0009, respectively, suggesting that this mutation may be disease - causing. two patients, iii7 and iv26, did not carry the c. 2081g > a mutation. further analysis of the phenotypes of these two patients revealed that they presented with certain symptoms different from those found in other patients in the family. the hearing curve for patient iii7 was different from other affected members in this family in two ways : (1) high - frequency hearing impairment at 2 khz and 4 khz was not obvious and thresholds were much better than at 8 khz, while other patients from family 686 had a more synchronized hearing decline, (2) low - frequency hearing loss occurred earlier than in other family members. hearing loss caused by tjp2 mutation presented as late - onset and typically involved the high frequencies first. however, iii7 had no characteristics typical of hearing loss caused by tjp2 mutation, making it difficult to elucidate its cause. therefore, hearing impairment of iii7 may have been caused by other reasons. in the case of patient iv26, hearing impairment began at the age of 32 years, and the hearing curve showed bilateral, severe hearing loss across all frequencies at the age of 35 years, progressing rapidly during the first 10 years of onset. the patient did not present with the pattern of high - frequency hearing being affected first, followed by all other frequencies. further investigation using targeted gene capture and ngs showed that this patient was a compound heterozygote for two mutations in the gjb2 gene, a condition that has been reported to cause hhi. the heterogeneity observed in one family was also reported in more than ten other families who experienced hearing loss. until date, three missense mutations in tjp2 and a tandem inverted genomic duplication that includes the tjp2 gene have been reported to be related with hhi. with the exception of iii7 and iv26, the phenotype of affected members of family 686 is similar to that of the previously studied tunisian family of jewish ancestry, in terms of age of onset and audiological results. in the jewish family, the ages of onset were in the fourth decade and hearing loss first involved high frequencies, followed by a gradual loss of hearing at mid and low frequencies. tjp2 is also predicted to be a candidate gene for age - related hearing loss (arhl). families with tjp2 mutations have patterns of hearing loss similar to those associated with arhl, in that the high frequencies are affected first. most cases of adnshhi are characterized by postlingual progressive sensorineural hearing loss, with the age of onset mostly being in the second or the 3 year, such as is the case with kcnq4, gjb3, and myh14. some other cases of adnshhi that are caused by genetic mutations do not manifest until the third or 4 year, such as coch and tjp2, which are thought to be related to arhl. however, both arhl and tjp2 mutation - related hearing impairments are most likely associated with apoptosis of hair cells in the inner ear. tjp2 is one of the three apoptosis - associated genes that have been reported in connection with adnshhi, in addition to dfna5 and msrb3. further research into tjp2 transcription and protein function may play a crucial role in understanding the molecular mechanism behind the pathogenesis of arhl, and in deciphering the possible theory of hair cell death and the connection between gene mutations and progressive hearing loss. traditionally, strategies for identifying genes associated with hhi have included linkage analysis and candidate gene screening, which has led to the mapping of more than 60 loci for adnshhi. however, only 34 genes have been identified since 1992 (http://hereditaryhearingloss.org). in this study, we located a gene responsible for hearing impairment on the short arm of chromosome 9 (9p13.2-p13.3) using traditional approaches, but without positive results after sequencing candidate genes in all of the identified family members. based on our experience, it is necessary to expand the physical scope of the location region moderately to avoid the deviation of the genetic distance calculation, which may be caused by neglecting some relatively small chromosomes exchange. this can lead to the real disease - causing gene being located near the targeted area instead of in the targeted region strictly. it is more difficult to identify the disease - causing gene than to map it onto a specific region on the chromosome, due to limitations in technology, the large size of many genes and the high - cost of sanger sequencing. since 2009, ngs has played a crucial role in studying the genetics of hhi, with more than 10 genes having been recently identified (http://hereditaryhearingloss.org). ngs, also known as high - throughput sequencing or massively parallel sequencing, is quickly replacing many single gene tests for hearing impairment. it is useful for assessing patients whose phenotypes are not clinically distinguishable and should be considered by clinicians if single gene tests yield no diagnosis. among the ngs technologies, targeted capture panel tests are superior to whole exome sequencing, which looks at 20,000 genes and possible missing parts of the genes, offering more depth of coverage in genes associated with hearing loss. in conclusion, through a series of studies over a period 10 years, we identified the co - occurrence of disease - causing genes tjp2 and gjb2 in family 686. the mechanism underlying its role in apoptosis and its association with arhl may be useful in understanding the pathogenesis of arhl. in addition, we have summarized the various strategies to study the pathogenesis of adnshhi in this chinese family. we concluded that none of the techniques are conclusive in every situation, and it is, therefore, necessary to combine the knowledge and skills from clinical medicine, genetics, bioinformatics and it, while applying lateral thinking and constantly proposing new solutions. therefore, it is necessary to address difficulties with an open mind and to use a combination of methods. supplementary information is linked to the online version of the paper on the chinese medical journal website. this work was supported by grants from the national key basic research program of china (no. 2014cb943001) and the national natural science foundation of china (no. 81120108009 and 81530032). to the best of our knowledge, this study is the third report on disease - causing missense mutations in tjp2 in patients with hhi. the c. 2081g > a mutation that was predicted to be pathogenic was detected in six patients from the family 686, and was absent in unaffected members. this mutation caused a transition from a guanine to adenine at nucleotide position 2081, causing a glycine to glutamic acid substitution at amino acid position 694 (p.g694e). however, the frequency of this mutation in dbsnp and 1k genome database was 0.0000 and 0.0009, respectively, suggesting that this mutation may be disease - causing. two patients, iii7 and iv26, did not carry the c. 2081g > a mutation. further analysis of the phenotypes of these two patients revealed that they presented with certain symptoms different from those found in other patients in the family. the hearing curve for patient iii7 was different from other affected members in this family in two ways : (1) high - frequency hearing impairment at 2 khz and 4 khz was not obvious and thresholds were much better than at 8 khz, while other patients from family 686 had a more synchronized hearing decline, (2) low - frequency hearing loss occurred earlier than in other family members. hearing loss caused by tjp2 mutation presented as late - onset and typically involved the high frequencies first. however, iii7 had no characteristics typical of hearing loss caused by tjp2 mutation, making it difficult to elucidate its cause. therefore, hearing impairment of iii7 may have been caused by other reasons. in the case of patient iv26, hearing impairment began at the age of 32 years, and the hearing curve showed bilateral, severe hearing loss across all frequencies at the age of 35 years, progressing rapidly during the first 10 years of onset. the patient did not present with the pattern of high - frequency hearing being affected first, followed by all other frequencies. further investigation using targeted gene capture and ngs showed that this patient was a compound heterozygote for two mutations in the gjb2 gene, a condition that has been reported to cause hhi. the heterogeneity observed in one family was also reported in more than ten other families who experienced hearing loss. until date, three missense mutations in tjp2 and a tandem inverted genomic duplication that includes the tjp2 gene have been reported to be related with hhi. with the exception of iii7 and iv26, the phenotype of affected members of family 686 is similar to that of the previously studied tunisian family of jewish ancestry, in terms of age of onset and audiological results. in the jewish family, the ages of onset were in the fourth decade and hearing loss first involved high frequencies, followed by a gradual loss of hearing at mid and low frequencies. tjp2 is also predicted to be a candidate gene for age - related hearing loss (arhl). families with tjp2 mutations have patterns of hearing loss similar to those associated with arhl, in that the high frequencies are affected first. most cases of adnshhi are characterized by postlingual progressive sensorineural hearing loss, with the age of onset mostly being in the second or the 3 year, such as is the case with kcnq4, gjb3, and myh14. some other cases of adnshhi that are caused by genetic mutations do not manifest until the third or 4 year, such as coch and tjp2, which are thought to be related to arhl. however, both arhl and tjp2 mutation - related hearing impairments are most likely associated with apoptosis of hair cells in the inner ear. tjp2 is one of the three apoptosis - associated genes that have been reported in connection with adnshhi, in addition to dfna5 and msrb3. further research into tjp2 transcription and protein function may play a crucial role in understanding the molecular mechanism behind the pathogenesis of arhl, and in deciphering the possible theory of hair cell death and the connection between gene mutations and progressive hearing loss. traditionally, strategies for identifying genes associated with hhi have included linkage analysis and candidate gene screening, which has led to the mapping of more than 60 loci for adnshhi. however, only 34 genes have been identified since 1992 (http://hereditaryhearingloss.org). in this study, we located a gene responsible for hearing impairment on the short arm of chromosome 9 (9p13.2-p13.3) using traditional approaches, but without positive results after sequencing candidate genes in all of the identified family members. based on our experience, it is necessary to expand the physical scope of the location region moderately to avoid the deviation of the genetic distance calculation, which may be caused by neglecting some relatively small chromosomes exchange. this can lead to the real disease - causing gene being located near the targeted area instead of in the targeted region strictly. it is more difficult to identify the disease - causing gene than to map it onto a specific region on the chromosome, due to limitations in technology, the large size of many genes and the high - cost of sanger sequencing. since 2009, ngs has played a crucial role in studying the genetics of hhi, with more than 10 genes having been recently identified (http://hereditaryhearingloss.org). ngs, also known as high - throughput sequencing or massively parallel sequencing, is quickly replacing many single gene tests for hearing impairment. it is useful for assessing patients whose phenotypes are not clinically distinguishable and should be considered by clinicians if single gene tests yield no diagnosis. among the ngs technologies, targeted capture panel tests are superior to whole exome sequencing, which looks at 20,000 genes and possible missing parts of the genes, offering more depth of coverage in genes associated with hearing loss. in conclusion, through a series of studies over a period 10 years, we identified the co - occurrence of disease - causing genes tjp2 and gjb2 in family 686. the mechanism underlying its role in apoptosis and its association with arhl may be useful in understanding the pathogenesis of arhl. in addition, we have summarized the various strategies to study the pathogenesis of adnshhi in this chinese family. we concluded that none of the techniques are conclusive in every situation, and it is, therefore, necessary to combine the knowledge and skills from clinical medicine, genetics, bioinformatics and it, while applying lateral thinking and constantly proposing new solutions. therefore, it is necessary to address difficulties with an open mind and to use a combination of methods. supplementary information is linked to the online version of the paper on the chinese medical journal website. this work was supported by grants from the national key basic research program of china (no. 2014cb943001) and the national natural science foundation of china (no. 81120108009 and 81530032).	background : there are more than 300 genetic loci that have been found to be related to hereditary hearing impairment (hhi), including 92 causative genes for nonsyndromic hearing loss, among which 34 genes are related to autosomal dominant nonsyndromic hhi (adnshhi). traditional linkage analysis and candidate gene sequencing are not effective at detecting the adnshhi, especially for the unconditional families that may have more than one pathogenic cause. this study identified two disease - causing genes tjp2 and gjb2 in a chinese family with unconditional adnshhi.methods:to decipher the genetic code of a chinese family (family 686) with adnshhi, different gene screening techniques have been performed, including linkage analysis, candidate genes screening, high - throughput sequencing and sanger sequencing. these techniques were done on samples obtained from this family over a period of 10 years.results:we identified a pathogenic missense mutation, c. 2081g > a (p.g694e), in tjp2, a gene that plays a crucial role in apoptosis and age - related hearing loss (arhl). the mutation was co - segregated in this pedigree in all, but not in the two patients who presented with different phenotypes from the other affected family members. in one of the two patients, we confirmed that the compound heterozygosity for p. y136 and p.g45e in the gjb2 gene may account for the phenotype shown in this patient.conclusions:we identified the co - occurrence of two genetic causes in family 686. the possible disease - causing missense mutation of tjp2 in family 686 presents an opportunity for further investigation into arhl. it is necessary to combine various genes screening methods, especially for some unconventional cases.
pregnant women and children under the age of 5 years are at risk of severe disease and mortality. the burden of malaria in pregnancy in sub - saharan africa is caused mainly by plasmodium falciparum. in malaria holo - endemic areas, malaria in pregnancy may remain asymptomatic despite sequestration of parasitized red blood cells in the placental micro - circulation with antecedent complications. the impact on the foetus is usually from maternal infection of the placenta and maternal anaemia resulting to low birth weight, prematurity, still birth, abortions, intra - uterine growth restriction, as well as congenital malaria [1, 2 ]. prevalence rates for malaria parasitaemia in pregnancy (using microscopy) in hospital - based surveys in different parts of nigeria included 3.1% in sokoto in the northwest, 9.0% in jos in the north - central 27.4% in lagos in the south - west and 39.1% in awka in the south - east. most of the available data were from urban areas, whereas most of nigeria is rural. this anomaly partly derives from the difficulty in conducting microscopy in rural areas because microscopists are few and power supply is erratic. however, rapid diagnostic test (rdt), now recommended by the world health organization (who) for diagnostic purposes where microscopy is unavailable [8, 9 ] is not yet widely used in surveys in nigeria. the objective of this study therefore was to determine the prevalence of asymptomatic malaria parasitaemia in pregnant unbooked women in a rural area using the rdt. the findings from this study will add to the body of knowledge of asymptomatic malaria parasitaemia in pregnant women living in rural areas as well as give information on the usefulness of rdt for malaria diagnosis. the study site was primary health care (phc), odo - aiye ; a rural community in okitipupa local government area (lga) located in ondo - south district, south - west nigeria. the lga lies within the equatorial evergreen rain forest of nigeria where malaria is stable with no seasonal variation. ethical clearance was obtained from the ethics and research committee, university of benin teaching hospital, benin city, nigeria. verbal permission to conduct the study in the phc the written informed consent was read, interpreted and explained in the local dialect by trained research assistance to the participants ; following which each participant then gave a verbal consent before being recruited in the study. recruitment of subjects was during the campaign for malaria control at the phc, odo - aiye. participants for this study included all pregnant women who were in attendance during the malaria campaign program. pregnant women who though were present during the program but had already booked at the phc or in any other organized health facility were excluded from this study. pregnant women who had fever or who received antimalaria drugs in the preceeding two weeks to the study and those who were on intermittent preventive treatment (ipt) were also excluded from the final analysis of the study. fever was defined as axillary body temperature 37.5c or history of fever in preceeding 7 days to this study. a total of 116 pregnant women attended the campaign program. in line with aforementioned criteria, 31 were excluded from the study because they had booked in the phc ; of whom 4 had received ipt for malaria and another 4 had fever during the period of recruitment for this study. a validated questionnaire was administered to the participants by one of the authors and assisted by a trained research assistance who verbally translated the required information in the questionnaire to respondents where necessary. the questionnaire contained questions about the participants ' demography, whether or not she had long lasting insecticide - treated net (llin), and whether she slept under the net. family social class was obtained by the method proposed by olusanya, okpere and ezimokhai, using level of education of the participant and the occupation of her husband. in this method of classification of social class, specific scores (0, 1, and 2 for the woman 's level of education ; and 1, 2 and 3 for her husband 's occupation) was allotted to each participant and the sum of these scores was used to describe social class as i, ii, iii, iv, and v. participants with scores i and ii were classified as upper class, iii - middle class ; iv and v as lower class. the gestational age of the participants was calculated from the first day of the last menstrual period (lmp). each subject was then tested for malaria using the rdt kit for malaria parasite- plasmodium falciparum (pf). the rdt was a one step invitro diagnostic test for qualitative detection of pf antigen known as the histidine - rich protein ii (hrp - ii) in capillary blood. the rdt kit used for this study was the standard diagnostic (sd) bioline malaria antigen pf test (kyonggi - do, korea). each pack of the rdt kit used in this study was from lot number 082048 (reference number 05fk50) and contained 25 cartridge kits or test devices, 25 un - opened alcohol swabs, 25 appropriately calibrated pipette loop and 25 sterile lancets. one pack was meant for testing malaria parasite for 25 people and a new test cartridge or test device was used for each participant. internal evaluation of the rdt kits which was certified by who showed that the sd bioline rdt kit has 100.0% sensitivity for blood with > 50 parasite/l and 98.0% specific for p. falciparum. the kits had storage temperature range of one to 40c which was within the average environmental temperature in ondo and the other southwestern region of nigeria. malaria parasite test was performed by one of the authors who had received certified training on the use of rdt for malaria diagnosis. procedure for the test was as follows : the pulp of the fourth finger of the subject was cleaned with an alcohol swab and when it was air dried, the cleaned area was pierced with the sterile lancet. a capillary pipette loop was used to collect 5.0 l of blood (a black line calibrated on the pipette loop provided in the test kit). four drops of provided buffer was then added to the buffer well window on the cartridge. malaria positive was by the presence of two colour bands (' t ' test line and ' c ' control line) within the result window, no matter which band appeared first and no matter the intensity of the colour band. negative result was by presence of only one colour band ' c ' (control line) within the result window. if the control band failed to appear within the result window or no colour band at both the ' t ' and ' c ' regions, the result was considered invalid and the test was repeated for that particular subject using a new test device. women with positive malaria parasite received the recommended anti - malarials in line with the national guideline for treatment of malaria in pregnancy. the data obtained for this research was entered into the statistical package for social sciences (spss) version 16.0 (spss inc chicago, illinois, usa) spread sheet where analysis was also done. associations with this variable were tested using chi - square and student t tests as appropriate. the data obtained for this research was entered into the statistical package for social sciences (spss) version 16.0 (spss inc chicago, illinois, usa) spread sheet where analysis was also done. associations with this variable were tested using chi - square and student t tests as appropriate. the level of significance of each test was set at p < 0.05. mean (sd) age of the 85 pregnant women recruited for this study was 26.1 4.7 years (age range 18 - 40 years) ; mean gestational age was 27.1 8.1 weeks (range 6 - 40 weeks). forty - five (52.9%) were in the third trimester, 35 (41.2%) were in second trimester and 5 (5.9%) were in the first trimester. forty - eight (56.5%) were multipara, 20 (23.5%) were primigravidae and 17 (20.0%) were grand multiparous women. fifty - four (63.5%) of the women had secondary education, 22 (25.9%) had primary, 2 (2.4%) had tertiary education and 7 (8.2%) had no formal education. table i shows the age group and social class of the respondents. majority (88.2%) of the women there was no significant difference in mean age of infected (26.5 5.1 years) and non - infected subjects (25.9 4.6 years) ; (t = 0.46, p = 0.65, 95%ci = -1.78, 2.85). only 3 (3.5%) out of the 85 women owned llin ; and only one of the three slept under a llin. reason given by the other two who did not sleep under the net was that the net was difficult to mount. malaria parasitaemia was not significantly associated with age, parity, gestational age and social class (tab. malaria parasitaemia and age group, gestation, parity and social class of the subjects. the high prevalence of asymptomatic malaria in pregnancy in this study supports similar findings in other stable malarial transmission areas [1, 3 - 6 ]. susceptibility to plasmodium parasitaemia has been linked to the level of antibodies to placental sequestrated parasites. the most preferentially adherence is to the chondroitin sulphate - a (csa) receptors expressed by the syncytiotrophoblasts in the placenata. this adherence is common in primigravidae and women in second pregnancy as the anti - adhesion antibodies against csa binding parasites usually develop in women after successive pregnancies. the consequence of untreated asymptomatic malaria parasitaemia in pregnancy is enormous [3 - 6 ], therefore adequate and prompt implementation of malaria control policy for pregnant women should be intensified to protect these women and their un - born babies from the antecedent complications of malaria disease. in most african countries, pregnant women usually book for antenatal care by the 2 trimester, there is the need for malaria parasite screening as a routine test at booking to identify and treat asymptomatic malaria parasitaemia with the recommended anti - malarial drug before the commencement of ipt. some authors have also suggested that in areas with high prevalence of asymptomatic malaria in pregnancy, it may be more appropriate to institute intermittent screening for malaria parasite using simple tools such as the rdt as well as treat the individual with presence of asymptomatic malaria parasitaemia as alternative to ipt especially in areas where sulphadoxine - pyrimethamine (sp) resistance is very high. though this study did not compare the prevalence of malaria parasitaemia by rdt and microscopy, the prevalence found (25.9%) was comparable to 27.4% observed in a study among pregnant women using microscopy in lagos, in the same southwest nigeria. malaria screening and diagnosis in pregnancy using rdt especially at booking may also be a cost effective malaria control strategy in pregnancy and should be an integral component of malaria control in pregnancy in holo - endemic regions. the use of the llin is now one of the most important preventive tools against malaria. llin has been shown to reduce the number of infective mosquito bites by 70.0 90.0% in a variety of ecologic settings as well as reduce the prevalence of malaria in children and adults [15, 16 ]. unfortunately, most women of reproductive age group in nigeria do not possess the llin and the few who had the llin barely sleep under the net. in addition, microscopy on the specimens could have offered an opportunity to assess the validity of the rdt results in the community. in addition, microscopy on the specimens could have offered an opportunity to assess the validity of the rdt results in the community. this study showed that one - quarter of the pregnant women had asymptomatic malaria parasitaemia and that rdt can be an invaluable instrument for malaria diagnosis in rural areas. the authors therefore recommended routine malaria parasite screening for all pregnant women at booking as well as scaling - up the availability of llin for vector control and the rdt kits for malaria diagnosis in rural areas. health education on the use of llin as important tool for control of malaria should be intensified in malaria control national advocacy, communication, and social mobilization strategic framework and implementation plan in nigeria.	summarybackground.malaria is a major contributor of maternal and peri - natal morbidity and mortality. the disease may be asymptomatic despite sequestration of parasitized red blood cells in the placental micro - circulation with antecedent complications. in such condition, it may also be difficult to identify the malaria parasite by the peripheral blood film microscopy, thus the need for use of simple but reliable tool for malaria parasite diagnosis.objective and method.to determine the prevalence of asymptomatic malaria parasitaemia using the rapid diagnostic test in pregnant unbooked women seen in a primary health centre during a malaria control campaign programme in rural ondo - south, district nigeria.results.prevalence of asymptomatic malaria parasitaemia was 25.9%. only 3 (3.5%) of the 85 women had the long lasting insecticide - treated nets. there was no significant association between malaria parasitaemia, and the age group, parity and gestation age.conclusion.given the high prevalence of asymptomatic malaria in pregnancy, routine screening for malaria at booking and scaling - up of other malaria control strategies such as the use of long lasting insecticidal - treated nets and intermittent preventive therapy for pregnant women are recommended.
the anterior region is a challenge for most clinicians to achieve optimal esthetics with dental implants. the provisional crown is a key factor in the success of obtaining pink esthetics around restorations with single implants, by soft tissue and inter - proximal papilla shaping. provisional abutments bring additional costs and make the treatment more expensive. since one of the aims of the clinician is to reduce costs and find more economic ways to raise patient satisfaction, this paper describes a practical method for chair - side fabrication of non - occlusal loaded provisional crowns used by the authors for several years successfully. twenty two patients (9 males, 13 females ; mean age, 36,72 years) with one missing anterior tooth were treated by using the presented method. metal definitive abutments instead of provisional abutments were used and provisional crowns were fabricated on the definitive abutments for all of the patients. the marginal fit was finished on a laboratory analogue and temporarily cemented to the abutments. the patients were all satisfied with the final appearance and no complications occurred until the implants were loaded with permanent restorations. the use of the definitive abutments for provisional crowns instead of provisional abutments reduces the costs and the same results can be obtained. dental implants are accepted as a valuable option for the restoration of missing teeth and various edentulous sites [1 - 3 ]. clinicians often face esthetic challenges when restoring implants in anterior regions [4 - 6 ]. especially when the restoration is in the maxillary anterior esthetic zone, the creation of a natural appearance is very important. the provisional crown is a key factor in the success of soft tissue and interproximal papilla shaping. the provisional crown should not induce extensive pressure on the gingiva, which could lead to recession. furthermore, the crown should mimic the natural color and shape of the symmetric tooth. a fixed provisional crown is more comfortable and less vulnerable to fracture or loss than a temporary removable denture. on the other hand, the soft tissue shaping can be accomplished by a composite layering technique on fixed provisional crowns (fig. 1). provisionalisation of abutments in the anterior regions may also be necessary when the final crowns are not delivered at the time the abutments are placed. this may be because of a need to alter the aesthetics and occlusion before completing the treatment. this article describes a practical method for provisionalisation of permanent metal abutments of implants placed in the maxillary anterior regions in several cases, as well as cases where these permanent metal abutments have been used instead of tooth colored zirconia abutments where excellent esthetic results could be obtained. patients with one missing anterior tooth who presented to the department of prosthodontics at a university clinic for prosthetic treatment were included in this case series. this treatment was not offered to heavy smokers, since smoking could be unfavorable for later pink esthetics. additionally, patients without a minimum of 1 mm of bone thickness surrounding the entire implant surface and patients with thin biotypes were excluded from the treatment group. in the end, twenty - two patients (9 males, 13 females ; mean age, 36,72 years) with one missing anterior tooth fulfilling the above mentioned criteria were treated with this method. an artificial tooth (optodent, bayer, leverkusen, germany) was positioned in the edentulous place by attaching it to the neighboring teeth with visible - light - cured dental restorative composite material (herculite xrv, kerr, orange, ca, usa) (fig. 2). a polyvinylsiloxane impression was made for provisional crown fabrication (brecision, bredent gmbh & co.kg, senden, germany). after placement of the dental implant (in total 14 astra - tech ab, mlndal, sweden and 8 straumann holding ag, basel, switzerland) in a 3-dimensionally correct position as a substitute for the missing anterior tooth (fig. 3), an abutment (straumann holding ag or direct abutment, astra - tech ab) was mounted (fig. 1), upon which a provisional crown was fabricated chair - side using the polyvinylsiloxane impression with an auto - polymerizing acrylic resin (dentalon, heraeus kulzer gmbh & co.kg, hanau, germany) (fig. an important step at this point was to control the marginal adaptation of the crown radiographically (fig. the temporary crown was used for gingival reshaping by composite addition to the marginal part of the acrylic crown (fig. the patients were all satisfied with the final appearance and no complications occurred until the implants were loaded with permanent restorations. pink esthetics as well as the marginal bone levels were satisfactory in all cases during the follow - up period up to 60 months. as progress in material and implant design continues noticeably over time, implant patients demand treatment protocols that take less time, require fewer surgeries, and have better esthetic outcomes. one of the most frequently mentioned requests by patients is that some type of crown can be put on a dental implant right after its placement, especially in the anterior region. following these expectations, most implant manufacturers have introduced a number of additional implant parts such as zirconia abutments for final restorations or tooth - colored plastic abutments for provisionalisation purposes. together with esthetics, these abutments also serve the important function of better shaping and maintaining the gum architecture around the tooth / implant. the provisional abutment is mainly used for the achievement of soft tissue modeling and shaping by the help of a temporary crown. the crown body is used to embody an ideal soft tissue contour of the gingiva as well as the interdental papillae, by adding composite or reducing the size. the zirconia abutments on the market are a good solution especially in thin biotyped cases, where a grayish permeability of the soft tissue could distort the whole esthetic outcome. as is well known, however, the above - mentioned abutment types cause a rise in the global treatment cost for the patient. studies showing the long - term results of these two methods are scarce and the number of cases is limited. in our experience, it seems that the use of temporary plastic abutments or zirconia abutments is not indispensable. for the achievement of esthetic results,, dental implants should be positioned 3-dimensionally in the correct position and primary stability must be obtained for loading, both of which were adequately achieved in the cases presented here. additionally, the socket walls where the implant will be placed must be intact to ensure later soft tissue esthetics. insufficient bone support and a thin biotype can often lead to undesirable consequences. if the bony support is defective, a dehiscence is present, or a thin biotype is detected prior or during the surgery, a grafting procedure that will delay the loading time is indispensable. if the clinician is dealing with a thick biotyped gingiva, a metal abutment can perfectly fulfill the esthetic demands, as shown in the present cases that were followed up for several years. another important risk factor is a history of aggressive periodontitis, especially combined with smoking. these cases can show an unpredictable soft tissue recession, making a metal abutment visible. the use of definitive abutments instead of provisional abutments both reduced the costs and maintained the health of peri - implant tissues just like provisional abutments in the 3 months follow - up period until the final restoration was made. all patients in this article who had been restored by metal abutments showed a stable soft tissue response and excellent esthetic outcome (fig. 8), indicating that in selected esthetic cases, costs can be reduced by the abdication of zirconia abutments for final restorations and temporary abutments for provisionalisation. it should be noted that in case of a deep subgingival margin, which is often found in cases with thick gingiva, residual cement may cause infection. to avoid this complication screw - retained restorations could be used. however, several implant systems, as in this case series, offer a variety of abutment heights making prevention of deep subgingival margins possible. proper indications, good surgical technique, and the use of a prosthetic protocol are very important for the esthetic success of dental implant therapy, especially in the anterior regions. nevertheless, there are also contraindications to the use of metal abutments in the esthetic zone. if even one among the patient - related factors of poor systemic health, a heavy smoking habit, poor oral hygiene, a thin biotype, or an infection in the extraction region is present, this treatment option should not be considered. based on the presented case series, it can be concluded that the use of the definitive abutments for provisional restorations reduce costs and the same result can be obtained. the use of zirconia abutments could be relinquished in accurately selected cases for cost reduction as well.	purposethe anterior region is a challenge for most clinicians to achieve optimal esthetics with dental implants. the provisional crown is a key factor in the success of obtaining pink esthetics around restorations with single implants, by soft tissue and inter - proximal papilla shaping. provisional abutments bring additional costs and make the treatment more expensive. since one of the aims of the clinician is to reduce costs and find more economic ways to raise patient satisfaction, this paper describes a practical method for chair - side fabrication of non - occlusal loaded provisional crowns used by the authors for several years successfully.methodstwenty two patients (9 males, 13 females ; mean age, 36,72 years) with one missing anterior tooth were treated by using the presented method. metal definitive abutments instead of provisional abutments were used and provisional crowns were fabricated on the definitive abutments for all of the patients. the marginal fit was finished on a laboratory analogue and temporarily cemented to the abutments. the marginal adaptation of the crowns was evaluated radiographically.resultsthe patients were all satisfied with the final appearance and no complications occurred until the implants were loaded with permanent restorations.conclusionsthe use of the definitive abutments for provisional crowns instead of provisional abutments reduces the costs and the same results can be obtained.
an exciting and challenging task in modern neuroscience is to gain a comprehensive understanding of how we learn and remember at the molecular, cellular and systems levels. as a versatile model organism for behavioral genetics, the fruit fly drosophila melanogaster has been successfully used to study individual genes and genetic pathways that are important for different aspects of memory (mcguire., 2005). notably, forward genetic screens for memory mutants in drosophila have resulted in the identification of molecular cascades that are critical to learning and memory, and also remarkably well - conserved among evolutionarily diverse animal species. in order to fully elucidate how these genes and genetic pathways control memory processes, we must understand their roles in the context of the neurons and neuronal circuits responsible for the acquisition, consolidation and retrieval of memories. to link neuronal circuits to memory processes, it is necessary to manipulate the activity of identified neurons within intact animals, and to analyze the direct effects of this manipulation on memory - based behavioral modifications. unfortunately, it is difficult to perform such experiments in drosophila melanogaster through surgical or electrophysiological means, mainly because of the small size of this animal. to circumvent this problem, we have developed a strategy that couples the drosophila dynamin mutant gene, shibire temperature - sensitive1 (shi), with the gal4/uas binary expression system, to rapidly and reversibly suppress synaptic neurotransmission from targeted neurons in intact free - moving animals, through a mild temperature shift (kitamoto, 2001, 2002). here we explain the principles of this strategy and discuss how it has been utilized to gain a better understanding of neuronal mechanisms of drosophila learning and memory. the drosophila gal4/uas system (brand and perrimon, 1993) has been widely used to drive the expression of uas - linked transgenes in specific sets of identified neurons in order to study their roles in behavior. for example, particular set of neurons can be eliminated by introducing a cell death - inducing gene. alternatively, functional properties of targeted neurons can be altered by expressing genetically engineered ion channels (to change neuronal excitability) or neurotoxins (to disrupt synaptic transmission). the relationships between particular neurons / neuronal functions and behavior can then be assessed using established assays. these non - conditional gene expression systems, however, have serious limitations with respect to studying the roles of neurons in behavior due to the inherent plasticity of the nervous system. when a group of neurons is eliminated or their functions are continuously altered, the rest of the nervous system is likely to adjust (either structurally or functionally) to the perturbation. such homeostatic responses from the nervous system may obscure the direct consequence of the targeted expression of uas - linked transgenes. a second problem is that most, if not all, gal4 driver lines exhibit substantial gal4 activity during development. thus, even if gal4-driven expression of a transgene is restricted to a small number of neurons in the adult brain, the behavioral changes observed in adult animals could nevertheless arise as a consequence of undefined expression of the transgene during development. these considerations underscore the importance of the inducibility of transgene expression for analyzing the neuronal basis of behavior using the gal4/uas system. only if a transgene exerts its expected function in the targeted neurons while the behavior is being examined, the observed behavioral changes can be directly link to the neurons expressing these genes. uas - linked transgenes can be conditionally expressed using the drug - inducible geneswitch gal4 system (osterwalder., 2001) or the temperature - dependent target system (mcguire., 2004), and these conditional gal4 systems are useful for eliminating any developmental effects of transgene expression. however, they are not suitable for analysis of the dynamic neuronal processes responsible for the temporal regulation of memory because of the lag time (several hours) between drug administration / temperature shift, respectively, and transgene expression. in addition, these conditional expression systems need several hours to terminate transgene activity once they are turned off. this kind of time resolution limits the usefulness of these systems for the functional study of neurons involved in temporally regulated memory processes. the drosophila gene shibire encodes dynamin (chen., 1991 ; van der bliek and meyerowitz, 1991), a protein that plays a critical role in synaptic vesicle recycling in nerve terminals (kosaka and ikeda, 1983). the dynamin molecule encoded by the mutant gene shibire temperature - sensitive1 (shi) has a single amino acid substitution in the gtpase domain, making it reversibly temperature - sensitive. shi mutants are paralyzed in a temperature - dependent manner, because the functions of shi - encoded dynamin are impaired at restrictive temperature. this results in depletion of synaptic vesicles at the nerve terminals, and thus leads to suppression of neurotransmission in the neurons responsible for motor control (figure 1). the shi allele is semidominant regarding the paralytic phenotype (kim and wu, 1990). therefore, it was expected that would be possible to perturb neurotransmission in a temperature - dependent manner by overexpressing shi in the presence of its endogenous wild - type counterpart. we have demonstrated that this is indeed the case, using cha - gal4 to drive uas - shi expression in the major excitatory (cholinergic) neurons in the drosophila nervous system (kitamoto, 2001). adult flies expressing shi in cholinergic neurons were apparently normal at permissive temperature, but became paralyzed within 2 min of being moved to the restrictive temperature ; activity was immediately regained when the flies were returned to permissive temperature. a more specific behavioral defect was observed when shi expression was directed to the photoreceptor cells ; both larvae and adults displayed a temperature - dependent defect in light - induced behavior, yet other behaviors were intact (kitamoto, 2001). these results demonstrated that uas - shi can be used as a molecular switch for synaptic transmission in targeted neurons (figure 1). a gal4 driver specific to neuronal subsets is crossed to the uas - shi line. when the temperature is shifted from permissive to restrictive, the shi product (temperature - sensitive dynamin) is rapidly inactivated and synaptic vesicle recycling is interrupted. as a result, behavioral consequences of spatial and temporal suppression of neurotransmission can be observed in free - moving animals. the shi product regains its activity and synaptic vesicles are restored immediately after the animals are returned to permissive temperature. drosophila has a remarkable ability to acquire, store and recall memory in various learning and memory paradigms. the most extensively studied of these paradigms is aversive olfactory conditioning (davis, 2005). previous studies indicated that single odor - shock training (single training) induces protein synthesis - independent memory that lasts for hours. the memory induced by single training is composed of at least three temporally distinct memory phases : short - term memory (stm), middle - term memory (mtm) and anesthesia - resistant memory (arm). stm and mtm are labile memories and disrupted by anesthetic treatment such as a cold shock, whereas arm, which develops during the first 30 min after training, is a form of consolidated memory and resistant to anesthetic treatment. two protocols that are more intensive than single training, known as mass training and spaced training, are used to study the consolidation of aversive olfactory memory. in mass training, paired odor - shock stimuli are repeatedly presented without intervening rest periods. as in single training, mass training generates stm, mtm and arm. spaced training, in which repeated odor - shock stimuli are presented at intervals, is used to generate long - term memory (ltm), a protein synthesis - dependent form of consolidated memory that lasts for at least several days. which neurons are involved in the regulation of distinct memory phases ? how does neuronal activity contribute to information processing relevant to the acquisition, consolidation and retrieval of memories ? the uas - shi transgene has been effectively used to gain important insights into these key questions. the primary brain region that has been studied in the context of olfactory memory is the mushroom body (mb) (heisenberg, 2003). previous studies showed that aversive olfactory memory is disrupted in flies with genetically or chemically ablated mbs (heisenberg., 1985 ; de belle and heisenberg, 1994), yet the mb - deficient flies are able to discriminate odors and respond to an electric shock. although these results indicated that mbs play an essential role in olfactory memory, little was known about how mb - intrinsic neurons contribute to memory processing. the mb - intrinsic neurons known as kenyon cells are broadly classified into three subtypes, the /, / and neurons, according to the projection patterns of their axons (lee., 1999) (figure 2). the use of uas - shi in combination with various gal4 drivers that are specific to different subsets of kenyon cells has revealed that these structurally distinct neurons are also functionally diverse in the temporal processing of olfactory memory. diverse roles of intrinsic and extrinsic mushroom body - associated neurons in the processing of aversive olfactory memory. (a) the mushroom body and associated neurons in one brain hemisphere are schematically represented (adapted from armstrong., 1998). the cell bodies of kenyon cells (mbcs), the mushroom body - intrinsic neurons, are located in the dorsal and posterior cortices of the brain. the axons of / and / neurons bifurcate to form vertical (and) and horizontal (and) lobes. the axons of neurons do not bifurcate, and form only a horizontal lobe (). the primary olfactory information received by the olfactory neurons is transmitted through the antennal nerve (an) to the first olfactory center antennal lobe (al), where the information is processed and further transmitted to the mushroom bodies by the projection neurons (pn). the dorsal paired medial (dpm) neuron extends an axon that branches and terminates in all lobes of the mushroom body. (b) conditioned stimuli (cs ; e. g. odors) and unconditioned aversive stimuli (us ; e.g. electric shock) are simultaneously presented to flies. the olfactory information received by the olfactory neurons (osn) is conveyed to the mushroom bodies (mb) through the first - order interneuron, the projection neurons (pn). the neuronal circuits that transmit the aversive sensory information include dopaminergic neurons (da). the information generated by cs and us converges at the mb, where aversive olfactory memory is formed. neurotransmission from dpm neurons to the / neurons, as well as that from the / neurons, contributes to the stabilization of memory. retrieval of both protein synthesis - independent, short - lasting memory and protein synthesis - dependent, long - term memory (ltm) require neurotransmission from the / neurons. the / neurons, which constitute approximately 50% of the kenyon cells, are labeled in the majority of the mb - specific gal4 lines (aso., 2009). when uas - shi was preferentially expressed in the / neurons and neurotransmission from these neurons was temporally blocked by maintenance at the restrictive temperature only during single training, the flies avoided the shock - associated odor during the test period, showing that memory is formed, stored and retrieved under these conditions. similarly, inhibiting / neuron output between the training and test periods did not significantly affect the memory - based avoidance behavior during testing. these results indicate that neurotransmission from the / neurons is dispensable for the acquisition and storage of olfactory memory. in marked contrast, blocking neurotransmission only during the test period (30 min to 3 h after training) eliminated the avoidance behavior, demonstrating that neurotransmission from the / neurons is required for the retrieval of aversive olfactory memory (dubnau., 2001 ; mcguire., an important implication of this finding is that any aversive olfactory memory formation (presumably as changes in structures and/or functions of cellular components) occurs at or upstream of the / output synapses of the neuronal circuits involved in the processing memory. memory is manifested as behavioral alterations in response to learned unconditioned stimuli (us) through neurotransmission from the / neurons in the mbs. either single or mass training can generate the consolidated form of memory known as arm. arm can be measured separately from stm and mtm by exposing animals to a cold shock 1 h after training, because labile stm and mtm are eliminated by this treatment. blocking the output of the / neurons using uas - shi has also been shown to impair arm, indicating that neurotransmission from the / neurons is necessary for the retrieval of three types of short - lasting, protein synthesis - independent memory (isabel., 2004). in contrast to arm, ltm is a protein synthesis - dependent, consolidated form of memory that is generated only after spaced training. recent optical imaging studies demonstrated that spaced training, but not single or mass training, results in a robust increase in calcium influx into the lobes, in a protein synthesis - dependent manner (yu., 2006). in addition, analysis of the alpha - lobes - absence (ala) mutants, which lack either the two vertical lobes (and) or two of the three horizontal lobes (and), suggested that ltm depends on the vertical lobes (pascual and preat, 2001). these results suggested that neurons forming vertical lobes, in particular those of the lobe, are important for ltm. experiments with uas - shi provided data in support of this notion. indeed, the retrieval of memory 24 h after spaced training is significantly impaired when neurotransmission from the / neurons is blocked only during testing, indicating that the output of the / neurons is required for the retrieval of ltm. as in the case of stm, a memory trace for ltm is likely formed, at least partly, at or upstream of the / neuron output synapses (isabel., 2004). the original model for aversive olfactory memory proposes that pairing conditioned stimuli (cs) with us leads to the sequential formation of stm and mtm, and that the latter is then processed into either arm and/or ltm, depending on the training protocol (dezazzo and tully, 1995). in this model, both arm and ltm derive from mtm, and arm and ltm coexist for 24 h after spaced training. interestingly, however, it was shown that spaced training that produces ltm leads to disappearance of arm in the ala mutants lacking vertical lobes (isabel., 2004). in addition, the experiments using uas - shi indicate that arm and ltm involve the same / subset of the mb neurons. these results suggest that arm and ltm are mutually exclusive. furthermore, normal arm can be detected in mutants defective for stm (rut) or mtm (amn). based on these collective results, it has been proposed that arm is processed largely independently of the sequential stm - mtm - ltm pathway, but stored in the common group of neurons in the mbs (isabel., 2004). with respect to aversive olfactory memory, the functional significance of the neurons, which account for approximately 30% of kenyon cells (aso., 2009), has thus far not been shown. the / neurons are relatively minor components of the mbs, comprising approximately 20% of the kenyon cells (aso., 2009). although they extend axons parallel to those of the / neurons, experiments using uas - shi demonstrated that the roles of the / neurons in olfactory memory processing are distinct from those of the / neurons. when neurotransmission from the / neurons was blocked only during the test period by introducing uas - shi in combination with gal4 drivers that preferentially label the / neurons, the memory - based avoidance behavior during the test period this result shows that, unlike neurotransmission from the / neurons, that from / neurons is dispensable for the retrieval of olfactory memory. interestingly, however, when the / neurons were blocked either during or after training, olfactory memory was severely impaired. thus, output from the / neurons appears to be required for the acquisition and stabilization of olfactory memory (krashes., 2007). an exciting line of future investigation will focus on how neurotransmission from the / neurons contributes to the processing the memory that eventually depends on output of the / neurons for its retrieval. the dorsal paired medial (dpm) neurons are two large neurons that project extensively to all the lobes and the base of the peduncle of mb neurons. they were first identified as primary cells in the adult brain that express the putative neuropeptide encoded by the amnesiac gene (amn) (waddell., 2000), whose product plays an important role in stabilizing memory (quinn., 1979). amn mutants can learn and form stm, but their memory decays abnormally rapidly, within 3060 min after training, and this results in severe defects in mtm. because the amn memory defect is rescued by expressing the wild - type amn gene product preferentially in dpm neurons, these neurons are considered to be the site at which amn acts to stabilize olfactory memory. results obtained from uas - shi experiments revealed the significance of dpm neuron output in memory stabilization. when neurotransmission from dpm neurons was constantly blocked (during the training, storage and test periods), the flies displayed defects in memory that could be measured 1 h after training, as was the case in amn mutants. however, temporally blocking neurotransmission from dpm neurons only during the training or test period did not affect olfactory memory. in contrast, blocking dpm output for 30 min during the storage period (between training and testing) resulted in a significant impairment of 1-h memory, demonstrating that the dpm neuron is critical for stabilizing memory (waddell., 2000). considering that neurotransmission from the / neurons is similarly required to stabilize memory during storage, and that dpm neurons heavily innervate the mb lobes including the and lobes it is possible that direct information flow from dpm neurons to the / neurons is essential to stabilizing the newly formed memory. this possibility was tested by using flies that express an isoform of drosophila down syndrome cell adhesion molecule (dscam17 - 2) in dpm neurons. the expression of dscam17 - 2 in dpm neurons affected their development such that projections of dmp neurons to the, and lobes are significantly reduced, but those to the and lobes are relatively intact. in these genetically engineered flies with limited dpm projections to the mb / neurons, aversive olfactory memory was not significantly affected. importantly, a temporal block in dpm neuron output to the / neurons, but not to most / and neurons, was enough to induce the amn - like memory phenotype (krashes., 2007). these results strongly suggest that stabilizing memory during the storage period mainly depends on neurotransmission from dpm neurons to a subset of mb neurons the / neurons. it has been proposed that aversive olfactory memory is formed in the mbs, where sensory information concerning conditioned odor stimuli and unconditioned aversive stimuli converges, and that this establishes an association between cs and us (davis, 2005). although significant progress has been made in understanding the molecular and neuronal mechanisms responsible for olfactory information processing (hallem and carlson, 2004), little is known about how unconditioned electric shocks are sensed as aversive stimuli, and which neurons are involved in conveying us information to the mbs. in other organisms, monoaminergic interneurons are thought to transmit such information. in drosophila, a study investigating mutants for dopa decarboxylase, which encodes an enzyme involved in the synthesis of both dopamine and serotonin, implicated these neurotransmitters in aversive olfactory learning (tempel., 1984). however, this result could not be reproduced by another study (tully, 1987). given the controversial nature of these findings, uas - shi was used to revisit this issue (schwaerzel., 2003). tyrosine hydroxylase (th) is the rate limiting enzyme for dopamine and expressed specifically in dopaminergic neurons. th - gal4, which was generated using regulatory dna of the th gene (friggi - grelin., 2003), was used to direct uas - shi to dopaminergic neurons. although blocking neurotransmission from dopaminergic neurons before and during testing did not affect memory, blocking them only during the training period severely impaired aversive olfactory memory (schwaerzel., 2003). interestingly, dopamine transmission was not required for appetitive olfactory learning using sugar as the us. these results provide strong support for the notion that dopaminergic neurons are part of the neuronal circuit for aversive us in drosophila. there are approximately 120 dopaminergic neurons in the adult fly brain, and some of which extensively innervate the mbs (riemensperger., future studies using gal4 lines specific to a subset of brain dopaminergic neurons should reveal functional differences between dopaminergic neurons within the brain, and identify the particular neuronal circuit involved in aversive olfactory memory. how does neuronal activity contribute to information processing relevant to the acquisition, consolidation and retrieval of memories ? the uas - shi transgene has been effectively used to gain important insights into these key questions. the primary brain region that has been studied in the context of olfactory memory is the mushroom body (mb) (heisenberg, 2003). previous studies showed that aversive olfactory memory is disrupted in flies with genetically or chemically ablated mbs (heisenberg., 1985 ; de belle and heisenberg, 1994), yet the mb - deficient flies are able to discriminate odors and respond to an electric shock. although these results indicated that mbs play an essential role in olfactory memory, little was known about how mb - intrinsic neurons contribute to memory processing. the mb - intrinsic neurons known as kenyon cells are broadly classified into three subtypes, the /, / and neurons, according to the projection patterns of their axons (lee., 1999) the use of uas - shi in combination with various gal4 drivers that are specific to different subsets of kenyon cells has revealed that these structurally distinct neurons are also functionally diverse in the temporal processing of olfactory memory. diverse roles of intrinsic and extrinsic mushroom body - associated neurons in the processing of aversive olfactory memory. (a) the mushroom body and associated neurons in one brain hemisphere are schematically represented (adapted from armstrong. the cell bodies of kenyon cells (mbcs), the mushroom body - intrinsic neurons, are located in the dorsal and posterior cortices of the brain. the axons of / and / neurons bifurcate to form vertical (and) and horizontal (and) lobes. the axons of neurons do not bifurcate, and form only a horizontal lobe (). the primary olfactory information received by the olfactory neurons is transmitted through the antennal nerve (an) to the first olfactory center antennal lobe (al), where the information is processed and further transmitted to the mushroom bodies by the projection neurons (pn). the dorsal paired medial (dpm) neuron extends an axon that branches and terminates in all lobes of the mushroom body. (b) conditioned stimuli (cs ; e. g. odors) and unconditioned aversive stimuli (us ; e.g. electric shock) are simultaneously presented to flies. the olfactory information received by the olfactory neurons (osn) is conveyed to the mushroom bodies (mb) through the first - order interneuron, the projection neurons (pn). the neuronal circuits that transmit the aversive sensory information include dopaminergic neurons (da). the information generated by cs and us converges at the mb, where aversive olfactory memory is formed. neurotransmission from dpm neurons to the / neurons, as well as that from the / neurons, contributes to the stabilization of memory. retrieval of both protein synthesis - independent, short - lasting memory and protein synthesis - dependent, long - term memory (ltm) require neurotransmission from the / neurons. the / neurons, which constitute approximately 50% of the kenyon cells, are labeled in the majority of the mb - specific gal4 lines (aso., 2009). when uas - shi was preferentially expressed in the / neurons and neurotransmission from these neurons was temporally blocked by maintenance at the restrictive temperature only during single training, the flies avoided the shock - associated odor during the test period, showing that memory is formed, stored and retrieved under these conditions. similarly, inhibiting / neuron output between the training and test periods did not significantly affect the memory - based avoidance behavior during testing. these results indicate that neurotransmission from the / neurons is dispensable for the acquisition and storage of olfactory memory. in marked contrast, blocking neurotransmission only during the test period (30 min to 3 h after training) eliminated the avoidance behavior, demonstrating that neurotransmission from the / neurons is required for the retrieval of aversive olfactory memory (dubnau. an important implication of this finding is that any aversive olfactory memory formation (presumably as changes in structures and/or functions of cellular components) occurs at or upstream of the / output synapses of the neuronal circuits involved in the processing memory. memory is manifested as behavioral alterations in response to learned unconditioned stimuli (us) through neurotransmission from the / neurons in the mbs. either single or mass training can generate the consolidated form of memory known as arm. arm can be measured separately from stm and mtm by exposing animals to a cold shock 1 h after training, because labile stm and mtm are eliminated by this treatment. blocking the output of the / neurons using uas - shi has also been shown to impair arm, indicating that neurotransmission from the / neurons is necessary for the retrieval of three types of short - lasting, protein synthesis - independent memory (isabel., 2004). in contrast to arm, ltm is a protein synthesis - dependent, consolidated form of memory that is generated only after spaced training. recent optical imaging studies demonstrated that spaced training, but not single or mass training, results in a robust increase in calcium influx into the lobes, in a protein synthesis - dependent manner (yu., 2006). in addition, analysis of the alpha - lobes - absence (ala) mutants, which lack either the two vertical lobes (and) or two of the three horizontal lobes (and), suggested that ltm depends on the vertical lobes (pascual and preat, 2001). these results suggested that neurons forming vertical lobes, in particular those of the lobe, are important for ltm. indeed, the retrieval of memory 24 h after spaced training is significantly impaired when neurotransmission from the / neurons is blocked only during testing, indicating that the output of the / neurons is required for the retrieval of ltm. as in the case of stm, a memory trace for ltm is likely formed, at least partly, at or upstream of the / neuron output synapses (isabel., 2004). the original model for aversive olfactory memory proposes that pairing conditioned stimuli (cs) with us leads to the sequential formation of stm and mtm, and that the latter is then processed into either arm and/or ltm, depending on the training protocol (dezazzo and tully, 1995). in this model, both arm and ltm derive from mtm, and arm and ltm coexist for 24 h after spaced training. interestingly, however, it was shown that spaced training that produces ltm leads to disappearance of arm in the ala mutants lacking vertical lobes (isabel. in addition, the experiments using uas - shi indicate that arm and ltm involve the same / subset of the mb neurons. furthermore, normal arm can be detected in mutants defective for stm (rut) or mtm (amn). based on these collective results, it has been proposed that arm is processed largely independently of the sequential stm - mtm - ltm pathway, but stored in the common group of neurons in the mbs (isabel., 2004). with respect to aversive olfactory memory, the functional significance of the neurons, which account for approximately 30% of kenyon cells (aso., 2009), has thus far not been shown. the / neurons are relatively minor components of the mbs, comprising approximately 20% of the kenyon cells (aso., 2009). although they extend axons parallel to those of the / neurons, experiments using uas - shi demonstrated that the roles of the / neurons in olfactory memory processing are distinct from those of the / neurons. when neurotransmission from the / neurons was blocked only during the test period by introducing uas - shi in combination with gal4 drivers that preferentially label the / neurons, the memory - based avoidance behavior during the test period was not affected. this result shows that, unlike neurotransmission from the / neurons, that from / neurons is dispensable for the retrieval of olfactory memory. interestingly, however, when the / neurons were blocked either during or after training, olfactory memory was severely impaired. thus, output from the / neurons appears to be required for the acquisition and stabilization of olfactory memory (krashes., 2007). an exciting line of future investigation will focus on how neurotransmission from the / neurons contributes to the processing the memory that eventually depends on output of the / neurons for its retrieval. the dorsal paired medial (dpm) neurons are two large neurons that project extensively to all the lobes and the base of the peduncle of mb neurons. they were first identified as primary cells in the adult brain that express the putative neuropeptide encoded by the amnesiac gene (amn) (waddell., 2000), whose product plays an important role in stabilizing memory (quinn., 1979). amn mutants can learn and form stm, but their memory decays abnormally rapidly, within 3060 min after training, and this results in severe defects in mtm. because the amn memory defect is rescued by expressing the wild - type amn gene product preferentially in dpm neurons, these neurons are considered to be the site at which amn acts to stabilize olfactory memory. results obtained from uas - shi experiments revealed the significance of dpm neuron output in memory stabilization. when neurotransmission from dpm neurons was constantly blocked (during the training, storage and test periods), the flies displayed defects in memory that could be measured 1 h after training, as was the case in amn mutants. however, temporally blocking neurotransmission from dpm neurons only during the training or test period did not affect olfactory memory. in contrast, blocking dpm output for 30 min during the storage period (between training and testing) resulted in a significant impairment of 1-h memory, demonstrating that the dpm neuron is critical for stabilizing memory (waddell., 2000). considering that neurotransmission from the / neurons is similarly required to stabilize memory during storage, and that dpm neurons heavily innervate the mb lobes including the and lobes it is possible that direct information flow from dpm neurons to the / neurons is essential to stabilizing the newly formed memory. this possibility was tested by using flies that express an isoform of drosophila down syndrome cell adhesion molecule (dscam17 - 2) in dpm neurons. the expression of dscam17 - 2 in dpm neurons affected their development such that projections of dmp neurons to the, and lobes are significantly reduced, but those to the and lobes are relatively intact. in these genetically engineered flies with limited dpm projections to the mb / neurons, aversive olfactory memory was not significantly affected. importantly, a temporal block in dpm neuron output to the / neurons, but not to most / and neurons, was enough to induce the amn - like memory phenotype (krashes., 2007). these results strongly suggest that stabilizing memory during the storage period mainly depends on neurotransmission from dpm neurons to a subset of mb neurons the / neurons. it has been proposed that aversive olfactory memory is formed in the mbs, where sensory information concerning conditioned odor stimuli and unconditioned aversive stimuli converges, and that this establishes an association between cs and us (davis, 2005). although significant progress has been made in understanding the molecular and neuronal mechanisms responsible for olfactory information processing (hallem and carlson, 2004), little is known about how unconditioned electric shocks are sensed as aversive stimuli, and which neurons are involved in conveying us information to the mbs. in other organisms, monoaminergic interneurons are thought to transmit such information. in drosophila, a study investigating mutants for dopa decarboxylase, which encodes an enzyme involved in the synthesis of both dopamine and serotonin, implicated these neurotransmitters in aversive olfactory learning (tempel., 1984). however, this result could not be reproduced by another study (tully, 1987). given the controversial nature of these findings, uas - shi was used to revisit this issue (schwaerzel., 2003). tyrosine hydroxylase (th) is the rate limiting enzyme for dopamine and expressed specifically in dopaminergic neurons. th - gal4, which was generated using regulatory dna of the th gene (friggi - grelin., 2003), was used to direct uas - shi to dopaminergic neurons. although blocking neurotransmission from dopaminergic neurons before and during testing did not affect memory, blocking them only during the training period severely impaired aversive olfactory memory (schwaerzel., 2003). interestingly, dopamine transmission was not required for appetitive olfactory learning using sugar as the us. these results provide strong support for the notion that dopaminergic neurons are part of the neuronal circuit for aversive us in drosophila. there are approximately 120 dopaminergic neurons in the adult fly brain, and some of which extensively innervate the mbs (riemensperger., 2005). future studies using gal4 lines specific to a subset of brain dopaminergic neurons should reveal functional differences between dopaminergic neurons within the brain, and identify the particular neuronal circuit involved in aversive olfactory memory. it is almost 30 years since the genetic analysis of learning and memory was initiated using drosophila melanogaster as a model system. since then, considerable progress has been made in identifying and characterizing genes and genetic interactions that are important for memory processes. currently the drosophila memory research field is aiming to attain a comprehensive understanding of the mechanisms that underlie learning and memory, by an approach that integrates molecular, cellular and systems analyses. since its introduction to drosophila behavioral research, the uas - shi transgene has contributed significantly to the studies of temporal and spatial regulation of the neuronal activities responsible for learning and memory. figure 2 summarizes the findings of the uas - shi studies regarding the roles of intrinsic and extrinsic neurons of the mbs in different aspects of aversive olfactory memory. this knowledge provides a strong foundation for detailed functional and structural analyses in the future. 2008) have generated thousands of transgenic drosophila lines in which gal4 expression is directed to distinct (most of them small) subsets of cells in the adult brain (pfeiffer., 2008). in the case of the mbs in particular, an increasing number of gal4 enhancer - trap strains have been identified that label specific subsets of its intrinsic and extrinsic neurons (tanaka., 2008). these new gal4 lines will be used in combination with uas - shi, as well as other equally useful uas - linked effector transgenes, to enable the conditional activation of identifiable neurons (lima and miesenbock, 2005 ; schroll., 2006) and the optical imaging changes in the levels of important messengers including ca, h and camp (yu., 2004). the same approach should be applied to other learning paradigms in drosophila that involve sensory stimuli and brain neurons distinct from those involved in aversive olfactory learning. with a handful of valuable molecular and genetic tools, drosophila research on memory and learning is poised to continue providing essential knowledge of the basic principles of learning and memory in the coming years. 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.	the fruit fly drosophila melanogaster is an excellent model organism to identify genes and genetic pathways important for learning and memory. however, its small size makes surgical treatment and electrophysiological manipulation technically difficult, hampering the functional analysis of neuronal circuits that play critical roles in memory processing. to circumvent this problem, we developed a unique experimental strategy that uses the temperature - sensitive allele of the drosophila dynamin gene, shibirets1 (shits1), in combination with the gal4/uas expression system. this strategy allows for rapid and reversible perturbation of synaptic neurotransmission in identifiable neurons, and analysis of the behavioral consequences of such manipulation in free - moving animals. since its introduction in 2001, this gal4/uas - shits1 strategy has been widely used to study the neuronal basis of learning and memory. this review focuses on how this strategy has revitalized drosophila memory research, and contributed to our understanding of dynamic neuronal processes that control various aspects of memory.
epidermolysis bullosa acquisita (eba) is an acquired, chronic subepidermal bullous disease affecting the skin and mucosa. it is characterized by autoantibodies to type vii collagen, the major component of anchoring fibrils that attaches the epidermis to the dermis. the two main phenotypic presentations of eba are the classic mechanobullous and the inflammatory vesiculobullous type. the mechanobullous phenotype is characterized by noninflammatory blisters on trauma - prone areas such as elbows, knees as well as the dorsum of hands and feet that often heals with scarring and milia formation. on the other hand, the inflammatory form of eba manifests as widespread blisters on an erythematous base that mimics other bullous diseases, including bullous pemphigoid (bp). histopathology of lesional skin shows a subepidermal blister with varying degree of dermal inflammatory infiltrate, depending on the clinical phenotype. by direct immunofluorescence (if) microscopy of perilesional skin, linear igg and/or c3 deposits are detected along the basement membrane zone (bmz). indirect if microscopy on 1 mol / l nacl - split skin demonstrates circulating autoantibodies that localize to the dermal side of the artificially split skin. it was initially thought to be rare in children ; however, an increasing number of cases of childhood eba have been reported over the last few years. we here report a child with the mechanobullous phenotype of eba with autoantibodies targeting the noncollagenous 1 domain (nc1) of type vii collagen. a 10-year - old girl, born out of nonconsanguineous marriage, presented with recurrent blisters all over the body since the age of 5 years. blisters developed initially on the legs and gradually progressed to involve the trunk and the upper limbs. birth and development history were unremarkable, and none of her family members had a similar skin problem. examination revealed multiple tense blisters (clear as well as hemorrhagic), crusted ulcers and erosions as well as milia, predominantly over legs, and dorsa of hands and feet [figure 1 ]. in the oral cavity, erosions on the hard palate and buccal mucosa were found. tense blisters, crusts, and milia (indicated by arrows) on the (a) dorsum of hands and (b) on lower legs direct if microscopy of a perilesional skin biopsy revealed linear staining of igg and c3 at the bmz. indirect if microscopy on 1 m nacl - split skin showed binding of igg along the dermal side of the split at a titer of 1:100 [figure 2 ]. when the serum was treated with a panel of hereditary epidermolysis bullosa (eb) skin samples, no bmz reactivity was seen with recessive dystrophic eb (rdeb) skin deficient for type vii collagen [figure 3 ]. enzyme - linked immunosorbent assay (elisa) based on the recombinant immunodominant nc1 domain of type vii collagen was positive (59 u / ml). by immunoblotting with dermal extracts and the recombinant nc1 domain of type vii collagen, a 290 kda band and 145 kda band she was treated with prednisolone 1 mg / kg body weight and dapsone 50 mg / day with good improvement of skin and mucous membrane lesions. direct immunofluorescence microscopy showing linear deposits of igg at the basement membrane zone (a) ; indirect immunofluorescence microscopy on salt - split skin revealing deposits of igg on the dermal side of the split (b) (fluorescein isothiocyanate, 200) indirect immunofluorescence on salt split skin with patient 's sera showing floor binding pattern (a) ; modified indirect immunofluorescence using patient 's sera and recessive dystrophic epidermolysis bullosa skin as a substrate showing absence of basement membrane zone staining (b) (fluorescein isothiocyanate, 200) western blotting of the patient 's serum with dermal extract showing binding, like in a control epidermolysis bullosa acquisita patient, to a 290 kda band representing full - length type vii collagen (upper arrow). another control serum from a patient with anti - p200 pemphigoid reacts with a 200 kda protein (lower arrow) (a). western bot analysis with the recombinant nc1 domain of type vii collagen shows reactivity with this 145 kda protein in both an epidermolysis bullosa acquisita control and the patient 's serum (b) it typically starts in the fourth or fifth decade of life ; childhood eba (16 years old or younger) is rare and < 50 cases have been reported in the literature. the inflammatory vesiculobullous phenotype is the most common mode of presentation of eba both in adult as well as in childhood. mucosal involvement is more common in children as compared to adults, and the prognosis in childhood eba seems to be much better than in adult eba. the adult form of the disease is known to be difficult to treat in contrast to childhood eba, which usually responds well to dapsone and low - dose prednisolone. our patient presented with the mechanobullous phenotype with tense blisters over trauma - prone areas which healed with scarring, atrophy, and milia formation. the routine screening test for diagnosing eba is direct and indirect if microscopy ; particularly, indirect if microscopy on salt - split skin can distinguish eba from bp. patients with eba have immune deposits on the dermal side of salt - split skin (floor pattern), whereas in bp, the deposits are seen on the epidermal side (roof pattern). binding diseases such as anti - p200 pemphigoid and anti - laminin 332 pemphigoid. in thin sections and with higher magnification (600-fold), labeling of the bmz appears as a u - serrated pattern with arches open at the top in eba skin. if this method, as in our patient, can not be applied, autoantigen identification using a panel of eb skin as the substrate may help to confirm the diagnosis. the advantage of this test is that it can be carried out in any laboratory with a facility for if microscopy. our patient 's serum did not demonstrate bmz staining in rdeb skin deficient in type vii collagen, thus confirming the diagnosis of eba. type vii collagen consists of a triple - helical domain flanked by the large 145 kda nc1 amino - terminal and a small 34 kda noncollagenous carboxyl - terminal (nc2) domain. reviewed 6 cases of childhood eba in whom immunoblotting was performed ; 2 children showed immunoreactivity against all 3 domains of type vii collagen while 2 other demonstrated reactivity against the triple - helical and nc2 domains only. one patient had antibodies targeting triple - helical domain only while the other child had antibodies directed against nc1 and nc2 domain of type vii collagen. this is in contrast to adult cases with classical eba where antibodies are directed against nc1. recently, several elisa systems based on the immunodominant nc1 domain of type vii collagen (alone or in combination with nc2) have been established with high sensitivity (66100%) and specificity (98.2100%). these elisa systems are useful both for the diagnosis of eba and evaluation of disease severity. in our case, elisa with the recombinant nc1 domain of type vii collagen confirmed the diagnosis of eba. in this report, we describe a case of childhood eba that presented with the classical mechanobullous phenotype. the diagnosis was confirmed by a combination of if microscopy, antigen identification using eb skin, elisa, and immunoblotting. although if tests on organ sections are useful for the diagnosis of eba, target antigens can be detected by indirect if microscopy on eb skin, elisa, and immunoblotting. among them, antigen identification using eb skin is a simple procedure which can be carried out in any laboratory with a facility for if microscopy. on the other hand, though immunoblotting is technically more demanding and expensive, it is useful to identify target epitopes within type vii collagen. commercially available elisa and indirect if tests based on the recombinant nc1 domain of type vii collagen have recently produced promising results. we report a case of childhood eba with mechanobullous phenotypefinal diagnosis was confirmed by a combination of tests which included indirect immunofluorescence microscopy, antigen identification using skin deficient in type vii collagen, elisa, and immunoblottingimmunoblotting studies in our case revealed antibodies targeting only nc1 domain of type vii collagen in contrast to previous reports of childhood eba. we report a case of childhood eba with mechanobullous phenotype final diagnosis was confirmed by a combination of tests which included indirect immunofluorescence microscopy, antigen identification using skin deficient in type vii collagen, elisa, and immunoblotting immunoblotting studies in our case revealed antibodies targeting only nc1 domain of type vii collagen in contrast to previous reports of childhood eba. we report a case of childhood eba with mechanobullous phenotypefinal diagnosis was confirmed by a combination of tests which included indirect immunofluorescence microscopy, antigen identification using skin deficient in type vii collagen, elisa, and immunoblottingimmunoblotting studies in our case revealed antibodies targeting only nc1 domain of type vii collagen in contrast to previous reports of childhood eba. we report a case of childhood eba with mechanobullous phenotype final diagnosis was confirmed by a combination of tests which included indirect immunofluorescence microscopy, antigen identification using skin deficient in type vii collagen, elisa, and immunoblotting immunoblotting studies in our case revealed antibodies targeting only nc1 domain of type vii collagen in contrast to previous reports of childhood eba. we report a case of childhood eba with mechanobullous phenotypefinal diagnosis was confirmed by a combination of tests which included indirect immunofluorescence microscopy, antigen identification using skin deficient in type vii collagen, elisa, and immunoblottingimmunoblotting studies in our case revealed antibodies targeting only nc1 domain of type vii collagen in contrast to previous reports of childhood eba. we report a case of childhood eba with mechanobullous phenotype final diagnosis was confirmed by a combination of tests which included indirect immunofluorescence microscopy, antigen identification using skin deficient in type vii collagen, elisa, and immunoblotting immunoblotting studies in our case revealed antibodies targeting only nc1 domain of type vii collagen in contrast to previous reports of childhood eba.	epidermolysis bullosa acquisita (eba) is an acquired subepidermal bullous disorder characterized by autoantibodies against type vii collagen. it usually affects adults ; childhood eba is rare. we describe a 10-year - old girl presenting with recurrent tense blisters predominantly on legs, dorsa of hands and feet accompanied by oral erosions since the age of 5 years. direct immunofluorescence (if) microscopy showed linear deposition of igg and c3 along the basement membrane zone (bmz) ; indirect if microscopy on salt - split skin revealed staining of igg to the dermal side of the split. the patient 's serum did not show bmz staining in recessive dystrophic epidermolysis bullosa skin deficient for type vii collagen, thus confirming autoantibody reactivity against type vii collagen. circulating antibodies against the immunodominant noncollagenous 1 domain of type vii collagen were detected by elisa and immunoblotting studies. the patient was treated with oral corticosteroids and dapsone with good improvement.
descrever os aspectos encontrados em tcar do trax de pacientes infectados pelo vrus influenza a (h1n1). foram analisadas retrospectivamente as tcar de 71 pacientes (38 femininos e 33 masculinos) com diagnstico confirmado de influenza a (h1n1) atravs da identificao laboratorial do vrus, estudados no perodo entre julho e setembro de 2009. a interpretao das tcar foi realizada por dois radiologistas torcicos de forma independente, e, em caso de discordncia, as decises foram tomadas por consenso. os achados de tcar mais comuns foram opacidades em vidro fosco (85%), consolidao (64%) ou a combinao de opacidades em vidro fosco e consolidao (58%). outros achados foram ndulos do espao areo (25%), espessamento das paredes brnquicas (25%), espessamento de septos interlobulares (21%), padro de pavimentao em mosaico (15%), espessamento perilobular (3%) e aprisionamento areo (3%). as alteraes foram frequentemente bilaterais (89%), com distribuio no especfica (68%). as alteraes predominantes foram opacidades em vidro fosco, consolidaes ou a combinao de ambas. o acometimento foi frequentemente bilateral e no houve predomnio quanto distribuio (axial ou craniocaudal). apesar de inespecficos, importante reconhecer os principais aspectos tomogrficos da infeco por influenza a (h1n1) a fim de incluir essa possibilidade no diagnstico diferencial de sintomas respiratrios. in april of 2009, there began an epidemic of acute febrile respiratory illness caused by a new virus : the influenza a (h1n1) virus. the first cases occurred in mexico, and the infection rapidly spread worldwide. by august of 2010, 214 countries had been hit by the infection and more than 18,000 deaths had been confirmed. although the number of cases of h1n1 infection has decreased significantly since the 2009 pandemic, various studies have reported that the virus is still circulating together with other seasonal viruses, with different prevalences. in brazil, by october of 2012, approximately 20,000 patients had been hospitalized for severe acute respiratory syndrome. of those, approximately 2,600 cases were caused by the influenza a (h1n1) post - pandemic virus. the most common clinical findings in the presentation of h1n1 infection are fever, cough, dyspnea, myalgia, and headache. gastrointestinal symptoms, such as nausea, vomiting, and diarrhea, have also been reported. in most cases, the symptoms are mild and run a self - limiting course ; however, a small proportion of individuals develop a severe course, which can result in respiratory failure and death. the most common laboratory test findings include increased serum lactate dehydrogenase levels (which can exceed 1,000 iu / l), bearing in mind that elevated lactate dehydrogenase levels are significantly associated with disease severity and icu admission ; increased c - reactive protein levels ; increased serum creatine kinase levels ; lymphopenia ; and thrombocytopenia. chest x - ray provides adequate information for defining the approach in most of the affected patients. however, hrct often becomes an important tool for determining the extent of pulmonary involvement, as well as being useful in the evaluation of complications and in the clarification of suspected mixed infections or failure to respond to therapy. although the diagnosis of viral infection is based on the clinical profile and on identification of the virus, the recognition of some imaging features of the disease can become useful, especially in patients with forme fruste or atypical clinical manifestations. therefore, the understanding of the imaging features of the disease becomes important in clinical practice. the objective of the present study was to evaluate, by means of a retrospective analysis of hrct scans of patients with confirmed h1n1 infection, the most common tomographic findings and the characteristics of their distribution in the lung parenchyma. the study was approved by the research ethics committee of the clementino fraga filho university hospital. this was a retrospective observational cross - sectional study in which we analyzed the hrct scans of 71 patients from several hospitals in different states in brazil between july and september of 2009. the study population included patients presenting with flu - like symptoms and diagnosed with h1n1 infection, regardless of age or gender. the inclusion criterion was the diagnostic confirmation of h1n1 infection through laboratory testing (viral culture or real - time pcr) of aspiration material or of nasopharyngeal or oropharyngeal swab specimens. another inclusion criterion was the required availability of complete chest hrct scans of each study patient, performed during the acute phase of the disease. patients with other pulmonary infections, confirmed through laboratory tests, were excluded, as were those with tomographic findings suggestive of pulmonary involvement resulting from chronic lung diseases. as multiple institutions were involved, imaging examination was performed with different tomography scanners, using the high - resolution technique, in accordance with the following protocol : patient in the supine position ; 1- to 2-mm slice thickness in increments of up to 10 mm, from the lung apices through the hemidiaphragm, at the end of a deep inhalation ; and a high spatial resolution reconstruction algorithm. parenchymal and mediastinal window settings (width = 1,400 - 1,600 hu ; level = 600 to 800 hu ; and width = 350 - 450 hu ; level = 15 - 25 hu, respectively) were used. the following tomographic characteristics were analyzed : pattern of the findings (airspace consolidations, ground - glass opacities, airspace nodules, interlobular septal thickening, crazy - paving pattern, air trapping - when identified in the inspiratory phase - and peribronchovascular interstitial thickening) ; and distribution of the lesions (central, peripheral, or random ; unilateral or bilateral ; upper, middle, or lower lung zone predominant, or any combination of the three). the hrct scans were independently interpreted by two experienced thoracic radiologists, and in case of disagreement, the decisions were made by consensus. consolidation was defined as increased attenuation of the lung parenchyma, resulting in the obscuration of the vascular outlines and adjacent airway walls ; ground - glass opacity was defined as slightly increased attenuation of the lung parenchyma that is unrelated to the obscuration of the vessels and adjacent airway walls ; interlobular septal thickening was defined as thin linear opacities, which correspond to the thickened peripheral connective septa ; a crazy - paving pattern was defined as interlobular septal thickening superimposed on ground - glass opacities ; airspace nodules were defined as ill - defined nodules smaller than 1 cm and tending to confluence ; peribronchovascular thickening was defined as an increase in connective tissue around the bronchi, pulmonary arteries, and lymphatic vessels ; a perilobular pattern was defined as thick and irregular polygonal opacities in the periphery of the secondary pulmonary lobule ; and air trapping was defined as decreased attenuation of the lung parenchyma, revealed by a lower - than - usual density. the bronchial walls were considered thickened if the bronchial lumen internal diameter was equal to or greater than 80% of its external diameter. the distribution of the lesions in the lung parenchyma was evaluated along the axial and craniocaudal axes. the craniocaudal distribution of the lesions was classified as upper, middle, or lower lung zone predominant, or any combination of the three. in addition, the predominance of the findings in one lung or the lack of predominance along the two axes was recorded. of the 71 patients, 38 (53.53%) and 33 (46.47%) were female and male, respectively. the mean age of the patients was 41.3 years (range, 16 - 92 years). the most common tomographic findings, in decreasing order, were as follows : ground - glass opacities, in 60 patients (85%) ; consolidations, in 46 (64% ; figures 1 and 2) ; airspace nodules, in 18 (25% ; figure 3) ; bronchial wall thickening, in 18 (25%) ; interlobular septal thickening, with or without ground - glass opacities, in 15 (21%) ; crazy - paving pattern, in 11 (15%) ; perilobular pattern, in 2 (3%) ; and air trapping, 2 (3% ; figure 4 and table 1). figure 1hrct scans. in a, areas of consolidation and ground - glass opacities in the upper lobes of both lungs. in b, areas of consolidation and ground - glass opacities in the lower lobes, with peripheral distribution of the lesions. figure 2axial hrct slices at the levels of the upper lobes (a) and lower lobes (b), as well as reformatted coronal image (c), showing extensive areas of consolidation with air bronchograms in both lungs. figure 3axial hrct slices at the levels of the upper lobes (a) and lower lobes (b) revealing multiple small centrilobular nodules, some of which have a tree - in - bud pattern, distributed principally in the middle lobe and lower lobes. note also bronchial wall thickening predominantly in the right lower lobe, as well as a small area of peripheral consolidation. figure 4axial hrct slice at the level of the lower lobes, obtained during the expiratory phase, showing bilateral areas of mosaic attenuation, as well as a well - defined area of air trapping in the lower lobe of the left lung. table 1frequency distribution of the tomographic findings in the 71 patients in the sample.findingfrequencyn%ground-glass opacities6085consolidations4664airspace nodules1825bronchial wall thickening1825interlobular septal thickening1521crazy - paving pattern1115perilobular pattern23air trapping23 the most common findings were ground - glass opacities and consolidations. a combination of ground - glass opacities and consolidations in the same patient was observed in 41 cases (58%). only 6 patients had no ground - glass opacities, consolidations, or a combination of both. of the patients who had ground - glass opacities, 11 (15%) also had interlobular septal thickening, characterizing a crazy - paving pattern. septal thickening, without ground - glass opacities, was a mild secondary finding in 4 patients (6%). airspace nodules were found in 18 cases (25%), and bronchial wall thickening was found in 18 (25%). bronchial wall thickening was observed in all 6 patients who had no ground - glass opacities, consolidations, or a combination of both. peribronchovascular interstitial thickening was observed in 8 cases (11%), and, in all of these cases, there were ground - glass opacities and consolidations. minimal pleural effusion was observed in 19 patients (27%), being bilateral in 10 (52%). parenchymal involvement was bilateral in 63 cases (89%) and unilateral in 8 (11%). in 55 patients (77%), the lesions were evenly distributed between the two lungs ; in 9 patients (13%), the lesions predominantly affected the right lung ; and, in 7 patients (10%), the lesions predominantly affected the left lung. in 7 of the 8 cases (87%) in which the involvement was unilateral, only one lobe was affected. an analysis of the craniocaudal distribution of the lesions by lung zone revealed the following : no preferential distribution, in 35 patients (49%) ; lower third predominance, in 26 (37%) ; middle third predominance, in 4 (6%) ; lower and middle third predominance, in 5 (7%) ; and upper third predominance, in only 1 (1.5%). an analysis of the axial distribution of the lesions revealed the following : no specific distribution, in 48 patients (68%) ; peripheral lung zone predominance, in 21 (29%) ; and central lung zone predominance, in 2 (3%). in 14 of the 21 patients (67%) with peripheral distribution, there were also lesions in the peribronchovascular interstitial space. in the present study, we retrospectively evaluated the hrct scans of 71 patients with confirmed h1n1 infection. the most common tomographic findings were ground - glass opacities, consolidations, and a combination of ground - glass opacities and consolidations in the same patient. these data are similar to literature data showing that these tomographic findings predominate in patients with h1n1 infection. a crazy - paving pattern, in which ground - glass opacities are associated with septal thickening, has been reported in few cases in the literature. in our study septal thickening without ground - glass opacities was a mild secondary finding, being observed in only 4 patients (6%). airspace nodules were present in 25% of the cases, and this was the dominant finding in only 1. this finding has not commonly been reported in the literature, and few studies have described the presence of airspace nodules in patients with h1n1 infection. this can be attributed to an alternative interpretation, by some authors, of airspace nodules being focal areas of consolidation, or it can even be attributed to the fact that airspace nodules can be obscured by other findings when there is extensive involvement by the disease. bronchial wall thickening was observed in one fourth of the patients in our sample. in the literature other authors suggested that these changes occur early in the disease course, a period when patients have not yet sought medical care, and is therefore poorly reported. tanaka. related the high frequency of these findings in their study (68%) to the fact that their patients had quick access to medical treatment. likewise, the immunocompromised patients in another study are probably patients who received prompt medical attention, given their comorbidities. in contrast, other authors reported air trapping in a female patient in a late phase, three months after the onset of the disease. in our sample, a mosaic attenuation pattern was observed in 3% of the cases and was interpreted as being air trapping because it was associated with bronchial wall thickening. as occurred in our sample, air trapping related to h1n1 infection has rarely been described in the literature. however, it should be emphasized that, in those studies, the expiratory phase, which increases sensitivity in the characterization of this pattern, was not performed. li. found a mosaic attenuation pattern due to air trapping in 13% of their patients and related it to the use of mechanical ventilation. this finding was seen in 2 of our patients (3%), and it had not been described in h1n1 infection. because patients affected by the pandemic virus can develop organizing pneumonia during the convalescence phase of the disease, a perilobular pattern is an expected finding in these cases. in the literature, the most common distribution of hrct findings is bilateral multifocal involvement predominantly in the lower lobes. however, in some studies, the distribution is diffuse, with no zonal predominance. in our sample, the hrct scans showed bilateral involvement in the vast majority of the cases (89%), and both lungs were similarly affected in 77% of them. the craniocaudal distribution of the lesions was random, with no zonal predominance in approximately half of the cases and with lower third predominance in 37%. the axial distribution of the findings showed no zonal predominance in most cases (68%), but, in approximately one third of the cases, it showed peripheral lung zone predominance. a central distribution was rarely observed. in the literature, data regarding the axial distribution of hrct findings are varied, there being reports of central or peripheral predominance. regarding the craniocaudal distribution, the literature reports lower lung zone predominance. pleural effusion, either unilateral or bilateral and typically minimal, has been described in some cases. in our sample, pleural effusion was observed in 27% of the cases, being bilateral in 52% and typically minimal. first, the study was retrospective, which made it impossible to establish a proper correlation between clinical and radiological findings. second, there were some differences in relation to the technical parameters in tomography image acquisition, since we evaluated hrct scans performed at various institutions. in conclusion, the most common tomographic findings were ground - glass opacities and airspace consolidations, or a combination of both. the findings were predominantly bilateral, with no axial or craniocaudal predominance in the distribution in most cases. when zonal predominance was present, it was more common in the lower thirds and peripheral regions of the lungs. although the major tomographic findings in h1n1 infection are nonspecific, it is important to recognize such findings in order to include infection with the h1n1 virus in the differential diagnosis of respiratory symptoms	objective : to describe aspects found on hrct scans of the chest in patients infected with the influenza a (h1n1) virus. methods : we retrospectively analyzed the hrct scans of 71 patients (38 females and 33 males) with h1n1 infection, confirmed through laboratory tests, between july and september of 2009. the hrct scans were interpreted by two thoracic radiologists independently, and in case of disagreement, the decisions were made by consensus. results : the most common hrct findings were ground - glass opacities (85%), consolidation (64%), or a combination of ground - glass opacities and consolidation (58%). other findings were airspace nodules (25%), bronchial wall thickening (25%), interlobular septal thickening (21%), crazy - paving pattern (15%), perilobular pattern (3%), and air trapping (3%). the findings were frequently bilateral (89%), with a random distribution (68%). pleural effusion, when observed, was typically minimal. no lymphadenopathy was identified. conclusions : the most common findings were ground - glass opacities and consolidations, or a combination of both. involvement was commonly bilateral with no axial or craniocaudal predominance in the distribution. although the major tomographic findings in h1n1 infection are nonspecific, it is important to recognize such findings in order to include infection with the h1n1 virus in the differential diagnosis of respiratory symptoms.
	radon is a well - established human carcinogen for which extensive data are available, extending into the range of exposures experienced by the general population. mounting epidemiologic evidence on radon and lung cancer risk, now available from more than 20 different studies of underground miners and complementary laboratory findings, indicates that risks are linear in exposure without threshold. radon is also a ubiquitous indoor air pollutant in homes, and risk projections imply that radon is the second leading cause of lung cancer after smoking. recommended control strategies in the united states and other countries, which include testing of most homes and mitigation of those exceeding guideline levels, have been controversial. further research is needed, drawing on molecular and cellular approaches and continuing the follow - up of the underground miner cohorts, and scientists should work toward constructing mechanistically based models that combine epidemiologic and experimental data to yield risk estimates with enhanced certainty.imagesfigure 1figure 2
the nervous system relies on conserved endocytic and synaptic vesicle (sv) recycling mechanisms to sustain high activity. defective endocytosis and sv recycling have long been implicated in neurodegeneration and neurodegenerative diseases, yet the molecular mechanisms remain unclear (esposito., 2012, heutink and verhage, 2012, saheki and de camilli, 2012, schreij., 2016). endophilin - a (henceforth endophilin) endocytic adaptors are key players in membrane dynamics at the neuronal synapse (ringstad., 1999,, 2002, milosevic., 2011, saheki and de camilli, 2012). endophilin-1 and 3 are brain - specific, whereas endophilin-2 is ubiquitous (ringstad., 1997, ringstad., 1999). all three contain a highly conserved bar domain and have a central role in both clathrin - mediated endocytosis (farsad., 2001,, 2001, verstreken., 2002, milosevic., 2011) and clathrin - independent endocytosis (boucrot., 2015). in clathrin - mediated endocytosis, endophilin recruits synaptojanin-1 to the necks of coated vesicles prior to scission by dynamin (milosevic., 2011). furthermore, availability of endophilin is rate limiting for vesicle uncoating and sv recycling, and neurons without endophilin show reduced neurotransmission (milosevic., 2011). total absence of endophilin is perinatally lethal, whereas mice lacking endophilin-1 and 2 (1,2 double knockout [dko ]) show ataxia, neurodegeneration, and early lethality (milosevic., 2011). it can not be easily explained by defective neurotransmission, because even mice with paralysis and abolished neurotransmission do not develop neurodegeneration (heutink and verhage, 2012). thus, it is unlikely that neurodegeneration in endophilin 1,2 dko mice is solely the result of defective sv recycling. mice without endophilin also show increased protein ubiquitination (cao., 2014), and neurodegeneration is frequently associated with impaired proteolytic systems, most notably the ubiquitin - proteasome system (ups) and autophagy (nakamura and lipton, 2009, nixon, 2013, menzies., 2015). endophilin has been linked to the ups through direct interaction with the parkinson s disease (pd)-related e3-ubiquitin ligase parkin (trempe., 2009) and as a key component of the cbl - cin85-endophilin complex known to downregulate receptors (soubeyran. additional links between endophilin and neurodegeneration have recently emerged. the most commonly disrupted gene in familial pd (cookson, 2010), lrrk2, encodes a protein that phosphorylates endophilin (matta., 2012). lrrk2 appears in lewy bodies and plays a role in apoptosis and autophagy (iaccarino. in addition, a number of case studies described synaptojanin-1 and auxilin mutations in early - onset parkinsonism (edvardson., 2012, quadri., 2013, krebs., 2013). auxilin is recruited to the clathrin - coated vesicles directly after the action of synaptojanin-1, which is brought to the neck of the clathrin - coated pit by endophilin (milosevic., 2011). interestingly, endophilin is upregulated in the brains of pd patients (shi., 2009). in addition, endophilin-1 and 3 have been shown to interact with ataxin-2, mutations in which lead to spinocerebellar ataxia type 2 (ralser., 2005, nonis., 2008). despite direct parkin - endophilin interaction and a striking upregulation of parkin in mice lacking all three endophilin genes (endophilin triple knockout [tko ] ; cao., 2014), elevated parkin we subjected endophilin mutants to rna sequencing and found upregulation of another e3-ubiquitin ligase, f - box - only protein 32 (fbxo32, also called atrogin-1/mafbx). fbxo32 has previously been studied in muscles, where its induction occurs prior to the onset of rapid atrophy (sandri., 2004). while studying how the fbxo32-endophilin - a interaction balances protein degradation and neuronal health in the brain, we discovered a direct role of endophilin - a in autophagosome formation. mice lacking all three endophilin genes die perinatally, whereas endophilin 1,2 dko animals show ataxia, neurodegenerative changes, and lethality around 3 weeks (movie s1 ; milosevic., 2011). whereas single knockout (ko) and heterozygous (1ht-2ht ; 1ht-2ht-3ht) mice appear phenotypically normal, endophilin 1ko-2ht, 1ko-2wt-3ko, and particularly 1ko-2ht-3ko mice exhibit shorter lifespans (19, 21, and 18 months, respectively ; 1ht-2ht mice lived over 27 months ; figure s1a). observing 1ko-2ht and 1ko-2ht-3ko mice revealed progressive impairments in motor coordination and increasingly obvious ataxia as these animals age. to systematically measure the progression of ataxia, we used a composite phenotype scoring system that incorporates hindlimb clasping, ledge walking, kyphosis, and gait (guyenet., 2010). when applied to four endophilin lines (1ko-2wt, 1ko-2ht, 1ko-2wt-3ko, and 1ko-2ht-3ko) and wild - type (wt) controls, it revealed phenotypic differences between endophilin mutants and within the same animals over the course of aging (figure 1a) ; i.e., the onset and progression of ataxia correlate with age and severity of endophilin deficiency. next, we systematically evaluated locomotion by accelerating rotarod (arr) performance test and gait analysis. whereas the performance of 1ko-2wt mice on the arr was comparable to wt, fall latency in 1ko-2ht-3ko mice was already significantly lower at a very young age (figure 1b). intermediate phenotypes were observed in 1ko-2ht and 1ko-2wt-3ko mice, where fall latency compared to wt was decreased significantly at 3 and 15 months, respectively. gait analysis revealed that ability to run on a motorized treadmill and walking pattern also depended on the number of missing alleles and age (figures 1c, 1d, and s1b ; see movie s2). 1ko-2ht-3ko gait was impaired at 3 months, whereas the littermate 1ko-2wt-3ko mice showed impaired gait at 15 months (figure 1d). overall, onset and progression of movement impairments correspond to age and the extent of endophilin deficiency. to evaluate whether neurodegenerative changes could cause these motor impairments, we inspected neuronal cell death and gliosis in our model. whereas overall neuronal cell morphology was unaltered in these mice (figure s1d), tunel assay revealed slightly increased cell death in endophilin 1ko-2wt and more prominently in 1,2 dko motor cortex and hippocampus at postnatal day (p)18 (figure 1e). increased cell death was also found in 18-month 1ko-2ht-3ko motor cortex and hippocampus (figure s1c). glial fibrillary acidic protein immunostaining revealed prominent upregulation of gliosis in 1,2 dko motor cortex (figure 1f). because endophilin tko mice die a few hours after birth, we probed for early markers of apoptosis in endophilin tko brains and found an increase in caspase 3 activity (figure 1 g). in summary, newborn endophilin tkos, juvenile 1,2 dko, and aged 1ko-2ht-3ko mice display signs of mild to moderate neurodegeneration. we were interested in the molecular mechanisms underlying neurodegeneration in our endophilin - deficient models, so we employed rna sequencing (experimental strategy in figure s2a). we first sequenced hippocampal rna from tko mice that show altered sv recycling and controls (n = 89 mice / condition). the data are of high technical quality (27 m aligned reads) and samples clustered well according to genotype (table s1 ; figures s2b and s2c). 182 differentially expressed genes (degs) between endophilin tko and littermate controls and 964 degs between tko and wt were detected (adjusted p value 0.4) (tables s2 and s3, respectively). given the pronounced movement defects of endophilin mutants, we first looked for movement - disorder - related genes or pathways. interestingly, tko versus wt degs showed significant enrichment of genes involved in ataxia and pd (figures 2a and 2b ; table s4). because endophilin 1,2 dko mice present first signs of neurodegeneration by the end of the 2 postnatal week, hippocampi from dko, littermate 1ko-2wt, and wt mice were extracted during the 3 week and subjected to rna sequencing and transcriptome analysis. the data are again of high technical quality (28 m reads ; table s1 ; samples clustered well according to genotype ; figures s2d and s2e). 1,479 degs between 1,2 dko and littermate controls, and 1,749 degs between dko and wt were detected (adjusted p value 0.4 ; tables s5 and s6, respectively). both 1,2 dko versus littermate and dko versus wt lists were significantly enriched for genes involved in ataxia and pd (table s4 ; figures 2c and 2d). ataxia - related genes include atm interactor (atmin), e2f1, and rad51 ; pd - related genes include park2 (encoding parkin), lrrk2, the tyrosine hydroxylase gene (th), and parl. these findings further support endophilin s association with neurodegeneration, ataxia, and pd (arranz., 2015, edvardson., 2012,, 2013, matta., 2012, milosevic., 2011, quadri., 2013, ralser., 2005, shi., 2009, trempe., 2009 we next analyzed tko versus both littermate and wt controls (genes significantly changed in the same direction in tko against both littermates and wts ; table s7 ; figure s2f) by ingenuity pathway analysis (ipa) software to identify altered pathways and networks. the resulting most - represented pathways were related to compromised synaptic transmission, protein homeostasis, phagosome maturation, mitochondrial function, dopamine metabolism, and apoptosis (figure 2e). altered protein homeostasis has been strongly linked to neurodegeneration (e.g., deger., 2015) and might be relevant to the ataxic and neurodegenerative phenotypes observed in endophilin - deficient mice. the differential expression of several genes was validated by quantitative real - time pcr in endophilin tko hippocampi versus wt, and a strong correlation was observed (r = 0.83 ; figure 2f). we also examined regulators of the transcriptional profile in endophilin tko hippocampi and found that, among those statistically enriched, fbxo32 presents the most - robust change in gene expression (1.9-fold increase ; table s8). this was interesting because fbxo32 is an e3-ubiquitin ligase linked to autophagy in skeletal and cardiac muscle (sandri. its expression is known to be regulated by the transcription factors foxo1 and foxo3a (sandri., 2004, webb and brunet, 2014) : whereas foxo1 was unchanged, foxo3a was significantly upregulated in tko hippocampi (table s8). given the observed changes in the foxo3a - fbxo32 network and that fbxo32 is known to promote cell death and is required for muscle atrophy (sandri., 2004, wu., 2011, xie., 2009), we decided to follow up on this network at the rna and protein level. as in the rna - sequencing data, foxo3a and fbxo32 were upregulated in tko hippocampi when measured by qpcr (figure 3a) whereas foxo1 was unchanged (figures s2 g and s2h). a striking increase in fbxo32 protein levels in tko whole - brain extracts relative to littermate controls and to wt mice was detected (figure 3b ; figure s2i displays elevated fbxo32 in the hippocampus). levels of foxo3a[ser253 ] phosphorylation (which represses foxo3a binding to dna) were also decreased (figure 3b), suggesting an increase in the foxo3a transcriptional activity, and the distribution of foxo3a[ser253]-p is altered in tko hippocampus in comparison to controls (figure 3c). to determine whether foxo3a activation is directly responsible for the upregulation of fbxo32, foxo3a was silenced in tko primary hippocampal neurons by small interfering rna (sirna) knockdown, using two sirna constructs. near - complete ablation of foxo3a affects neuronal survival, so we used a milder condition (45% reduction) to probe for fbxo32 levels (figure 3d). the elevated fbxo32 levels in tko neurons were lowered upon foxo3a knockdown, implying that foxo3a is a sufficient regulator of fbxo32 in this system (figure 3d). fbxo32 was also strongly upregulated in 1,2 dko brain samples, both at p0 (whole brain) and 3 weeks (cortex ; figure 3e), as well as in the cortex of 1ko-2ht-3ko mice at 18 months (figure 3f). thus, the foxo3a - fbxo32 network originally described in muscle atrophy is operative in the brain of endophilin mutant mice. given the reported role of fbxo32 in the regulation of protein homeostasis via the ups, we investigated whether the ubiquitin system is also affected in endophilin mutants. the pd - related e3-ubiquitin ligase parkin monoubiquitinates endophilin, and tko brains show increased levels of parkin and ubiquitinated proteins (trempe., 2009, cao., 2014 however, 1,2 dko mice in which the parkin gene park2 was also knocked out displayed a similar neurological phenotype to 1,2 dko mice (cao., 2013), suggesting that other ubiquitin ligases might be involved. given that fbxo32 is also an e3-ubiquitin ligase, we hypothesized that its robust upregulation in endophilin - deficient brain might contribute to the loss of protein homeostasis in endophilin knockouts. first, we tested whether fbxo32 can (mono)ubiquitinate endophilin-1 and 2, but this was not the case (figures s3a and s3b). we observed that 1,2 dko brains also display increased levels of ubiquitinated proteins (figure 4a), similar to tko brains (cao., 2013). to distinguish whether this was due to proteasome - related or localization-/signaling - related ubiquitination, we measured k48-(proteasome - related) and k63-(signaling - related)-linkage - specific polyubiquitination (herhaus and dikic, 2015). whereas k48-linked ubiquitination was prominently increased in 1,2 dko cortical extracts (figure 4b), there was no significant change in the levels of k63-linked ubiquitination (figure 4c), suggesting that the accumulation of ubiquitinated proteins is associated with defective processing by the proteasome. given the apparent inability of the proteasome to cope with the level of ubiquitinated proteins in endophilin knockouts, we probed the proteasomal system using a ubiquitinated substrate that is targeted to the proteasome and degraded (ubiquitin - g76v - gfp ; dantuma., 2000), as well as by measuring the enzyme activity of the proteasome itself. we observed an accumulation of gfp - fluorescence originating from the substrate in tko primary hippocampal neurons as well as fibroblasts (mouse embryonic fibroblasts [mefs ]) in comparison to respective controls (figures 4d and 4e), denoting proteasome saturation in the endophilin ko cells. interestingly, we noticed that the increase in ubiquitinated proteins elicited by the expression of ubiquitin - g76v - gfp results in significantly more apoptosis in the tko mefs than in controls (figure 4 g). altogether, these results suggest a causal link between proteasome saturation in endophilin mutants, higher levels of ubiquitinated proteins, and increased cell death susceptibility. to determine whether the proteasome saturation was due to decreased activity of the proteasome, we measured the proteasome enzyme activity. while there was a trend toward a decrease in proteasomal activity, we observed no significant difference in the proteasome activity per se in the brain extracts (figure 4f). thus, the proteasome system in tko brains seems functional but unable to keep up with the high demand. in light of the upregulation of fbxo32 in the absence of endophilin first, we tested whether fbxo32 interacts with endophilin family members by immunoprecipitation and pull - downs. an interaction between fbxo32 and all three endophilins in vivo was revealed by specific enrichment of fbxo32 in anti - gfp immunoprecipitates from hela expressing fbxo32-mcherry and endophilin 1, 2, or 3-egfp (figures 5a5c). similar results were obtained by glutathione s - transferase (gst) pull - downs from mouse brain lysate using the gst - fbxo32 as bait (figure s3e). we demonstrated that fbxo32 is able to pull - down purified endophilin-1 protein (figure 5d), suggesting a direct interaction. furthermore, the interaction is mediated through endophilin s sh3 domain, as revealed by another set of pull - downs from mouse brain lysate using the gst - sh3 domains of endophilin-1 and 3 as baits (an enrichment of fbxo32 with stronger binding to endophilin-1 and 3 sh3s was observed ; figure s3f). we examined the intracellular localization of fbxo32 and its overlap with endophilins in cultured clonal cells (hela and mefs were used to control for cell line variability) and primary hippocampal neurons. it is noteworthy that expressing fbxo32 (either fbxo32-egfp or fbxo32-mcherry) induced cell death : 50% of fbxo32-transfected hela and 40% of fbxo32-transfected mefs survived 30 hr post - transfection ; less than 15% of fbxo32-transfected primary neurons were detected on day in vitro (div) 14 (detailed below). fbxo32 distribution showed substantial cell - to - cell variability : although mainly cytosolic, fbxo32 was enriched at dynamic organelle - like structures of various sizes and often accumulated in the nucleus (movie s3). native fbxo32 distribution was similar to that of expressed fbxo32 : immunostaining against fbxo32 revealed nuclear as well as cytosolic protein distribution in hela cells (figure s4a). surprisingly, both in hela cells and mefs, fbxo32-labeled tubular structures were observed (examples in figures 5f, 5i, and s4c ; see also movie s5). given that fbxo32 interacts with endophilins, and the propensity of endophilins to tubulate membranes, we examined the colocalization of fbxo32 with endophilin 1, 2, and 3. the distribution of endophilins is mainly cytosolic : sparse puncta were attributed to endophilin s role in endocytosis (milosevic., 2011, perera., 2006, boucrot., 2015, when endophilin-1 and fbxo32 were coexpressed at similar levels, fbxo32 protein distribution changed : fbxo32 became predominantly cytoplasmic and excluded from the nucleus (figures s4b and s6e ; also see figures 7b and 7d). interestingly, we also observed that cell death due to fbxo32 overexpression was abolished upon endophilin coexpression, which we detail further below. fbxo32 and endophilin-1 were also found to colocalize sparsely at dynamic organelle - like structures (figures 5l and s4b). these were found throughout the cytoplasm and not only in the vicinity of the plasma membrane (as would be expected for endophilin-1 when being recruited to endocytic structures). when endophilin-2 and fbxo32 were coexpressed, they colocalized transiently at both organelle - like structures and tubuli (figures 5e, 5l, and s4c ; movie s5). we further observed that endophilin-2 colocalized with fbxo32 on dynamic tubulated structures, although endophilin-2 was not needed for the recruitment of fbxo32 to tubuli (figure 5f at 24 s ; movie s5). similarly to endophilins, fbxo32 also directly binds and tubulates membranes in vitro (b. kroppen, m. meinecke, and i.m. lastly, fbxo32 and endophilin-3 colocalized partially on organelle - like structures (figures 5l and s4d). we examined the overlap between fbxo32 and endophilin-1 or endophilin-2 in primary hippocampal cultures. fbxo32-egfp and endophilin-1-mrfp or endophilin-2-mrfp signal overlapped partially both in the cell bodies and in neuronal processes (figures 5 g and 5h ; again fbxo32 colocalized to a greater extent with endophilin-2 than endophilin-1). in sum, the data show that fbxo32 interacts directly with endophilin - a proteins (possibly through their sh3 domain), both proteins are involved in membrane tubulation, and both transiently colocalized on dynamic organelle - like structures of various sizes. in addition to being an e3-ubiquitin ligase, fbxo32 is known to regulate autophagy in muscle, raising the question of whether the fbxo32-labeled organelle - like structures were autophagosomes. we coexpressed fbxo32-egfp together with lc3-mrfp and observed a prominent colocalization of the two probes in starved cells (figures 5i and 5 m ; movie s4). a similar result was observed when fbxo32 was immunostained (figure s4a), irrespective of type of cell (hela cells were also used) or fluorescent labeling (figure s4e). next, when endophilin-2 was coexpressed with the autophagosomal marker lc3, sparse but significant colocalization was observed in starved hela cells, suggesting that endophilin-2, like fbxo32 albeit to a lesser extent, transiently associates with autophagosomes (figures 5j and 5 m). similar observations were made for endophilin-1 and endophilin-3, which were also occasionally seen on lc3-labeled autophagosomes (figures 5 m, s4f, and s4 g ; please note that the values represent colocalization above coincidental colocalization, which is prominent in this system given that endophilins are cytosolic proteins). for example, a kymograph from endophilin-3- and lc3-expressing hela cell shows that endophilin-3 colocalizes with an lc3-labeled autophagosome transiently (figure 5n ; arrows indicate colocalization). importantly, fbxo32, lc3, and endophilin-2 could be simultaneously detected at the same dynamic organelle (movie s6). notably, endophilin-2 showed much stronger overlap with atg5 (an early autophagosomal marker) than with lc3 (figure 5k ; 43% colocalization ; see movie s7), implying a role for endophilin - a in autophagosome formation. given that both fbxo32 and endophilins - a were occasionally found on lc3-positive autophagosomes, we examined whether autophagy was altered in brains of tko and 1,2 dko mice. notably, both lc3b - ii (lower band is the active form), a classical marker for autophagosomes, and atg5, a marker for early autophagosomes, were prominently decreased in tko brains (figure 6a) and in aged endophilin 1ko-2ht-3ko mice (figures 6c and 6d). interestingly, the atg5 protein level in endophilin 1ko-2ht-3ko mice decreases progressively between 3 and 18 months of age (figure 6d). a reduction in lc3b - ii and atg5 was also observed in 1,2 dko cortex (figure 6b), suggesting that endophilin-3 can not effectively compensate for the loss of endophilin-1 and 2 in autophagy. we prepared primary neuronal cultures from endophilin tko and wt hippocampi and labeled autophagosomes either by mildly expressing lc3-egfp (figures 6e and s5c) or by immunostaining for lc3 (figure s5e). we detected an overall decrease in the number of lc3-labeled autophagosomes when autophagy was simultaneously induced by starvation and mtor inhibitor torin-1 (figures 6e, 6f, s5c, and s5d). interestingly, the effect was more obvious at the synapses, as apparent from colocalization of lc3 and synaptobrevin-2, a presynaptic marker (figure 6e ; insets in figure s5c). altogether, the biochemical and imaging data suggest that autophagy depends on endophilin in vivo and in vitro and imply a role for endophilin in the formation and/or maturation of autophagosomes. notably, endophilin - b has a known role in autophagosome formation, and its cdk5-mediated phosphorylation is required for induced autophagy in models of pd (wong., 2011). endophilin - a proteins are closely related to endophilin - b : they share an identical domain structure with a high homology, and both protein subfamilies are enriched at the synapses and able to tubulate membranes (farsad., 2001, thus, we tested the levels of endophilin - b, cdk5, and several autophagy initiators. no change in the protein level of endophilin - b in endophilin - a tko brains was detected (figure 6 g), whereas the levels of cdk5 were decreased (figure 6a). however, in 1,2 dko brains, the cdk5 level was unchanged (figure 6b). protein levels of uvrag, beclin, atg3, and hdac6 were not altered, either in endophilin tko brains or endophilin 1,2 dko cortex (figures s5a and s5b). these results imply that endophilin - b can not compensate for autophagosome formation in the absence of endophilin - a and that autophagy is not altered due to repression of signaling pathways, because the key autophagy initiation regulators are not affected by absence of endophilin - a. the key autophagy factors atg9a and atg16l have been shown to undergo clathrin - mediated endocytosis (ravikumar., 2010a, puri., 2014). in the light of endophilins well - established role in endocytosis, it is possible that the observed phenotype is an indirect effect of defective endocytosis in endophilin mutants. however, we found that both atg9a and atg16l were not significantly changed in our mutants (figures 6h and s5f), indicating that availability of these two factors does not influence the findings we describe here. to further characterize the role of fbxo32 in autophagy, we employed the drosophila melanogaster neuromuscular junction (nmj) to see whether fbxo32 is relevant for autophagy in neuronal cells in vivo. when fbxo32 is silenced in drosophila in vivo, there was a very obvious decrease in the number of autophagosomes detected at the nmj (figures s6a s6c ; quantification in figure s6d). thus, fbxo32 seems necessary for autophagosome formation at the fruit fly nmj. given that, in the endophilin mutant brains there is more fbxo32 and yet still less autophagosome formation, the interaction between endophilin and fbxo32 may play a key role in this process. to determine whether upregulation of fbxo32 is an important step in the brain pathology of endophilin mutants, we explored the consequences of fbxo32 upregulation in clonal (hela and mef) and primary (neurons) cell lines. it has been reported that fbxo32 elevation induces apoptosis in cardiomyocytes and cancer cells (wu., 2011, xie., 2009). we observed that expression of fbxo32 causes substantial cell death in hela and mefs, as determined by the number of remaining egfp - positive cells and by the activity of caspases 3 and 7 (figures 7a7c, 7e, and s6e). the detrimental effects of fbxo32 were independent of its fluorescent tag and cell - transfection method (electroporation / lipofectamine / fugene). strikingly, when endophilin-1 was coexpressed with fbxo32 in mefs, fbxo32-induced cell death was rescued, as demonstrated by the number of egfp - positive cells (normalized [norm. ] to total dapi count) 30 hr after transfection and healthy cell appearance (figure 7b ; also in hela cells : figure s6e), the quantification of cell survival (figure 7c), and the activity of caspases 3 and 7 (figure 7e). importantly, the levels of fbxo32 expression were elevated when fbxo32 and endophilin-1 were coexpressed, implying that the rescue of fbxo32-induced apoptosis is not due to a reduction in fbxo32 levels (figures 7a and 7b). however, coexpression with endophilin-1 seemed to relocate fbxo32-egfp out of the nucleus (figures 7d and s6e) in agreement with the data presented in figure 5. thus, fbxo32-induced apoptosis is associated with aberrant intracellular fbxo32 localization, and both the localization and the apoptosis susceptibility can be rescued by simultaneous upregulation of endophilin-1. these data show that fbxo32 and endophilin-1 interact not only at the protein but also at the genetic level. in neurons, when either wt primary hippocampal or cortical neurons were transfected with fbxo32-egfp, less than 15% of all transfected neurons were detected at div14 (figures 7f7h). the surviving fbxo32-expressing neurons displayed a lower number of processes and appeared unhealthy (figure s6f). tko hippocampal and cortical neurons in culture were comparable to wt and littermate endophilin 1ko-2wt-3ko cells and did not show increased cell death under standard culturing conditions. however, when tko cortical neurons were transfected with fbxo32, less than 2% of transfected neurons were detected at div14 (in comparison to egfp - expressing tko neurons), with the surviving cells presenting a reduction in the number of processes and vacuoles in cell bodies. lastly, coexpression of endophilin-1 or endophilin-2 was able to rescue fbxo32-induced apoptosis in cortical neurons : when endophilin-1-mrfp or endophilin-2-mrfp were coexpressed with fbxo32-egfp, either in wt and endophilin tko cortical neurons, the majority of neurons thrived (over 80% and 67% of all fbxo32-transfected neurons, respectively ; figures 7f7h). imbalance in fbxo32/endophilin - a levels is likely a key mechanistic step in the brain pathology of endophilin mutants, and fbxo32-endophilin interplay seems important for maintaining neuronal health and overall protein homeostasis in the brain. to date, the known roles of endophilin - a have been limited to endocytosis and sv recycling. here, we show that endophilin also affects the ups and autophagy in the brain. the robust induction of the foxo3a / fbxo32 network (known to mediate muscle atrophy) in the brain of mice with endophilin deficiency promotes apoptosis and likely mediates brain pathology. similarly to muscle, the foxo / fbxo32 network seems relevant for regulating protein degradation in the brain and for maintaining neuronal health. if fbxo32 is expressed in cell lines and primary neurons, it causes apoptosis that can be rescued by simultaneous coexpression of endophilin. we also report that partial endophilin absence in mice results in neurodegeneration and in perturbations of a significant number of ataxia- and pd - related genes. these observations corroborate a number of recent findings that indirectly link endophilin to neurodegeneration and pd (arranz., 2015, edvardson., 2012, krebs., 2013, matta., 2012, milosevic., 2011, endophilin models may also be of relevance for neurodegeneration research because they may provide insights into signaling pathways affected prior to overt manifestation of neurodegeneration. fbxo32 was thus far studied mainly in skeletal muscle, where this e3-ubiquitin ligase was found to mediate rapid atrophy as a result of foxo3a transcriptional activation. interestingly, foxo3a activation is associated with cellular stress responses and neurodegeneration (webb and brunet, 2014). foxo3a stimulates autophagy in neurons (xu., 2011), and fbxo32 is necessary for autophagy ; activation of the foxo - fbxo32 axis should thus lead to increased autophagy. nevertheless, this is not the case when endophilin is lacking : the number of autophagosomes is sharply decreased. also, endophilin colocalizes transiently with mature and even more impressively with early autophagosomes, suggesting that endophilin - a has a previously unreported role in the formation and/or maturation of autophagosomes. this is supported by the recent identification of endophilin - a as one of the regulators of autophagosome formation (strohecker., 2015). interestingly, mice without endophilin also display the same pattern of perinatal lethality observed in mice lacking proteins necessary for autophagosome formation (kuma., 2004). given endophilin s involvement in endocytic processes, the decrease in the number of autophagosomes might also result from compromised endocytosis. specifically, it has been shown that blockage of endocytosis upstream of clathrin coat assembly results in decreased autophagosome formation (ravikumar.. however, endophilin deficiency results in the accumulation of clathrin - coated vesicles, and the levels of clathrin are not altered (milosevic., 2011). thus, clathrin is available to interact with atg16l1, and rna and protein levels of atg16l1 are not altered in this model. in addition, the rna and protein levels of atg9, a key autophagic protein known to traffic via the plasma membrane and endosomal compartments (mari., 2010, puri., 2014) are not altered in endophilin mutant brains. altogether, whereas we can not entirely exclude endophilin s role in endocytosis also affecting autophagy, we consider other mechanism(s) besides endocytic defects to be underlying the decrease in autophagosomes in endophilin mutants. decreased autophagosome availability and, consequently, lower autophagic flux are known to affect protein homeostasis (korolchuk., 2009). we observed an accumulation of k48-linked ubiquitinated proteins in the cytoplasm, suggesting saturation of the ups (due to overloading rather than deficiencies in the proteasome). there is possibly a double hit on this pathway : decreased autophagy and increased availability of ubiquitin ligases (fbxo32 and parkin). both fbxo32 and parkin are involved in selective autophagy of ubiquitinated proteins (zaglia., 2014), and both function under the regulation of foxos (webb and brunet, 2014). upregulation of fbxo32 and parkin may thus be a compensatory response to defective autophagy, an attempt to shunt more of the burden to the ups, or possibly both. interestingly, the lower abundance of autophagosomes in the absence of endophilin - a can not be (fully) compensated for by a related protein with the same domain organization, endophilin - b, an established mediator of autophagosome formation (takahashi., 2011). endophilin - a primarily operates in the endocytic processes at the plasma membrane, whereas endophilin - b has prominent roles in apoptosis, mitochondrial membrane dynamics, and autophagosome formation (kjaerulff., 2011). furthermore, cdk5-mediated phosphorylation of endophilin - b is required for inducing autophagy in models of pd (wong., 2011). more research is needed to understand endophilin - a and endophilin - b links to autophagy, neurodegeneration, and pd. we propose a model in which endophilin s absence, likely due to its membrane - curvature - inducing / stabilizing properties, leads to reduced availability of membranes for autophagosome formation and decreased autophagic activity. interestingly, fbxo32 and endophilin interact at both the gene and protein level and both are required for the autophagy process. like endophilin, fbxo32 can tubulate membranes and is recruited to membrane tubules in vivo. thus, endophilin and fbxo32 membrane - tubulating activity may contribute to the formation / maturation of autophagosomes. this model is in concordance with the reported role of endophilin - a as necessary and sufficient for macroautophagy at presynaptic terminals of fruit flies (soukup. in summary, our study addresses the origin of neurodegeneration in a key mammalian endocytic model and reveals that the foxo / fbxo32/endophilin network may play a role in maintaining neuronal health by balancing autophagy and the ups in the brain. the results are consistent with the following plausible scenario : reduced endophilin availability results in impaired capacity to form autophagosomes, resulting in decreased autophagy, accumulation of ubiquitinated proteins in the cytoplasm, proteasome saturation, and further accumulation of unwanted proteins over time that eventually affect neuronal function and health, leading to neurodegeneration. accordingly, we show that imbalanced autophagy also develops in aged endophilin heterozygote mice that display age - dependent progressive ataxia. our data reveal a role in autophagy for a key endocytic adaptor previously thought to be strictly dedicated to endocytosis and invite further research addressing the physiological and pathological implications of these findings. supplemental experimental procedures detail gene - targeting and cloning strategies and list the antibodies and plasmids used. animal experiments were conducted according to the european guidelines for animal welfare (2010/63/eu) with approval by the lower saxony landesamt fr verbraucherschutz und lebensmittelsicherheit (laves), registration number 14/1701. for rotarod experiments, 620 males / group were tested on a setup by tse systems (3375 - 5). a training phase consisted of four trainings over 2 days (intertrial interval [iti ] was 25 hr ; 180 s at 5 rpm). the testing phase consisted of four tests over 2 days (iti 25 hr). mice were placed on the rod, which then accelerated from 5 to 40 rpm in 180 s. fall latency was measured. a simple composite phenotype scoring system for evaluating mouse models of ataxia was applied as described (guyenet., eight to twelve mice were used per condition, and scoring was performed by two researchers independently. ventral plane videography for gait analysis was performed using instrumentation (digigait) and software from mouse specifics, according to manufacturer s instructions. for each group, tunel assay was performed on cryosections of the brains of perfused mice to test for apoptotic and necrotic cells. mice were anesthetized with chloral hydrate (40 mg / ml) before cardiac perfusion with 4% paraformaldehyde (pfa). after slow dehydration with sucrose, brains were embedded in oct (tissue tek) and frozen in liquid n2. sagittal brain sections were cut with a cryostat (10 m thick). sections were post - fixed and permeabilized with 0.1% in situ cell death detection kit (fluorescein) was used according to manufacturer s instructions. slides were counterstained with dapi, embedded with mowiol 4 - 88, and imaged with a lsm 710 confocal setup (zeiss). immunofluorescence of frozen brain sections and cultured neurons (div 1420) was carried out as described (ringstad., 2001, milosevic., 2011 live mefs, hela cells, and neurons were imaged either using a zeiss axioobserver z1 total internal reflection fluorescence (tirf) microscope with an evolve charge - coupled device (ccd) camera (photometrics) and 100 plan apo zeiss objective or perkin elmer ultraview spinning - disk confocal setup that consists of nikon ti - e eclipse inverted microscope equipped with perfect focus, 60 cfi plan apo vc nikon objective, and 14-bit electron - multiplied ccd camera (hamamatsu c9100). autophagy induction and colocalization analysis was performed as detailed in the supplemental experimental procedures and below. whole - brain or cortex samples for western blotting were prepared by homogenization of brain tissue (whole brain for p0 animals ; one cortex for p14p21 animals and aged animals) as detailed in supplemental experimental procedures. for trap - gfp immunoprecipitation, hela cells were transfected with endophilin (1, 2, or 3)-egfp and mcherry / fbxo32-mcherry using lipofectamine 3000 (invitrogen). we used chromotek trap - gfp agarose beads (chromotek) and followed manufacturer s protocol. alternatively, hela cells were transfected with fbxo32-egfp and coupled to chromotek trap - gfp agarose beads. purified endophilin-1 was added, and all fractions (input, non - bound, and bound) were collected. primary hippocampal and cortical cultures were prepared as previously described (ferguson., 2007). primary neurons expressed constructs, following amaxa (lonza)-based transfection, under the control of the chicken--actin promoter to allow for long - term expression. a minimum of 15 images from three to five experiments were analyzed for each genotype / condition. given that the majority of respective fluorescent signals were cytosolic, colocalization was evaluated semi - automatically using object - based overlap analysis and imagej jacop plugin (bolte and cordelires, 2006). in short, the image was first segmented into objects and background (i.e., bright fluorescent objects are segmented from the image), and then their spatial relationship (overlap) was measured. the same analysis was performed with the mirror image, and random colocalization was subtracted in each case., circles were superimposed on bright fluorescent spots in the fbxo32 (or lc3 or atg5) channel and transferred to identical image locations in the endophilin channel. if the fluorescence intensity maximum in the endophilin channel was located in the same circle and the morphology of the signal resembled that of the fbxo32 signal, the circle was rated as positive (colocalized) ; if not, it was rated as negative (not colocalized). to correct for accidental colocalization, circles were also transferred to a mirror image of the endophilin channel (detailed in supplemental experimental procedures). we used wt and endophilin tko mefs, immortalized with e6/e7 viral proteins, grown on supplemented dmem at 5% co2 at 37c. cells were pelleted and resuspended with n - dodecylmaltoside 1.5% in pbs with complete protease inhibitor cocktail (roche) and phosstop phosphatase inhibitor cocktail (roche) and lysed at 4c in a rotator for 30 min. the lysate was then centrifuged to pellet debris and placed in 96-well glass - bottomed plates for use in the assay. the fluorimetric proteasome assay (proteasome 20s activity assay kit) was performed according to manufacturer s instructions using a synergy h1 plate reader (biotek) at 37c. caspase 3/7 apoptosis assay was performed with the apotox - glo triplex assay (promega) kit according to a modified version of the manufacturer s protocol for 96-well plates, detailed in supplemental experimental procedures. figures 2e [with n.r. ], 3a, 3b, 3e, and 6a6c ; c.m.r. : figures 1 [with i.m. ], 3c, 3f, 4a4c, 6d, 6 g, and 6h) ; supervision, p.v.	summaryendophilin - a, a well - characterized endocytic adaptor essential for synaptic vesicle recycling, has recently been linked to neurodegeneration. we report here that endophilin - a deficiency results in impaired movement, age - dependent ataxia, and neurodegeneration in mice. transcriptional analysis of endophilin - a mutant mice, complemented by proteomics, highlighted ataxia- and protein - homeostasis - related genes and revealed upregulation of the e3-ubiquitin ligase fbxo32/atrogin-1 and its transcription factor foxo3a. fbxo32 overexpression triggers apoptosis in cultured cells and neurons but, remarkably, coexpression of endophilin - a rescues it. fbxo32 interacts with all three endophilin - a proteins. similarly to endophilin - a, fbxo32 tubulates membranes and localizes on clathrin - coated structures. additionally, fbxo32 and endophilin - a are necessary for autophagosome formation, and both colocalize transiently with autophagosomes. our results point to a role for endophilin - a proteins in autophagy and protein degradation, processes that are impaired in their absence, potentially contributing to neurodegeneration and ataxia.
fasting in ramadan, the ninth month of islamic lunar hijri calendar, is practiced safely by the majority of muslims with type 2 diabetes (t2 dm) across the globe. the common practice is to consume one large meal after sunset and one lighter meal before dawn. in many cases, sedentary lifestyle and change in sleep pattern during ramadan may alter energy metabolism in people with t2 dm. the epidemiology of diabetes and ramadan (epidiar) study showed that in some people with t2 dm, during ramadan fasting, the risk of severe hypoglycemia (defined as hospitalization due to hypoglycemia) increased 7.5-fold (from 0.4 to 3 events/100 people / month). the study also revealed that excessive reduction in dosage of insulin to prevent hypoglycemia, and dietary indiscretion practised by some patients during non - fasting periods, lead to postprandial glycemic excursion / severe hyperglycemia (before ramadan 0.01 0.05 episodes, during ramadan 0.05 0.35 episodes per patient per month, p 300 mg / dl or 16.6 mmol / l), patients should break the fast if they are symptomatic or if the ketosis is present or if the hyperglycemia is due to reasons other than a heavy meal. otherwise, patients should be advised to repeat bg testing after 1 to 2 hrs and break the fast if hyperglycemia persists or worsens. blood glucose monitoring (bgm) : it is essential for patients to have the ability to monitor their blood glucose levels multiple times daily. it must be emphasized to patients that testing via bgm does not break the fast. indeed, insulin dose titration and dosage adjustment should be based on pre - meal blood glucose levels. the frequency of bgm for people on low - ratio premix insulin is dependent on the risk of hypoglycemia and their level of glycemic control : high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. whenever symptoms of hypoglycemia occur a midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur whenever symptoms of hypoglycemia occur diet : the diet during ramadan should be a healthy balanced diet. prayers should be considered as part of the daily exercise regimen because they involve standing, bowing, prostrating, and sitting. breaking the fast : the standard advice as per the ada studies have shown that many people continue to fast even after they experience a hypoglycemic episode. patients should be educated on breaking the fast if they experience any hypoglycemic episode. in case of hyperglycemia (bg > 300 mg / dl or 16.6 mmol / l), patients should break the fast if they are symptomatic or if the ketosis is present or if the hyperglycemia is due to reasons other than a heavy meal. otherwise, patients should be advised to repeat bg testing after 1 to 2 hrs and break the fast if hyperglycemia persists or worsens. trial fast in pre - ramadan period a trial fast for 3 consecutive days before ramadan should be advised. a trial fast mimicking ramadan can be helpful in detecting hypoglycemia risk and creates the possibility for guiding the self - titration of premix insulin dosage, using a titration algorithm. though it may give an insight into the patient 's response to premix insulin during ramadan, findings should be carefully extrapolated to the ramadan fasting, because of the difference in duration of fast. therefore, these fasts could be used as a trial prior to the ramadan fast. the management plan during ramadan should be individualized. the dosage of premix insulin during ramadan will depend on the meal size as well as composition, post meal fasting period and according to individual blood glucose targets. although, the majority of patients are on conventional twice daily premix insulin regimen, few patients on premix insulin analogues may be using once daily dosing usually before supper. studies have looked into the preference between analogue and human premix insulin during the fasting month. (2009), in 152 patients showed that analogue mix 30 significantly reduced all glycemic indices, i.e. fasting plasma glucose (fpg), 2-hour postprandial plasma glucose (2-hr ppg), and glycosylated hemoglobin (hba1c). there were no significant changes in body weight and body mass index (bmi), with significant reduction in hypoglycemic episodes in patients after three months of treatment. similarly, another study which compared human insulin 30/70 with analogue mix 25 during ramadan, found that premix analogue resulted in better average daily glycemia and blood glucose control. this favorable outcome in ramadan fasting could be related to the pharmacodynamics of low - ratio premix analogues compared to human insulin, as well as to the meal time flexibility where unlike for human insulin a person, after a long fasting day can eat immediately after analogue insulin injection. the following points are considered as general practical advice : if the patient is on once daily premix insulin in combination with oral glucose lowering drugs (oglds) in the pre - ramadan period, then same dosage should be given at the sunset meal (iftar) and can be titrated further as per the algorithm listed below [table 1 ]. the dose of oglds should be optimized as per the standard recommendations of ada. table 1algorithm for self - titration of premix insulin during ramadan if the patient on once daily premix insulin is uncontrolled, then dosage should be uptitrated as per the algorithm given below [table 1 ]. if after titration, the pre - meal / fasting blood glucose is not controlled, advise the patient to break the fast and start premix insulin twice daily.if, during the pre - ramadan period, the patient is on twice daily premix insulin then prescribe the usual morning dose at the sunset meal (iftar) and half the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 10 units at pre-suhur.if the patient on twice daily premix insulin is uncontrolled and has before dinner blood glucose > 300 mg / dl (16.6 mmol / l) in the pre - ramadan period, then prescribe the usual morning dose at the sunset meal (iftar) and the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 20 units at pre-suhur.if the patient is on thrice daily insulin, omit the afternoon dose and titrate the pre - iftar and pre - suhur dose as described above for twice daily premix regimen.dosage adjustments should be done based on home bgm data.the preference of premix insulin analogue instead of human insulin can be considered when immediate injection before a meal is preferred orfrequent hypoglycemia is a concern orthere are marked postprandial blood glucose excursions. while switching from human premix to analogue premix insulin, the dose of analogue insulin at pre - iftar should be 20 to 30% lower than the morning human insulin dose pre - ramadan. pre - suhur dose should be 40% lower than the evening dose pre - ramadan. if the patient is on once daily premix insulin in combination with oral glucose lowering drugs (oglds) in the pre - ramadan period, then same dosage should be given at the sunset meal (iftar) and can be titrated further as per the algorithm listed below [table 1 ]. the dose of oglds should be optimized as per the standard recommendations of ada. table 1algorithm for self - titration of premix insulin during ramadan algorithm for self - titration of premix insulin during ramadan if the patient on once daily premix insulin is uncontrolled, then dosage should be uptitrated as per the algorithm given below [table 1 ]. if after titration, the pre - meal / fasting blood glucose is not controlled, advise the patient to break the fast and start premix insulin twice daily. if, during the pre - ramadan period, the patient is on twice daily premix insulin then prescribe the usual morning dose at the sunset meal (iftar) and half the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 10 units at pre - suhur. if the patient on twice daily premix insulin is uncontrolled and has before dinner blood glucose > 300 mg / dl (16.6 mmol / l) in the pre - ramadan period, then prescribe the usual morning dose at the sunset meal (iftar) and the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 20 units at pre - suhur. if the patient is on thrice daily insulin, omit the afternoon dose and titrate the pre - iftar and pre - suhur dose as described above for twice daily premix regimen. the preference of premix insulin analogue instead of human insulin can be considered when immediate injection before a meal is preferred orfrequent hypoglycemia is a concern orthere are marked postprandial blood glucose excursions. immediate injection before a meal is preferred or frequent hypoglycemia is a concern or there are marked postprandial blood glucose excursions. while switching from human premix to analogue premix insulin, the dose of analogue insulin at pre - iftar should be 20 to 30% lower than the morning human insulin dose pre - ramadan. pre - suhur dose should be 40% lower than the evening dose pre - ramadan. it is advisable to titrate insulin dose every 3 days.the lowest of the three readings on three consecutive days should be considered to up titrate the insulin dose. the timing of bgm is described in the algorithm [table 1].hypoglycemia is defined as blood sugar below 70 gm / dl (3.9 mmol / l) or symptoms of hypoglycemia.if hypoglycemia is noted on two (any time of the day) out of three consecutive days, fasting must be stopped and insulin dose should be reduced as described in the algorithm [table 1 ]. fasting should be stopped even if the hypoglycemia occurs close to the time of iftar [figure 1 ]. figure 1avoiding hypoglycemic episodes while on premix insulin during ramadan if blood glucose is > 300 mg / dl or 16.6 mmol / l, ketones in blood or urine should be checked and the patient should break the fast [figure 1].patients should avoid fasting on sick days. the lowest of the three readings on three consecutive days should be considered to up titrate the insulin dose. hypoglycemia is defined as blood sugar below 70 gm / dl (3.9 mmol / l) or symptoms of hypoglycemia. if hypoglycemia is noted on two (any time of the day) out of three consecutive days, fasting must be stopped and insulin dose should be reduced as described in the algorithm [table 1 ]. fasting should be stopped even if the hypoglycemia occurs close to the time of iftar [figure 1 ]. figure 1avoiding hypoglycemic episodes while on premix insulin during ramadan avoiding hypoglycemic episodes while on premix insulin during ramadan if blood glucose is > 300 mg / dl or 16.6 mmol / l, ketones in blood or urine should be checked and the patient should break the fast [figure 1 ]. in patients taking low - ratio premix insulin who intend fasting, the following recommendations should be considered : a pre - ramadan preparation period of at least 23 months is advisable in order to ensure safe fasting. this will include : pre - ramadan individualized medical assessment the working group advises observance of the current ada recommendations with specific attention to patients overall well - being and the importance of good glycemic control, blood pressure, and lipid management. the understanding of patients experiences of fasting, amendments of medications, extent of success, and problems encountered during a previous ramadan is of paramount importance. special attention should be devoted to an individualized risk assessment of patient with t2 dm on low - ratio premix insulin who wishes to fast during ramadan. structured education for patients on low - ratio premix insulin focus should be placed on factors like frequency of glucose monitoring during fasting and non - fasting hours, meal planning, meal choices, and physical activity to avoid hypoglycemia as well as post - prandial hyperglycemia. it is prudent to impart education both to the patient and his / her family. all means of communication of the relevant information could be used, including the media, the internet and written information as well as individual and group counselling by diabetes educators and physicians. the structured education will include the advice on the importance of home blood glucose monitoring, when to break the fast, meal, and exercise planning. blood glucose monitoring (bgm) : it is essential for patients to have the ability to monitor their blood glucose levels multiple times daily. it must be emphasized to patients that testing via bgm does not break the fast. indeed, insulin dose titration and dosage adjustment should be based on pre - meal blood glucose levels. the frequency of bgm for people on low - ratio premix insulin is dependent on the risk of hypoglycemia and their level of glycemic control : high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur diet : the diet during ramadan should be a healthy balanced diet. the fluid choice should be water or low - calorie drinks.exercise : regular light and moderate exercise is advisable. prayers should be considered as part of the daily exercise regimen because they involve standing, bowing, prostrating, and sitting.breaking the fast : the standard advice as per the ada studies have shown that many people continue to fast even after they experience a hypoglycemic episode. patients should be educated on breaking the fast if they experience any hypoglycemic episode. in case of hyperglycemia (bg > 300 mg / dl or 16.6 mmol / l), patients should break the fast if they are symptomatic or if the ketosis is present or if the hyperglycemia is due to reasons other than a heavy meal. otherwise, patients should be advised to repeat bg testing after 1 to 2 hrs and break the fast if hyperglycemia persists or worsens. blood glucose monitoring (bgm) : it is essential for patients to have the ability to monitor their blood glucose levels multiple times daily. it must be emphasized to patients that testing via bgm does not break the fast. indeed, insulin dose titration and dosage adjustment should be based on pre - meal blood glucose levels. the frequency of bgm for people on low - ratio premix insulin is dependent on the risk of hypoglycemia and their level of glycemic control : high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. whenever symptoms of hypoglycemia occur a midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur whenever symptoms of hypoglycemia occur diet : the diet during ramadan should be a healthy balanced diet. prayers should be considered as part of the daily exercise regimen because they involve standing, bowing, prostrating, and sitting. breaking the fast : the standard advice as per the ada studies have shown that many people continue to fast even after they experience a hypoglycemic episode. patients should be educated on breaking the fast if they experience any hypoglycemic episode. in case of hyperglycemia (bg > 300 mg / dl or 16.6 mmol / l), patients should break the fast if they are symptomatic or if the ketosis is present or if the hyperglycemia is due to reasons other than a heavy meal. otherwise, patients should be advised to repeat bg testing after 1 to 2 hrs and break the fast if hyperglycemia persists or worsens. trial fast in pre - ramadan period a trial fast for 3 consecutive days before ramadan should be advised. a trial fast mimicking ramadan can be helpful in detecting hypoglycemia risk and creates the possibility for guiding the self - titration of premix insulin dosage, using a titration algorithm. though it may give an insight into the patient 's response to premix insulin during ramadan, findings should be carefully extrapolated to the ramadan fasting, because of the difference in duration of fast. therefore, these fasts could be used as a trial prior to the ramadan fast. a pre - ramadan preparation period of at least 23 months is advisable in order to ensure safe fasting. this will include : pre - ramadan individualized medical assessment the working group advises observance of the current ada recommendations with specific attention to patients overall well - being and the importance of good glycemic control, blood pressure, and lipid management. the understanding of patients experiences of fasting, amendments of medications, extent of success, and problems encountered during a previous ramadan is of paramount importance. special attention should be devoted to an individualized risk assessment of patient with t2 dm on low - ratio premix insulin who wishes to fast during ramadan. structured education for patients on low - ratio premix insulin focus should be placed on factors like frequency of glucose monitoring during fasting and non - fasting hours, meal planning, meal choices, and physical activity to avoid hypoglycemia as well as post - prandial hyperglycemia. it is prudent to impart education both to the patient and his / her family. all means of communication of the relevant information could be used, including the media, the internet and written information as well as individual and group counselling by diabetes educators and physicians. the structured education will include the advice on the importance of home blood glucose monitoring, when to break the fast, meal, and exercise planning. blood glucose monitoring (bgm) : it is essential for patients to have the ability to monitor their blood glucose levels multiple times daily. it must be emphasized to patients that testing via bgm does not break the fast. indeed, insulin dose titration and dosage adjustment should be based on pre - meal blood glucose levels. the frequency of bgm for people on low - ratio premix insulin is dependent on the risk of hypoglycemia and their level of glycemic control : high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur diet : the diet during ramadan should be a healthy balanced diet. the fluid choice should be water or low - calorie drinks.exercise : regular light and moderate exercise is advisable. prayers should be considered as part of the daily exercise regimen because they involve standing, bowing, prostrating, and sitting.breaking the fast : the standard advice as per the ada studies have shown that many people continue to fast even after they experience a hypoglycemic episode. patients should be educated on breaking the fast if they experience any hypoglycemic episode. in case of hyperglycemia (bg > 300 mg / dl or 16.6 mmol / l), patients should break the fast if they are symptomatic or if the ketosis is present or if the hyperglycemia is due to reasons other than a heavy meal. otherwise, patients should be advised to repeat bg testing after 1 to 2 hrs and break the fast if hyperglycemia persists or worsens. blood glucose monitoring (bgm) : it is essential for patients to have the ability to monitor their blood glucose levels multiple times daily. it must be emphasized to patients that testing via bgm does not break the fast. indeed, insulin dose titration and dosage adjustment should be based on pre - meal blood glucose levels. the frequency of bgm for people on low - ratio premix insulin is dependent on the risk of hypoglycemia and their level of glycemic control : high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur high risk group : this includes people with higher risk of hypoglycemia and/or poor glycemic control. whilst these patients should be advised against fasting during ramadan, bgm should be done at the following times should they insist on fasting : pre - suhur2 hours post - suhurmiddaypre - iftar2 hours post - iftarwhenever symptoms of hypoglycemia occura midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. whenever symptoms of hypoglycemia occur a midnight blood glucose measurement could also be considered for many people with t2 dm which may help in optimising premix insulin dose. low risk group : it is advisable to do bgm at the following times during a day pre - suhurmiddaypre - iftarwhenever symptoms of hypoglycemia occur whenever symptoms of hypoglycemia occur diet : the diet during ramadan should be a healthy balanced diet. prayers should be considered as part of the daily exercise regimen because they involve standing, bowing, prostrating, and sitting. studies have shown that many people continue to fast even after they experience a hypoglycemic episode. patients should be educated on breaking the fast if they experience any hypoglycemic episode. in case of hyperglycemia (bg > 300 mg / dl or 16.6 mmol / l), patients should break the fast if they are symptomatic or if the ketosis is present or if the hyperglycemia is due to reasons other than a heavy meal. otherwise, patients should be advised to repeat bg testing after 1 to 2 hrs and break the fast if hyperglycemia persists or worsens. trial fast in pre - ramadan period a trial fast for 3 consecutive days before ramadan should be advised. a trial fast mimicking ramadan can be helpful in detecting hypoglycemia risk and creates the possibility for guiding the self - titration of premix insulin dosage, using a titration algorithm. though it may give an insight into the patient 's response to premix insulin during ramadan, findings should be carefully extrapolated to the ramadan fasting, because of the difference in duration of fast. therefore, these fasts could be used as a trial prior to the ramadan fast. the dosage of premix insulin during ramadan will depend on the meal size as well as composition, post meal fasting period and according to individual blood glucose targets. although, the majority of patients are on conventional twice daily premix insulin regimen, few patients on premix insulin analogues may be using once daily dosing usually before supper. studies have looked into the preference between analogue and human premix insulin during the fasting month. (2009), in 152 patients showed that analogue mix 30 significantly reduced all glycemic indices, i.e. fasting plasma glucose (fpg), 2-hour postprandial plasma glucose (2-hr ppg), and glycosylated hemoglobin (hba1c). there were no significant changes in body weight and body mass index (bmi), with significant reduction in hypoglycemic episodes in patients after three months of treatment. similarly, another study which compared human insulin 30/70 with analogue mix 25 during ramadan, found that premix analogue resulted in better average daily glycemia and blood glucose control. this favorable outcome in ramadan fasting could be related to the pharmacodynamics of low - ratio premix analogues compared to human insulin, as well as to the meal time flexibility where unlike for human insulin a person, after a long fasting day can eat immediately after analogue insulin injection. the following points are considered as general practical advice : if the patient is on once daily premix insulin in combination with oral glucose lowering drugs (oglds) in the pre - ramadan period, then same dosage should be given at the sunset meal (iftar) and can be titrated further as per the algorithm listed below [table 1 ]. the dose of oglds should be optimized as per the standard recommendations of ada. table 1algorithm for self - titration of premix insulin during ramadan if the patient on once daily premix insulin is uncontrolled, then dosage should be uptitrated as per the algorithm given below [table 1 ]. if after titration, the pre - meal / fasting blood glucose is not controlled, advise the patient to break the fast and start premix insulin twice daily.if, during the pre - ramadan period, the patient is on twice daily premix insulin then prescribe the usual morning dose at the sunset meal (iftar) and half the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 10 units at pre-suhur.if the patient on twice daily premix insulin is uncontrolled and has before dinner blood glucose > 300 mg / dl (16.6 mmol / l) in the pre - ramadan period, then prescribe the usual morning dose at the sunset meal (iftar) and the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 20 units at pre-suhur.if the patient is on thrice daily insulin, omit the afternoon dose and titrate the pre - iftar and pre - suhur dose as described above for twice daily premix regimen.dosage adjustments should be done based on home bgm data.the preference of premix insulin analogue instead of human insulin can be considered when immediate injection before a meal is preferred orfrequent hypoglycemia is a concern orthere are marked postprandial blood glucose excursions. while switching from human premix to analogue premix insulin, the dose of analogue insulin at pre - iftar should be 20 to 30% lower than the morning human insulin dose pre - ramadan. pre - suhur dose should be 40% lower than the evening dose pre - ramadan. if the patient is on once daily premix insulin in combination with oral glucose lowering drugs (oglds) in the pre - ramadan period, then same dosage should be given at the sunset meal (iftar) and can be titrated further as per the algorithm listed below [table 1 ]. the dose of oglds should be optimized as per the standard recommendations of ada. table 1algorithm for self - titration of premix insulin during ramadan algorithm for self - titration of premix insulin during ramadan if the patient on once daily premix insulin is uncontrolled, then dosage should be uptitrated as per the algorithm given below [table 1 ]. if after titration, the pre - meal / fasting blood glucose is not controlled, advise the patient to break the fast and start premix insulin twice daily. if, during the pre - ramadan period, the patient is on twice daily premix insulin then prescribe the usual morning dose at the sunset meal (iftar) and half the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 10 units at pre - suhur. if the patient on twice daily premix insulin is uncontrolled and has before dinner blood glucose > 300 mg / dl (16.6 mmol / l) in the pre - ramadan period, then prescribe the usual morning dose at the sunset meal (iftar) and the usual evening dose at pre - dawn (suhur), e.g. if 30/70 premix insulin is prescribed 30 units in the morning and 20 units in the evening before ramadan, then the recommended dose during ramadan will be 30 units pre - iftar and 20 units at pre - suhur. if the patient is on thrice daily insulin, omit the afternoon dose and titrate the pre - iftar and pre - suhur dose as described above for twice daily premix regimen. the preference of premix insulin analogue instead of human insulin can be considered when immediate injection before a meal is preferred orfrequent hypoglycemia is a concern orthere are marked postprandial blood glucose excursions. immediate injection before a meal is preferred or frequent hypoglycemia is a concern or there are marked postprandial blood glucose excursions. while switching from human premix to analogue premix insulin, the dose of analogue insulin at pre - iftar should be 20 to 30% lower than the morning human insulin dose pre - ramadan. pre - suhur dose should be 40% lower than the evening dose pre - ramadan. it is advisable to titrate insulin dose every 3 days.the lowest of the three readings on three consecutive days should be considered to up titrate the insulin dose. the timing of bgm is described in the algorithm [table 1].hypoglycemia is defined as blood sugar below 70 gm / dl (3.9 mmol / l) or symptoms of hypoglycemia.if hypoglycemia is noted on two (any time of the day) out of three consecutive days, fasting must be stopped and insulin dose should be reduced as described in the algorithm [table 1 ]. fasting should be stopped even if the hypoglycemia occurs close to the time of iftar [figure 1 ]. figure 1avoiding hypoglycemic episodes while on premix insulin during ramadan if blood glucose is > 300 mg / dl or 16.6 mmol / l, ketones in blood or urine should be checked and the patient should break the fast [figure 1].patients should avoid fasting on sick days. the lowest of the three readings on three consecutive days should be considered to up titrate the insulin dose. hypoglycemia is defined as blood sugar below 70 gm / dl (3.9 mmol / l) or symptoms of hypoglycemia. if hypoglycemia is noted on two (any time of the day) out of three consecutive days, fasting must be stopped and insulin dose should be reduced as described in the algorithm [table 1 ]. fasting should be stopped even if the hypoglycemia occurs close to the time of iftar [figure 1 ]. figure 1avoiding hypoglycemic episodes while on premix insulin during ramadan avoiding hypoglycemic episodes while on premix insulin during ramadan if blood glucose is > 300 mg / dl or 16.6 mmol / l, ketones in blood or urine should be checked and the patient should break the fast [figure 1 ]. this document provides practical guidance for usage of low - ratio premix insulin during ramadan (summarized in figure 2). patients on low - ratio premix insulin who insist on fasting should be assessed before ramadan and educated about physical activity, meal planning, blood glucose monitoring, dosage and timing of medications. this document provides a simple titration algorithm which will help patients to perform home bgm and self - titration. moreover, continued efforts are needed towards formulation of evidence based guideline for the use of low - ratio premix insulin during ramadan. while this document is based on the use of low - ratio premix insulin, higher ratios (e.g. premix 50/50) may be preferable during fasting, since the higher quick - acting component will cover the suhur meal and the lower intermediate - acting component (compared to the 70% or 75% low - ratio premix) will minimize the risk of afternoon hypoglycemia.	the challenge of insulin use during ramadan could be minimized, if people with diabetes are metabolically stable and are provided with structured education for at least 23 months pre - ramadan. although, american diabetes association (ada) recommendations 2010 and south asian consensus guideline 2012 deal with management of diabetes in ramadan and changes in insulin dosage, no specific guidance on widely prescribed low - ratio premix insulin is currently available. hence, the working group for insulin therapy in ramadan, after collective analysis, evaluation, and opinion from clinical practice, have formulated a practical advice to empower physicians with pre - ramadan preparation, dose adjustment, and treatment algorithm for self - titration of low - ratio premix insulin.
pancreatoblastoma, though a rare childhood tumor, is the most common malignant pancreatic tumor in children. clinicoradiological and histopathological resemblance to hepatoblastoma can make its diagnosis and management challenging. to the best of our knowledge, there have been no reports of a pancreatoblastoma diagnosed and initially treated as hepatoblastoma. we hereby report a 4-year - old boy with pancreatoblastoma masquerading as hepatoblastoma and the treatment outcome. a 4-year - old boy presented with complaints of abdominal fullness and low - grade fever for the duration of 1 month. on examination a 12 cm 10 cm, firm, nontender mass was present in the right hypochondrium in continuation with the liver. ultrasonography suggested a necrotic mass in the inferior surface of the right lobe of the liver. computed tomography (ct) scan of the abdomen revealed an 8.5 cm 8.3 cm 7.2 cm nonenhancing necrotic mass in segment v of the liver, closely abutting the inferior vena cava, portal vein, and superior mesenteric vein [figure 1 : ct scan showing a well - defined mass with necrotic, nonenhancing areas within arising exophytically from segment v of the liver. laboratory investigations revealed hypochromic microcytic anemia, normal liver function test, lactate dehydrogenase (ldh) of 330 u / l, and serum alpha - fetoprotein (afp) was 3,971 ng / ml. a diagnosis of nonmetastatic pretreatment tumor extension (pretext) i hepatoblastoma was made and the patient was stratified to standard risk (sr) as per the international childhood liver tumours strategy group (siopel) system. computed tomography scan showing a well - defined mass with necrotic, nonenhancing areas within arising exophytically from segment v of the liver. the lesion displaces the portal and superior mesenteric vein (arrows) the patient received two cycles of single agent cisplatin as per the siopel 3 sr protocol. although after the first cycle of chemotherapy the serum afp level dropped to 3217 ng / ml, it increased to 4,260 ng / ml after the second cycle. in view of poor biochemical response, the chemotherapy was changed to cisplatin and doxorubicin (plado). at the end of two cycles of plado, the serum afp level was 2,170 ng / ml ; however, there was negligible change in the mass on ct scan. a lobulated mass was found arising from the head of the pancreas with flimsy adhesions with the liver and ascending colon [figure 2a : intraoperative image showing the mass arising from the head of pancreas. intraoperatively a decision for pancreaticoduodenectomy (whipple 's procedure) was taken and a complete resection was accomplished. the postoperative course was uneventful and the patient was discharged on the sixth postoperative day. (a) intraoperative image showing the mass arising from the head of the pancreas. duodenum is seen draping the mass (b) cut - open specimen showing the cystic and lobulated solid areas (arrow : gallbladder, arrowhead : proximal jejunum (c) pancreatoblastoma composed of sheets and nests of small, round, uniform cells. haematoxylin and eosin staining, 20 on gross examination, a solid globular tumor measuring 10 cm 8.2 cm 6 cm was present in the head of the pancreas with submucosal extension into the duodenum. the cut surface was cystic with lobulated gray white areas [figure 2b : the cut - open specimen showing the cystic and lobulated solid areas (arrow : gallbladder, arrowhead : proximal jejunum ]. microscopically, the tumor was predominantly composed of nests of uniform round cells separated by fibrovascular stroma. [figure 2c : pancreatoblastoma composed of sheets and nests of small, round, uniform cells. were conspicuous acinar and focal glandular structures ; however, typical squamous morules were absent. based on the morphology a diagnosis of pancreatoblastoma was made. the focal parts of the tumor showed hepatoid morphology, which seemed a variation of the cell type within the tumor. on immunohistochemistry, the tumor showed positivity for ck7 and ck19 highlighted interspersed ductal elements. chromogranin was focally positive while synaptophysin, afp, and beta - catenin were negative. the patient has been on regular follow - up and remains disease - free 4 years after completion of therapy. pancreatoblastoma is a rare childhood tumor but the most common malignant pancreatic tumor in young children with the median age of diagnosis being 5 years. it originates from the epithelial exocrine cells of the pancreas and can develop in any part of the pancreas but commonly affects the head. pancreatoblastoma may be diagnosed incidentally with the child being asymptomatic. however, being a slow - growing tumor, pancreatoblastomas are often clinically occult until quite large, when symptoms due to the mass effect in the upper abdomen occur. in the present case, the child was investigated for abdominal fullness, which revealed a necrotic mass probably arising from the inferior surface of the right lobe of the liver on imaging. the large size of the tumor posed a diagnostic difficulty in determining the organ of origin on ct scan, which mimicked an exophytic growth from segment v of the liver. pancreatoblastomas show resemblance with hepatoblastoma in many characteristics ; they arise from the same primitive cells and can therefore, have increased serum afp levels. histologically pancreatoblastoma consists of small round cells, which together with an elevated afp and the close spatial relation with the liver may pose a challenge in differentiating with hepatoblastoma. surgical resection is the mainstay of treatment, which may entail a whipple 's procedure or a distal pancreatectomy with or without splenectomy, depending on the location of tumor. long - term survival is possible with complete resection of the tumor though pancreatoblastoma has a high recurrence rate. the role of chemotherapy is not precise ; however, it has been used for unresectable or metastatic disease and cisplatin, carboplatin, cyclophosphamide, doxorubicin, and etoposide - based chemotherapy regimens have been found to be effective. the italian tumori rari in eta pediatrica (the italian trep project) has provided guidelines for pancreatoblastoma. the recommended chemotherapy regimen is cisplatin at 80 mg / m as a continuous 24-h intravenous infusion followed by doxorubicin 60 mg / m over 48 h (plado) for four cycles for completely resected patients and six cycles for resection with microscopic residual or nodal involvement. for cases with initial biopsy only or metastatic disease, four cycles of preoperative, cisplatin, and doxorubicin are recommended followed by surgery and two further cycles of chemotherapy. however, it has been often utilized after incomplete resection, positive surgical margins, or tumor spillage. in conclusion, the difficulty in detecting the organ of origin, coupled with raised serum afp levels in pancreatoblastoma may make diagnosis challenging ; therefore, it is pertinent for the clinician to be aware of the differentials so that effective therapy can be delivered.	clinicoradiological and histopathological differentiation of pancreatoblastoma from hepatoblastoma can often be a challenge in clinical practice owing to their peculiar resemblance. we report a case of a 4-year - old boy with a right hypochondriac region mass, which was diagnosed as hepatoblastoma on the basis of imaging, raised tumor marker, and biopsy ; however, pancreatic origin of the mass was ascertained on exploration and pancreatoblastoma was confirmed on histopathology.
the mitochondrial aldehyde dehydrogenase 2 (aldh2) plays a key role in removal of not only ethanolderived acetaldehyde, but also other toxic aldehydes such as 4hydroxy2nonenal (4hne) derived from lipid peroxidation.1, 2 up to 40% of east asians carry a variant aldh2 or aldh2 2 (glu504lys) genotype with deficient activity and suffer from alcohol (ethanol) flushing syndrome (afs) including facial flushing, headache, nausea, and palpitation, in response to a small amount of alcohol intake attributed to accumulation of acetaldehyde.2, 3, 4, 5 recent studies indicate that carriers of aldh2 2 genotype have a higher risk for acute myocardial infarction (ami).6, 7, 8, 9, 10, 11 coronary spasm is not rarely demonstrated in ami patients in japan,12, 13 and we have reported that aldh2 2 genotype is associated with coronary spastic angina (csa).14 aldh2 protects against myocardial ischemia / reperfusion (i / r) injury by removing toxic aldehydes such as 4hne in experimental ami models.2, 15, 16, 17 however, the underlying mechanisms linking aldh2 to ami in humans remain to be elucidated. in the present study, we examined the clinical features of ami patients with aldh2 2 by comparing to those with wildtype aldh2 1/1 in acute stsegment elevation myocardial infarction (stemi) patients who underwent reperfusion therapy with primary percutaneous coronary intervention (pci). two hundred two patients (156 men and 46 women with a mean age of 67.312.0) who underwent primary pci with stent implantation successfully within 12 hours after symptoms and consented to be genotyped for aldh2 gene polymorphism were the subjects of this study. these patients were recruited from the consecutive 235 japanese patients admitted and diagnosed as stemi at our institutions between december 2009 and october 2015. not included in the study were 26 patients who died during hospitalization (11 with pci and 15 without pci attributed to delay of admission), 10 patients with unsuccessful pci (thrombolysis in myocardial infarction [timi ] grade 180/110 mm hg (n=6) acute systemic illness, and hepatic or renal insufficiency or other severe conditions (n=12) were excluded from the study. all vasoactive medications, including calciumchannel blockers, betareceptor blockers, angiotensinconverting enzyme inhibitors, angiotensin ii receptors blockers, and statins, were withdrawn for at least 3 days before angiography except for nitroglycerin used for attacks. coronary spasm was defined as a transient total or subtotal occlusion or severe diffuse vasoconstriction of an epicardial coronary artery associated with ischemic changes on ecg with or without chest discomfort. the final diagnosis was determined with the consensus of 3 investigators blinded to genotype / flushing phenotype. coronary spasm was induced by the intracoronary injection of acetylcholine (ach ; daiichisankyo co., tokyo, japan) after diagnostic catheterization in the morning.14 details of the provocation test were previously reported.13, 14 briefly, ach dissolved in 5 ml of warmed 0.9% saline was infused manually in 20 seconds into the left coronary artery (lca) in incremental doses of 10, 20, 50, and 100 g and then 10, 20, and 50 g into the right coronary artery (rca) depending on the vascular reactivity under continuous monitoring of 12lead ecg and blood pressure with a temporary pacemaker in place in the morning. coronary angiography was performed 1 minute after each ach injection or when ischemic ecg changes appeared. figure 1 shows a typical case of coronary spasm with stent implantation in the provocation test by ach 6 months postami. a representative case of coronary spasm induced by intracoronary ach infusion. severe diffuse spasm appeared associated with stsegment elevation on ecg postach infusion at the distal segments of stent implanted in the left anterior descending artery and disappeared after nitroglycerin. coronary spasm induced by this method usually disappeared spontaneously within 1 to 2 minutes and both the lca and rca could be examined separately, unless severe spasm occurred in the lca and necessitated the prompt injection of nitroglycerin (gtn) or isosorbide dinitrate (isdn) into the artery. the number of study patients needed was calculated based on an uncorrected chisquared statistic at the 2tailed test 5% significance level and 80% power, assuming the prevalence of coronary spasm to be 80% in the variant aldh2 2 mi patient group and 50% in the wild aldh2 1/1 mi patient group based on our previous work and others.12, 13, 14 subjects were asked to fill out a simple questionnaire concerning alcohol flushing on alcohol intake, alcohol drinking habit, and smoking. habitual drinker was defined as an alcohol drinker more than 5 days a week. alcohol flushing was defined as a current or a history of facial flushing immediately after drinking a glass of beer. blood samples for measurement of clinical chemistry and other data were collected on admission and repeatedly at least 3 to 4 times later with patients in the supine position. total creatine phosphokinase (cpk) was measured with a standard enzymatic method on a unicel dxc800 multi analyzer 2700 (beckman coulter clinical diagnostics, brea, ca). ctnt was assessed by an electrochemoluminescence immunoassay using the cobas system troponin t kit provided by the manufacturer on a cobas e 411 disk system analyzer (roche diagnostics, indianapolis, in). blood samples for total cpk and lactate dehydrogenase (ldh) were measured on admission and repeated every 4 hours during the first 12 hours and every 12 hours up to 72 hours. ctnt was measured on admission and repeated 3 to 6 hours later and further several times for 1 to 5 days depending on patient conditions. allele frequencies were determined by direct gene counting, and genotype distributions were checked for departure from hardyweinberg equilibrium using the pearson chisquare test. baseline clinical data were expressed as meansd or median (25th or 75th percentile) for continuous variables, and withingroup differences were evaluated with an unpaired t test or the mann data were expressed as counts and percentages and analyzed with the chisquare or fisher 's exact test. analyses were done using the stata software program (stata 11.0 ; statacorp, college station, tx). two hundred two patients (156 men and 46 women with a mean age of 67.312.0) who underwent primary pci with stent implantation successfully within 12 hours after symptoms and consented to be genotyped for aldh2 gene polymorphism were the subjects of this study. these patients were recruited from the consecutive 235 japanese patients admitted and diagnosed as stemi at our institutions between december 2009 and october 2015. not included in the study were 26 patients who died during hospitalization (11 with pci and 15 without pci attributed to delay of admission), 10 patients with unsuccessful pci (thrombolysis in myocardial infarction [timi ] grade 180/110 mm hg (n=6) acute systemic illness, and hepatic or renal insufficiency or other severe conditions (n=12) were excluded from the study. all vasoactive medications, including calciumchannel blockers, betareceptor blockers, angiotensinconverting enzyme inhibitors, angiotensin ii receptors blockers, and statins, were withdrawn for at least 3 days before angiography except for nitroglycerin used for attacks. coronary spasm was defined as a transient total or subtotal occlusion or severe diffuse vasoconstriction of an epicardial coronary artery associated with ischemic changes on ecg with or without chest discomfort. the final diagnosis was determined with the consensus of 3 investigators blinded to genotype / flushing phenotype. coronary spasm was induced by the intracoronary injection of acetylcholine (ach ; daiichisankyo co., tokyo, japan) after diagnostic catheterization in the morning.14 details of the provocation test were previously reported.13, 14 briefly, ach dissolved in 5 ml of warmed 0.9% saline was infused manually in 20 seconds into the left coronary artery (lca) in incremental doses of 10, 20, 50, and 100 g and then 10, 20, and 50 g into the right coronary artery (rca) depending on the vascular reactivity under continuous monitoring of 12lead ecg and blood pressure with a temporary pacemaker in place in the morning. coronary angiography was performed 1 minute after each ach injection or when ischemic ecg changes appeared. figure 1 shows a typical case of coronary spasm with stent implantation in the provocation test by ach 6 months postami. a representative case of coronary spasm induced by intracoronary ach infusion. severe diffuse spasm appeared associated with stsegment elevation on ecg postach infusion at the distal segments of stent implanted in the left anterior descending artery and disappeared after nitroglycerin. coronary spasm induced by this method usually disappeared spontaneously within 1 to 2 minutes and both the lca and rca could be examined separately, unless severe spasm occurred in the lca and necessitated the prompt injection of nitroglycerin (gtn) or isosorbide dinitrate (isdn) into the artery. the number of study patients needed was calculated based on an uncorrected chisquared statistic at the 2tailed test 5% significance level and 80% power, assuming the prevalence of coronary spasm to be 80% in the variant aldh2 2 mi patient group and 50% in the wild aldh2 1/1 mi patient group based on our previous work and others.12, 13, 14 subjects were asked to fill out a simple questionnaire concerning alcohol flushing on alcohol intake, alcohol drinking habit, and smoking. habitual drinker was defined as an alcohol drinker more than 5 days a week. alcohol flushing was defined as a current or a history of facial flushing immediately after drinking a glass of beer. blood samples for measurement of clinical chemistry and other data were collected on admission and repeatedly at least 3 to 4 times later with patients in the supine position. total creatine phosphokinase (cpk) was measured with a standard enzymatic method on a unicel dxc800 multi analyzer 2700 (beckman coulter clinical diagnostics, brea, ca). ctnt was assessed by an electrochemoluminescence immunoassay using the cobas system troponin t kit provided by the manufacturer on a cobas e 411 disk system analyzer (roche diagnostics, indianapolis, in). blood samples for total cpk and lactate dehydrogenase (ldh) were measured on admission and repeated every 4 hours during the first 12 hours and every 12 hours up to 72 hours. ctnt was measured on admission and repeated 3 to 6 hours later and further several times for 1 to 5 days depending on patient conditions. allele frequencies were determined by direct gene counting, and genotype distributions were checked for departure from hardyweinberg equilibrium using the pearson chisquare test. baseline clinical data were expressed as meansd or median (25th or 75th percentile) for continuous variables, and withingroup differences were evaluated with an unpaired t test or the mann data were expressed as counts and percentages and analyzed with the chisquare or fisher 's exact test. a 2tailed value of p<0.05 was considered to be statistically significant. analyses were done using the stata software program (stata 11.0 ; statacorp, college station, tx). genotype distributions did not depart from the hardyweinberg equilibrium for aldh2 genes in the whole group (=0.057 ; p=0.972). frequencies of aldh2 1/1, aldh2 1/2, and aldh2 2/2 genotype were 49.0% (99 of 202), 42.3% (86 of 202), and 8.4% (17 of 202), respectively. frequency of the aldh2 2 allele was thus 29.7% (120 of 404) in the stemi patients in this study. this is high, as compared with that of the general population in japan (23.5% [247 of 1050 ] in kumamoto and 24.3% [183 of 752 ] in tokyo, japan).3 we have recently reported that frequencies of aldh2 1/1, aldh2 1/2, and aldh2 2/2 genotype were 57.5% (272 of 473), 36.6% (173 of 473), and 5.9% (28 of 473), respectively, and thus frequency of the aldh2 2 allele was 24.2% (229 of 946) in subjects who had no histories or ecg signs of ischemic heart disease recruited from a health screening program, with age and sex comparable to those of stemi patients in the present study (age : 68.87.6 vs 67.312.0 ; p=0.054 and male ratio : 72.7% [344 of 473 ] vs 77.2% [156 of 202 ] ; p=0.222).21 frequency of the aldh2 2 allele was therefore significantly higher in stemi patients than in those without apparent ischemic heart disease (29.7% [120 of 404 ] vs 24.2% [229 of 946 ] ; odds ratio [or]=1.32 [95% ci, 1.011.73 ] ; p=0.035). the findings are consistent with those of previous studies6, 7, 8, 9, 10, 11 and indicate that aldh2 2 is associated with ami, being randomly assigned at conception independently of the possible confounding factors (mendelian randomization).22 the study also indicates that the variant aldh2 2 genotype exists mainly as the heterozygote (aldh2 1/2), and we combined heterozygote (aldh2 1/2) and homozygote of (aldh2 2/2) as a single category of variant aldh2 2 and compared them with the wild homozygote aldh2 1/1 in the following analysis. accordingly, 51.0% (103 of 202) of stemi patients (82 men and 21 women ; mean age, 67.112.2) had variant aldh2 2 and the remaining 99 (74 men and 25 women ; mean age, 67.511.8) or 49.0% wildtype aldh2 1/1 as shown in table. comparison of clinical characteristics between variant and wild type of aldh2 afs indicates alcohol flushing syndrome ; aldh2, aldehyde dehydrogenase type 2 ; bmi, body mass index ; bnp, btype natriuretic peptide ; cpk, creatine phosphokinase ; hscrp, high sensitivity creactive protein ; ctnt, cardiac troponin t ; dbp, diastolic blood pressure ; ef, ejection fraction ; egfr, estimated glomerular filtration rate ; hdlc, highdensity lipoproteincholesterol ; lad, left anterior descending artery ; lcx, left circumflex artery ; ldh, lactate dehydrogenase ; ldlc, lowdensity lipoproteincholesterol ; lmt, left main trunk ; rca, right coronary artery ; sbp, systolic blood pressure ; totalc, totalcholesterol. table compares the clinical characteristics of patients between the variant aldh2 2 group and wild aldh2 1/1 group. there were no differences in age, sex, body mass index (bmi), blood pressure, heart rate, cardiac index, pulmonary capillary wedge pressure (pcwp), smoking habit, and plasma levels of lowdensity lipoprotein (ldl) cholesterol, triglycerides, glucose, creactive protein, infarctrelated artery, percentage of multivessel disease, and other variables between the two groups. however, the frequencies of afs and coronary spasm were higher (94.3% vs 17.6% ; p<0.001 and 88.6% vs 56.1% ; p=0.001, respectively) and alcohol habit was lower (14.6% vs 51.5% ; p<0.001) in the aldh2 2 group than in the aldh2 1/1 group (table and figure 2a). thus, aldh2 2 is associated with coronary spasm as well as afs (or=6.1 ; 95% ci [1.823.4 ] and or=77.8 ; 95% ci [17.9441.8 ], respectively), being randomly assigned at conception independently of the possible confounding factors (mendelian randomization).22 accordingly, afs had 94.3% (50 of 53) sensitivity and 82.4% (42 of 51) specificity for aldh2. conversely, aldh2 2 had an 84.7% (50 of 59) sensitivity and 93.3% (42 of 45) specificity for afs and 62.9% (39 of 62) sensitivity and 78.2% (18 of 23) specificity for csa, respectively. afs may therefore be a useful clinical marker for aldh2 2 in the absence of genotyping. comparison of (a) frequency of cs and afs by aldh2 genotype and (b) levels of peak cpk. frequencies of cs and afs (a) and peak cpk levels (b) were higher in aldh2 2 compared with aldh2 1/1 carriers. horizontal bar indicates a median and the vertical bar 25th and 75th percentile, respectively (b). afs indicates alcohol flushing syndrome ; aldh2, aldehyde dehydrogenase 2 ; cpk, creatine phosphokinase. we performed sexspecific analyses to make sure that we did not miss a sexspecific effect. in men, coronary spasm were higher (91.9% vs 59.4% ; or=7.8 ; 95% ci [1.846.3 ] ; p=0.001) in the aldh2 2 group than in the aldh2 1/1 group. however, the number of women was small (n=46) for statistical significance (71.4% vs 44.4% ; p=0.28). peak plasma levels of total cpk were significantly higher (p=0.002) (table and figure 2b) and those of ldh, ctnt, and btype natriuretic peptide (bnp) tended to be higher and left ventricular ejection fraction (lvef) tended to lower in the aldh2 2 than in the aldh2 1/1 carrier group (table). this suggested that myocardial injury may be more severe in ami patients with aldh2 2 as compared to those with aldh2 1 1. genotype distributions did not depart from the hardyweinberg equilibrium for aldh2 genes in the whole group (=0.057 ; p=0.972). frequencies of aldh2 1/1, aldh2 1/2, and aldh2 2/2 genotype were 49.0% (99 of 202), 42.3% (86 of 202), and 8.4% (17 of 202), respectively. frequency of the aldh2 2 allele was thus 29.7% (120 of 404) in the stemi patients in this study. this is high, as compared with that of the general population in japan (23.5% [247 of 1050 ] in kumamoto and 24.3% [183 of 752 ] in tokyo, japan).3 we have recently reported that frequencies of aldh2 1/1, aldh2 1/2, and aldh2 2/2 genotype were 57.5% (272 of 473), 36.6% (173 of 473), and 5.9% (28 of 473), respectively, and thus frequency of the aldh2 2 allele was 24.2% (229 of 946) in subjects who had no histories or ecg signs of ischemic heart disease recruited from a health screening program, with age and sex comparable to those of stemi patients in the present study (age : 68.87.6 vs 67.312.0 ; p=0.054 and male ratio : 72.7% [344 of 473 ] vs 77.2% [156 of 202 ] ; p=0.222).21 frequency of the aldh2 2 allele was therefore significantly higher in stemi patients than in those without apparent ischemic heart disease (29.7% [120 of 404 ] vs 24.2% [229 of 946 ] ; odds ratio [or]=1.32 [95% ci, 1.011.73 ] ; p=0.035). the findings are consistent with those of previous studies6, 7, 8, 9, 10, 11 and indicate that aldh2 2 is associated with ami, being randomly assigned at conception independently of the possible confounding factors (mendelian randomization).22 the study also indicates that the variant aldh2 2 genotype exists mainly as the heterozygote (aldh2 1/2), and we combined heterozygote (aldh2 1/2) and homozygote of (aldh2 2/2) as a single category of variant aldh2 2 and compared them with the wild homozygote aldh2 1/1 in the following analysis. accordingly, 51.0% (103 of 202) of stemi patients (82 men and 21 women ; mean age, 67.112.2) had variant aldh2 2 and the remaining 99 (74 men and 25 women ; mean age, 67.511.8) or 49.0% wildtype aldh2 1/1 as shown in table. comparison of clinical characteristics between variant and wild type of aldh2 afs indicates alcohol flushing syndrome ; aldh2, aldehyde dehydrogenase type 2 ; bmi, body mass index ; bnp, btype natriuretic peptide ; cpk, creatine phosphokinase ; hscrp, high sensitivity creactive protein ; ctnt, cardiac troponin t ; dbp, diastolic blood pressure ; ef, ejection fraction ; egfr, estimated glomerular filtration rate ; hdlc, highdensity lipoproteincholesterol ; lad, left anterior descending artery ; lcx, left circumflex artery ; ldh, lactate dehydrogenase ; ldlc, lowdensity lipoproteincholesterol ; lmt, left main trunk ; rca, right coronary artery ; sbp, systolic blood pressure ; totalc, totalcholesterol. table compares the clinical characteristics of patients between the variant aldh2 2 group and wild aldh2 1/1 group. there were no differences in age, sex, body mass index (bmi), blood pressure, heart rate, cardiac index, pulmonary capillary wedge pressure (pcwp), smoking habit, and plasma levels of lowdensity lipoprotein (ldl) cholesterol, triglycerides, glucose, creactive protein, infarctrelated artery, percentage of multivessel disease, and other variables between the two groups. however, the frequencies of afs and coronary spasm were higher (94.3% vs 17.6% ; p<0.001 and 88.6% vs 56.1% ; p=0.001, respectively) and alcohol habit was lower (14.6% vs 51.5% ; p<0.001) in the aldh2 2 group than in the aldh2 1/1 group (table and figure 2a). thus, aldh2 2 is associated with coronary spasm as well as afs (or=6.1 ; 95% ci [1.823.4 ] and or=77.8 ; 95% ci [17.9441.8 ], respectively), being randomly assigned at conception independently of the possible confounding factors (mendelian randomization).22 accordingly, afs had 94.3% (50 of 53) sensitivity and 82.4% (42 of 51) specificity for aldh2. conversely, aldh2 2 had an 84.7% (50 of 59) sensitivity and 93.3% (42 of 45) specificity for afs and 62.9% (39 of 62) sensitivity and 78.2% (18 of 23) specificity for csa, respectively. afs may therefore be a useful clinical marker for aldh2 2 in the absence of genotyping. comparison of (a) frequency of cs and afs by aldh2 genotype and (b) levels of peak cpk. frequencies of cs and afs (a) and peak cpk levels (b) were higher in aldh2 2 compared with aldh2 1/1 carriers. horizontal bar indicates a median and the vertical bar 25th and 75th percentile, respectively (b). afs indicates alcohol flushing syndrome ; aldh2, aldehyde dehydrogenase 2 ; cpk, creatine phosphokinase. we performed sexspecific analyses to make sure that we did not miss a sexspecific effect. in men, coronary spasm were higher (91.9% vs 59.4% ; or=7.8 ; 95% ci [1.846.3 ] ; p=0.001) in the aldh2 2 group than in the aldh2 1/1 group. however, the number of women was small (n=46) for statistical significance (71.4% vs 44.4% ; p=0.28). peak plasma levels of total cpk were significantly higher (p=0.002) (table and figure 2b) and those of ldh, ctnt, and btype natriuretic peptide (bnp) tended to be higher and left ventricular ejection fraction (lvef) tended to lower in the aldh2 2 than in the aldh2 1/1 carrier group (table). this suggested that myocardial injury may be more severe in ami patients with aldh2 2 as compared to those with aldh2 1 1. east asians show rapid, intense afs after drinking a small amount of alcohol in up to 40% of the population.2, 3, 4, 5 this is caused by the presence of the variant of mitochondrial aldh2 genotype with a substitution of glutamate to lysine at position 504 (glu504lys) or aldh2 2, which is present only in east asians but is virtually absent in other populations of the world.2, 3, 4, 5 aldh2 2 exerts a dominant negative effect over wildtype homozygote aldh2 1/1, and heterozygote aldh2 1/2 shows a severely reduced and homozygote aldh2 2/2 negligible aldh2 activity.4 carriers of aldh2 2 thus manifest the characteristic afs caused by accumulation of ethanolderived acetaldehyde. aldh2 is a key enzyme that removes toxic aldehydes such as 4hne derived from lipid peroxidation as well as acetaldehyde.1, 2 previous studies reported that aldh2 2 genotype was associated with ami in east asians including japanese, koreans, and chinese.6, 7, 8, 9, 10 takeuchi. identified the genetic locus of aldh2 2 (rs671) as the strongest predictor for ami on a genomewide association study in japanese,10 and their findings were confirmed recently by the study of hirokawa.11 the present study shows that aldh2 2 genotype is significantly associated with stemi, and the results are consistent with those of previous studies.6, 7, 8, 9, 10, 11 because the genotype is assigned randomly at conception independently of the possible confounding factors according to mendel 's law (mendelian randomization),22 it is reasonable to think that aldh2 2 genotype is causally associated with stemi. the study therefore identifies deficient aldh2 activity and hence increased reactive aldehydes as a causative risk factor for stemi. however, the underlying mechanisms linking aldh2 2 to stemi remain to be elucidated. stemi is assumed to be triggered by plaque rupture or erosion resulting in coronary thrombotic occlusion.23, 24, 25 the present study also shows that aldh2 2 is causally associated with coronary spasm as well as afs in patients with stemi, which is in agreement with our previous study in patients with csa.14 coronary spasm is associated with increased reactive oxygen species (ros), endothelial impairment, and thrombogenicity.26, 27, 28, 29, 30, 31 it is therefore likely that coronary spasm may trigger plaque rupture or erosion and eventually result in coronary occlusion and stemi,18, 19, 23, 24, 25 particularly in those with aldh2 2. it is to be noted in this connection that coronary spasm has a circadian variation with a peak incidence occurring in the early morning, which corresponds to that of stemi.31, 32 it is also possible that stemi with stent implantation may conversely predispose to coronary spasm.12, 13 interestingly, there are currently almost no genetic data pertaining directly to stemi except for abo blood groups in the populations other than east asians.33 previous studies treated the phenotypes of myocardial infarction (mi) and coronary artery disease (cad) as synonymous on the basis that mi is precipitated by rupture of an atheromatous plaque followed by thrombotic occlusion superimposed on significant coronary atherosclerosis.34 the association of genes with both cad and mi are mainly accounted for by significant atherosclerosis rather than mi.33 however, the genetic variants contributing to plaque rapture or thrombosis may be distinct from those contributing to coronary atherosclerosis.34 timely reperfusion by direct pci is mandatory to salvage ischemic myocardium from infarction, but reperfusion per se paradoxically contributes to myocardial injury.19, 35, 36 toxic aldehydes such as 4hne are abundantly produced as a result of myocardial i / r injury, and aldh2 activity protects against i / r injury by eliminating toxic aldehydes in experimental models.2, 15, 16 in the present study, peak plasma total cpk levels were significantly higher, those of ldh, ctnt, and bnp tended to be higher, and lvef lower in patients with aldh2 2 than those with aldh2 1 1. these findings suggest that the carriers of aldh2 2 suffer moresevere myocardial injury postami as compared to those of aldh2 1/1. it is intriguing in this connection to note that the inhospital mortality rate for ami is the highest in japan among the developed countries of the organization for economic cooperation and development (oecd) member nations37 and that the adjusted inhospital mortality rates for ami are higher in asianamericans than white americans.38 however, recent studies indicate that 4hne may paradoxically evoke hormetic protective effects against oxidative injury in the relatively low concentrations through activating antioxidative systems such as the nrf2 pathway in experimental models.16, 39, 40, 41, 42 thus, further studies are required to determine whether carriers of aldh2 2 suffer moresevere myocardial injury postami. oxidative degradation of lipid membrane (lipid peroxidation) generates numerous reactive aldehydes and causes oxidative damage in various organs and tissues including the cardiovascular system.1, 2, 15, 16, 39, 40, 41, 42 aldh2 removes not only acetaldehyde but also other toxic aldehydes, including 4hne and malondialdehyde from lipid peroxidation or acrolein in tobacco smoke, and thereby protects tissues and cells from oxidative damage.1, 2 conversely, aldh2 activity is suppressed by ros and/or aldehydes.2, 41, 42 it is thus likely that carriers of aldh2 2 have increased reactive aldehydes as risk factors for coronary spasm and ami. the present study therefore identifies reactive aldehydes as the target to be intervened for the treatment and prevention of coronary spasm and ami. using genetically modified aldh2 models, ren. have shown that aldh2 also has a cardioprotective role through counteracting cardiac remodeling and myocardial dysfunction after alcohol intake.43 aldh2 may thus possess important therapeutic potential against alcoholic and other forms of myocardial damage as well.43 the study also indicates that afs is a sensitive clinical marker for aldh2 2. we thus propose that all patients should be screened for afs or at least have it documented in the patient 's health records. association of variant aldh2 2 with coronary spasm and acute myocardial infarction. variant aldh2 2 with deficient aldh2 activity leads to increased reactive aldehydes, such as 4hne, associated with increased reactive oxygen species. increased reactive aldehydes and reactive oxygen species cause coronary artery injury, which may lead to coronary spasm and acute myocardial infarction. carriers of variant aldh2 2 also have increased acetaldehyde caused by deficient aldh2 activity on alcohol intake and may thereby suffer from alcohol flushing syndrome. aldh2 also plays an essential role in bioactivation of gtn widely used for treatment of ischemic heart disease.2, 44, 45 however, continued administration of gtn leads to tolerance or even cardiac events through inactivation of aldh2 enzyme and an increased ros.2, 44, 45, 46 accordingly, carriers of aldh2 2 genotypes are less responsive to gtn and are also susceptible to gtn tolerance and oxidative damage.2, 44, 45, 46 calciumchannel blockers are the mainstay in treatment of coronary spasm at present.31, 47 the results of the present study thus imply that calciumchannel blockers also should be considered for treatment of postami patients, particularly those with aldh2 2.12, 13, 47 recently, chen. showed that a novel small molecule activator of aldh2, alda1, enhanced activity of aldh2 and effectively restored activity of variant aldh2 2 to wildtype levels in animal models.2, 15 accordingly, it is expected that this class of drug may serve as new therapeutics for ami and coronary spasm, particularly in those with aldh2 2 in the future. the number of study patients was small because the study included also the invasive intervention. the study subjects were a select population of stemi who underwent pci with stent implantation, and this may have altered the relationship with the aldh2 variant. the study subjects were limited to japanese ami patients because of the genetic association study,22 and thus the results of this study may not be necessarily applicable to other populations. frequency of afs was assessed on questionnaire survey, and recall subjective biases may have influenced the results. aldehydes such as 4hne were not measured because aldehydes are highly reactive and large parts of them exist as adducts with proteins, dna, or lipids in tissues,1, 41, 42 and clinically applicable methods are not yet available. in the present study, the patients with severe coronary atherosclerosis and those with severe hypertension were excluded, although the rs671 variant has been reported to associate with both cad and essential hypertension in the previous studies.9, 10, 11, 48 however, the risk factors for, and the predilection sites of, coronary spasm are distinct from those of coronary atherosclerosis and hypertension is not a risk factor for coronary spasm.49, 50 we have shown that stable angina, a prototype of advanced coronary atherosclerosis, is not associated with aldh2 2 (rs671), but mi is.21 oxidative degradation of lipid membrane (lipid peroxidation) generates numerous reactive aldehydes and causes oxidative damage in various organs and tissues including the cardiovascular system.1, 2, 15, 16, 39, 40, 41, 42 aldh2 removes not only acetaldehyde but also other toxic aldehydes, including 4hne and malondialdehyde from lipid peroxidation or acrolein in tobacco smoke, and thereby protects tissues and cells from oxidative damage.1, 2 conversely, aldh2 activity is suppressed by ros and/or aldehydes.2, 41, 42 it is thus likely that carriers of aldh2 2 have increased reactive aldehydes as risk factors for coronary spasm and ami. the present study therefore identifies reactive aldehydes as the target to be intervened for the treatment and prevention of coronary spasm and ami. using genetically modified aldh2 models, ren. have shown that aldh2 also has a cardioprotective role through counteracting cardiac remodeling and myocardial dysfunction after alcohol intake.43 aldh2 may thus possess important therapeutic potential against alcoholic and other forms of myocardial damage as well.43 the study also indicates that afs is a sensitive clinical marker for aldh2 2. we thus propose that all patients should be screened for afs or at least have it documented in the patient 's health records. variant aldh2 2 with deficient aldh2 activity leads to increased reactive aldehydes, such as 4hne, associated with increased reactive oxygen species. increased reactive aldehydes and reactive oxygen species cause coronary artery injury, which may lead to coronary spasm and acute myocardial infarction. carriers of variant aldh2 2 also have increased acetaldehyde caused by deficient aldh2 activity on alcohol intake and may thereby suffer from alcohol flushing syndrome. aldh2 also plays an essential role in bioactivation of gtn widely used for treatment of ischemic heart disease.2, 44, 45 however, continued administration of gtn leads to tolerance or even cardiac events through inactivation of aldh2 enzyme and an increased ros.2, 44, 45, 46 accordingly, carriers of aldh2 2 genotypes are less responsive to gtn and are also susceptible to gtn tolerance and oxidative damage.2, 44, 45, 46 calciumchannel blockers are the mainstay in treatment of coronary spasm at present.31, 47 the results of the present study thus imply that calciumchannel blockers also should be considered for treatment of postami patients, particularly those with aldh2 2.12, 13, 47 recently, chen. showed that a novel small molecule activator of aldh2, alda1, enhanced activity of aldh2 and effectively restored activity of variant aldh2 2 to wildtype levels in animal models.2, 15 accordingly, it is expected that this class of drug may serve as new therapeutics for ami and coronary spasm, particularly in those with aldh2 2 in the future. the number of study patients was small because the study included also the invasive intervention. the study subjects were a select population of stemi who underwent pci with stent implantation, and this may have altered the relationship with the aldh2 variant. the study subjects were limited to japanese ami patients because of the genetic association study,22 and thus the results of this study may not be necessarily applicable to other populations. frequency of afs was assessed on questionnaire survey, and recall subjective biases may have influenced the results. aldehydes such as 4hne were not measured because aldehydes are highly reactive and large parts of them exist as adducts with proteins, dna, or lipids in tissues,1, 41, 42 and clinically applicable methods are not yet available. in the present study, the patients with severe coronary atherosclerosis and those with severe hypertension were excluded, although the rs671 variant has been reported to associate with both cad and essential hypertension in the previous studies.9, 10, 11, 48 however, the risk factors for, and the predilection sites of, coronary spasm are distinct from those of coronary atherosclerosis and hypertension is not a risk factor for coronary spasm.49, 50 we have shown that stable angina, a prototype of advanced coronary atherosclerosis, is not associated with aldh2 2 (rs671), but mi is.21 the study identified deficient aldh2 activity and hence reactive aldehydes as the risk factors to be targeted and intervened for treatment and prevention of coronary spasm and ami. this study was supported, in part, by the japan heart foundation, tokyo, and the japan vascular disease research foundation, kyoto, japan.	backgroundmitochondrial aldehyde dehydrogenase 2 (aldh2) plays a key role in removing toxic aldehydes. deficient variant aldh2 2 genotype is prevalent in up to 40% of the east asians and reported to be associated with acute myocardial infarction (ami). to elucidate the mechanisms underlying the association of aldh2 2 with ami, we compared the clinical features of ami patients with aldh2 2 to those with wildtype aldh2 1/1.methods and resultsthe study subjects consisted of 202 japanese patients with acute stsegment elevation myocardial infarction (stemi) (156 men and 46 women ; mean age, 67.312.0) who underwent primary percutaneous coronary intervention (pci). in 85 patients, provocation test for coronary spasm was also done 6 month postpci. aldh2 genotyping was performed by direct application of the taqman polymerase chain system. of the 202 patients, 103 (51.0%) were carriers of aldh2 2 and 99 (49.0%) those of aldh2 1/1. there were no differences in clinical features between aldh2 2 and aldh2 1/1 carrier groups except higher frequencies of coronary spasm and alcohol flush syndrome (afs) (88.6% vs 56.1% ; p=0.001 and 94.3% vs 17.6% ; p<0.001), lessfrequent alcohol habit (14.6% vs 51.5% ; p<0.001), and higher peak plasma creatine phophokinase levels (2224 vs 1617 mg / dl ; p=0.002) in the aldh2 2 than the aldh2 1/1 carrier group.conclusions aldh2 2 is prevalent (51.0%) among japanese stemi patients, and those with aldh2 2 had higher frequencies of coronary spasm and afs and moresevere myocardial injury compared to those with aldh2 1/1.
microspheres can be prepared by different methods. in the present study, microspheres were prepared by spray drying method [1, 2 ]. spray drying is advantageous as it is a well - established technology and the equipment is capable of high product output. thermosensitive substances can be coated successfully by this method because the time of exposure to elevated temperature is extremely short. the biological half - life of metformin hydrochloride (mh) is 2.5 to 5 hr and gastrointestinal absorption is incomplete with an absolute bioavailability of 5060% (under fasting condition). reduction of gastrointestinal motility enhances the absorption of mh. the rate of absorption is slower than that of elimination. the plant dillenia indica l. (family : dilleniaceae ; synonym : dillenia speciosa thunb.) is known as elephant apple / indian catmon (english) ; bhavya, bharija (sanskrit) ; ou tenga (assamese). the fruits of dillenia indica (di) are used by tribal and folk communities of various regions as vegetable. (figure 1), with excrescent calyx, 12.515.0 cm in diameter, green when young and yellowish and sweet - scented when ripe ; seeds many, compressed, embedded in hairy cells [3, 4 ]. the leaf, bark, and fruit of this plant are used traditionally as medicine in different forms for their therapeutic values. description of d. indica is available in various ancient indian literatures about its important common and medicinal uses [5, 6 ]. there are also reports that the plant parts exhibited antileukemic, anti - inflammatory, antioxidant, antiproliferative, antidiabetic, antimicrobial, antifungal, antidiarrheal, cytotoxic, and hepatoprotective activities [79 ]. earlier, extraction, characterization of the mucilage, and compatibility study of the mucilage with metformin hydrochloride was reported. the mucilage was extracted by heating with water at 6070c and the filtrate was precipitated by acetone. quantitative estimation of pectin was carried out after detection of the presence of pectin in the mucilage by colorimetric method. dsc, ftir, and xrd studies were carried out alone and in combination with metformin hydrochloride. metformin hydrochloride loaded microspheres were prepared using bora rice flour in combination with sodium alginate. in another study, mucilage of di fruit, mucilage of abelmoschus esculentus pods and bora rice flour were used for formulation of metformin hydrochloride loaded microspheres. in this study, metformin hydrochloride loaded microspheres were prepared by spray drying method using the mucilage of di fruits alone. the effect of the mucilage concentration on the properties of the microspheres was studied. metformin hydrochloride (mh) was received as gift sample from ozone pharmaceutical ltd., the mucilage of d. indica fruit was extracted by acetone precipitation method as described above [10, 13 ]. all chemicals used in this study were of analytical grade and were procured commercially (e - merck, india). the intestinal segment of goat (ileum), used for mucoadhesive study, was procured from local slaughter house. metformin hydrochloride loaded microspheres with the mucilage of di fruits were prepared by spray drying method. aqueous dispersions of the mucilage of di fruits of three different concentrations (1.0%, 2.0%, and 3.0% w / v) were prepared by heating at 60 0.5c for 1 hr. the dispersions were allowed to cool to room temperature and then mh was added to dispersions at a concentration of 1.5% w / v and stirred to dissolve mh., the mixture was spray dried using lab spray dryer (lu-222 advanced labultima, india) with 0.7 mm nozzle. parameters of spray dryer were changed and products were collected separately in each run. the composition and various processing parameters of the method used the prepared microspheres were evaluated for particle size, particle shape and surface topography, liquid uptake capacity, drug entrapment efficiency, mucoadhesive property, and in vitro drug release property. the shape and surface topography of the microspheres was investigated by using (s-3600n, hitachi) scanning electron microscope (sem) at 15 kv. the liquid uptake capacity of microspheres was determined in three aqueous media (water, 0.1 m hcl and phosphate buffer ph 7.4) at room temperature (37.5c 0.5c). weighed quantities of the samples were taken into test tubes separately with fixed volume of media in undisturbed condition for 6 hrs. the liquid was then decanted off carefully and the final weight of microspheres was taken. the percentage liquid uptake was calculated using the following relation : (1)uptake(%)=wtw0w0100, where w0 and wt are the initial weight and final weight of sample after 6 hrs, respectively [15, 16 ]. microspheres equivalent to 50.0 mg of the drug were taken and the amount of drug entrapped was estimated by dispersing the crushed microspheres in 100.0 ml of phosphate buffer (ph 7.4) by shaking on a mechanical shaker (remi motors, mumbai, india) for 12 hr. the solution was filtered and the filtrate was diluted 25 times with phosphate buffer (ph 7.4) and the absorbance was measured spectrophotometrically (uv-1700, shimadzu, japan) at 233 nm against a blank. the amount of drug entrapped in the microspheres was calculated in percentage from the ratio of actual drug content to theoretical drug content as given in the following equation [17, 18 ] : (2)drug entrapment efficiency% = actual drug contenttheoretical drug content100. the mucoadhesive property of microspheres was evaluated by in vitro wash off test method [1921 ]. freshly excised pieces of goat intestinal mucosa (2 2 cm) were washed with phosphate buffer (ph 7.4) mounted on to glass slides with cotton thread. then immediately thereafter, the slides were hung to usp tablet disintegration test apparatus (lab. when the test apparatus was operated, the sample was subjected to slow up and down movement in the test fluid (phosphate buffer ph 7.4) at 37c contained in a 1 l vessel of the apparatus. after 5 hrs, the apparatus was stopped and the number of microspheres still adhering to mucosal surface was counted with the help of microscope. this process was repeated for three times (n = 3) and the average number of microspheres adhered was calculated. the release of drug from the microspheres was studied in phosphate buffer (ph 7.4) as dissolution medium, using usp paddle - type dissolution test apparatus (six vessels, tl 06, electro lab, india) at 37 0.5c with a rotating speed of 100 rpm. the liquid uptake capacity and mucoadhesive property were found to be more in phosphate buffer (ph 7.4) and therefore the in vitro drug release study was carried out using phosphate buffer media (ph 7.4). a sample of microspheres equivalent to 50.0 mg of metformin hydrochloride was used in each test. 10.0 ml of the sample from each dissolution test vessel was withdrawn at predetermined time intervals (0, 0.5, 1, 1.5, 2, 4, 6, 8, and 10 hr) for 10 hrs and the same volume of media was replaced. the withdrawn samples were filtered through a membrane filter (0.45 m) and were analyzed for drug content spectrophotometrically at 233 nm using the uv - visible spectrophotometer (uv-1700, shimadzu, japan) [2224 ]. the mean particle sizes of the formulations were found to be in the range of 62 6.24 to 98 2.13 m in the formulations from sd1 to sd12. the particle size was found to be increased, when mucilage concentration was increased at constant inlet temperature, feed flow rate, and aspirator flow rate as observed in formulations sd3, it was observed from the sem photograph (figure 2) of the samples of microspheres that the surfaces of the microspheres were rough with deepening on the surface. this was due to rapid loss of water from the surface on exposure to high temperature (table 1). the liquid uptake capacity of microspheres was determined in water, phosphate buffer ph 7.4, and in 0.1 m hcl. the liquid uptake was found to be within the range of 10 0.82 to 28 0.14% in 0.1 m hcl ; 16 0.23 to 54 0.13% in water, and 42 1.07 to 66 0.05% in phosphate buffer (ph 7.4). maximum water uptake was observed in formulations containing higher amounts of the mucilage (sd3, sd6, sd9, and sd12) when inlet temperature, feed flow rate, and aspirator flow rate were kept constant (tables 1 and 2). the uptake was low in 0.1 m hcl and in water but higher in phosphate buffer (ph 7.4). the acidic mucilage (ph 5.0) developed network structures in basic ph (ph 7.4) due to which more liquid uptake was observed in comparison to water and 0.1 m hcl. the entrapment efficiency was found to be in the range of 43.67 0.58 to 55.12 1.83 (sd1 to sd12). highest entrapment efficiency was found to be 55.12 1.83 (sf-12) in the formulation containing higher amount of the mucilage of dillenia indica. the low entrapment efficiency was due to the aqueous solubility of mh and low water uptake capacity of the mucilage which in turn the drug was made available in the bulk solution before it was entrapped by the mucilage. the increased concentration of mucilage increased water uptake due to which more amount of the drug was available in the network structure of the mucilage. during spray drying process, the water evaporated leaving the drug within the mucilage network. the results of in vitro wash - off test of the spray dried microspheres prepared using the mucilage of dillenia indica are presented in figure 3. it was observed that the particles of formulations sd3, sd6, sd8, sd9, sd10, and sd12 remained adhered to the mucosal surface of the intestinal membrane up to 3.5 hrs. the mucoadhesive property was found to be increased at increased concentration of mucilage in the formulations sd3, sd6, sd9, and sd12 (table 1). in vitro drug release study of the microspheres the results exhibited maximum drug release from most of the formulations within 4 hrs during the 10 hrs study except the formulations sd2, sd5, sd8, and sd11, which exhibited maximum drug release within 6 hrs during the 10 hrs study. these four formulations contained intermediate concentration of the mucilage (2.0 g) for a particular operating condition of spray dryer (table 1). the in vitro drug release of the formulations was best explained by higuchi kinetics with the highest linearity in formulations sd8, sd10, and sd12. the correlation coefficient (r) of the above formulations was 0.917, 0.863, and 0.909, respectively, which revealed that the drug diffused at a comparatively slower rate as the diffusional path length was increased. however, a direct correlation between the mucilage concentrations with the drug release kinetics could not be established due to the variation in the operating condition of the spray dryer. sd2, sd3, sd7, sd8, sd9, and sd11 exhibited fickian diffusion, whereas, sd4, sd5, sd6, sd10, and sd12 exhibited non - fickian diffusion. this revealed that the mechanism was not directly related to the mucilage concentration but was related to the process parameters. the concentration versus time profile of the formulations (y error = 2%) is presented in figure 4. the microspheres prepared using the mucilage of di by spray drying method exhibited poor entrapment efficiency but better mucoadhesive property. the liquid uptake was the maximum in alkaline media and the minimum in 0.1 m hcl. maximum amount of drug was released within 2 hrs and a decrease of concentration was observed during the study period of 10 hrs. however, there are options for improvement of the properties of microspheres for release of drug in a more controlled manner for a prolonged period of time.	natural materials are preferred over synthetic counterparts because of their biodegradable and biocompatible nature. the present work was proposed to utilize mucilage from natural source for the development of controlled release formulation of metformin hydrochloride. natural mucilaginous substance extracted from dillenia indica l. (di) fruit was used in fabricating controlled release microspheres. the microspheres were prepared by spray drying method under different formulation parameters. the prepared microspheres were studied for particle size, drug excipient compatibility, particle shape and surface morphologies, drug entrapment efficiency, mucoadhesivity, and in vitro drug release properties. the prepared microspheres exhibited mucoadhesive properties and demonstrated controlled release of metformin hydrochloride. the study reveals that the natural materials can be used for formulation of controlled release microspheres and would provide ample opportunities for further study.
fetal and neonatal thyrotoxicosis are names given to the same disease manifesting at different periods of life. when it manifests in utero it is called fetal and when it manifests after the baby is born it is called neonatal thyrotoxicosis. usually, fetal thyrotoxicosis continues as neonatal thyrotoxicosis after birth. the prevalence of grave 's disease in pregnancy is 0.2%. of those pregnant patients with grave 's disease thus, the prevalence of neonatal thyrotoxicosis is 1/4000 - 1/50000 pregnancies. in patients who require treatment for grave 's disease in the last trimester of pregnancy the mortality is 12 - 20% due to heart failure, but other complications are tracheal compression, infections, thrombocytopenia. thus neonatal thyrotoxicosis is uncommon, but not a rare disease which can be fatal. this disease is caused by thyroid stimulating hormone (tsh) receptor stimulating antibodies (tshr). during pregnancy in grave 's disease, patient 's thyroid stimulating antibodies can cross the placenta like all immunoglobulin g (igg) antibodies and stimulate the fetal thyroid triggering fetal thyrotoxicosis, which lasts until the maternal antibodies disappear from the fetal circulation. the prevalence of fetal thyrotoxicosis is low because pregnancy is a state of generalized immunosuppression and levels of thyroid receptor antibodies (trab) are reduced in pregnancy and only women who have three to five times normal levels of thyroid stimulating immunoglobulins (tsis) result in fetal and neonatal thyrotoxicosis. although transplacental passage of maternal antibodies (igg class) to the fetus does occur early in gestation, the fetal concentration is low until the end of second trimester. placental permeability then increases such that in the last trimester, fetal levels are equivalent to maternal. this change in permeability as well as ability of the fetal thyroid to respond to tsh and trab explains why fetal hyperthyroidism occurs in the second half of pregnancy. even women of grave 's disease who are euthyroid due to anti - thyroid medication or hypothyroid due to thyroidectomy or radioiodine therapy can have high levels of trab in their sera, which can cause fetal or neonatal thyrotoxicosis. the guidelines of the american thyroid association (ata) for the diagnosis and management of thyroid disease during pregnancy and postpartum published in 2011 recommend measurement of trab during 24 - 28 weeks of pregnancy and if the value is over three times normal, close follow for fetal thyrotoxicosis is recommended. first are assays that detect trab in patient 's sera by their ability to compete for binding of tsh receptor with a known tsh receptor ligand, which is tsh or tsh receptor antibody. the other etiology of neonatal thyrotoxicosis is due to activating mutations in the tsh receptor and activating mutations of the stimulatory g prpotein in mcune albright syndrome. this entity should be suspected if there are more than two generations affected with thyrotoxicosis or there are first degree relatives with thyrotoxicosis. inherited mutations leading to activation of the tsh receptor activating mutations of tsh receptor result in permanent thyrotoxicosis needing definitive treatment, which is thyroidectomy. a few de novo germline mutations activating the tsh receptor have been described, they also cause persistent thyrotoxicosis and need thyroidectomy. somatic mutations activating the tsh receptor have been described as the most common mechanism for hot nodules. these usually present in adults and older children but one single case of fetal onset has been reported. the most important clue to the diagnosis of fetal thyrotoxicosis is the mother 's history and clinical findings. the mother has active grave 's disease or has suffered from grave 's thyrotoxicosis in the past and treated with radioiodine, anti - thyroid drugs or thyroidectomy either the signs of thyrotoxicosis are present or the past stigmata of grave 's in form of ophthalmopathy or scar of thyroid surgery are present. one study even described that mothers who have severe or recurrent thyrotoxicosis and those with grave 's ophthalmopathy are most likely to present with fetal or neonatal thyrotoxicosis. fetal outcome is related to control of maternal thyrotoxicosis and complications are increased in mothers who remain thyrotoxic in the third trimester. a history of previous fetal loss is an important clue. in one case, which was treated by the author, the importance of history and physical examination is illustrated. a patient of grave 's disease treated with subtotal thyroidectomy had five pregnancies complicated with fetal thyrotoxicosis. the mother had stigmata of grave 's in the form of grave 's ophthalmopathy, goiter and the presence of clinical and biochemical thyrotoxicosis at time of presentation in the fourth and fifth pregnancy. the autopsy of the fetus in the first pregnancy revealed macerated still birth with autolysis of all organs. in her second pregnancy fetal tachycardia of 188 beats / min was documented and autopsy of the fetus showed goiter, pleural effusion and ascites. in the third pregnancy there was documented fetal tachycardia of 188/min and at 26 weeks gestation a macerated stillbirth. fetal tachycardia in the fourth and the fifth pregnancies was documented by ultrasound (fetal heart rate 160 - 180/min). in the fourth pregnancy and the fifth pregnancy treatment of fetal thyrotoxicosis the fourth pregnancy resulted in a live birth, but the child developed severe neonatal thyrotoxicosis, which proved fatal. this history and physical examination findings leave no doubt about the diagnosis of fetal thyrotoxicosis.. a consistent resting fetal heart rate of above 160 beats / min documented by doppler or ultrasound is indicative of fetal thyrotoxicosis. the normal fetal heart rate is 120 - 160 beats / min. an important feature of fetal tachycardia due to fetal thyrotoxicosis, is a reactive non stress testing and variability in heart rate is maintained. this differentiates it from the other causes of fetal tachycardia. in our country where tsi assay is not available, fetal goiter can also be present in fetal hypothyroidism due to transplacental passage of anti - thyroid drugs given to the mother. this iatrogenic fetal goiter regresses on reduction of doses of anti - thyroid drugs while that of fetal thyrotoxicosis does not. the other features of this disease are fetal tachycardia, fetal goiter and history of spontaneous abortions and findings of goiter, ascites, craniosynostosis, fetal growth retardation, maceration and hydrops at fetal autopsy. 5 - 7% off springs of mothers receiving medical or surgical treatment for thyrotoxicosis and 24% of offspring of untreated hyperthyroid mothers end in intrauterine death. preterm delivery occurs in 4 - 11% of mothers treated for thyrotoxicosis during pregnancy and 53% of mothers who remain untreated. umbilical cord blood sampling or cordocentesis or funipunture and measurement of fetal cord blood levels is the gold standard for the diagnosis of fetal thyrotoxicosis, but carries a risk of fetal hemorrhage, bradycardia, infection and death. it should be done only if absolutely necessary and only by doctors highly experienced in doing this. this procedure should only be performed in those cases where diagnosis of fetal thyrotoxicosis is still in doubt after ultrasound. measurement of tsh receptor antibodies i.e. thyroid receptor binding immunoglobulins and tsis is necessary in 24 - 28 weeks of gestation. the ata guidelines do not discuss the assays to be used to determine the presence of trab in the mother. some believe that a screening traumatic brain injury (tbi) assay should be done and if positive followed by a tsi assay. some believe that only a tsi assay should be performed, but this carries a risk of missing tshr blocking antibodies. thus, in a pregnant women who has received definitive treatment both tsi and tbi tests have a complimentary role. fetal thyrotoxicosis is a rare disease and a high index of suspicion is needed to diagnose it. the presence of autoimmune thyroid disease in the mother active or euthyroid on treatment, fetal tachycardia or fetal goiter on ultrasonography, should alert us. strong correlation has been found between maternal and fetal tsi and thyrotropin binding inhibiting immunoglobulins (tbii) levels. maternal tsi > 350 - 500% (n 40 - 70% (n 3 times normal tsis in 24 - 28 weeks of pregnancy.clinical thyrotoxicosis in third trimester or history of thionamide treatment in third trimester.family history of tsh receptor mutation andfeatures of fetal hyperthyroidism in the fetus. > 3 times normal tsis in 24 - 28 weeks of pregnancy. one study found that if trab were more than three times upper limits of normal on day 1 - 7 in infants who developed neonatal hyperthyroidism. cord blood levels should be taken in cases of suspected neonatal thyrotoxicosis for free thyroxine (ft) 3, ft4/tsh and cord trab levels. symptoms and signs of neonatal thyrotoxicosis can be apparent at birth or may be delayed due to the effect of transplacental passage of maternal anti - thyroid drugs or effect of coexisting blocking antibodies, but they are apparent by 10 days of life, rarely they can be delayed up to 45 days. cardiovascular system signs are tachycardia, arrhythmias, cardiac failure, systemic and pulmonary hypertension. signs of hypermetabolism include voracious appetite, weight loss, diarrhea, sweating, flushing. other signs are persisting acrocyanosis, hepatosplenomegaly, lymphadenopathy, thymic enlargement. bruising and petechial hemorrhage are secondary to thrombocytopenia. advanced bone age, craniosynostosis and microcephaly may be evident both in fetus and newborn. duration of neonatal throtoxicosis secondary to maternal grave 's disease is determined by transplacentally acquired tsi and is usually 8 - 20 weeks, sometimes 48 weeks. daneman and howard found craniosynostosis in six out of eight children and intellectual impairment in 4/6 children. physical growth was normal in all children whereas another long - term follow - up study by hollingsworth and mabry reported poor growth in three of four patients and intellectual impairment in all four, but these cases had persistent thyrotoxicosis and autosomal dominant and probably were due to gene mutation in the tsh receptor. these two drugs do not affect neonatal thyroid function, there is no contraindication for both drugs, but ptu is preferred. the goal of the treatment is to normalize thyroid functions as quickly as possible, to avoid iatrogenic hypothyroidism while providing management and supportive therapy for the infant 's specific signs and symptoms. the treatment of neonatal thyrotoxicosis is on the same principles as that of a thyrotoxic crisis in the adult. the dose of carbimazole is 0.5 - 1.5 mg / kg / day as a single dose and that of ptu is 5 - 10 mg / kg / day. lugol 's iodine acts by blocking the synthesis of thyroid hormones as well as blocking the release of the hormone stored in the colloid. lugol 's iodine, which contains 5% potassium iodide is used in a dose of one drop 8 hourly. beta blockers, which control the adrenergic symptoms effectively as well as inhibit the peripheral iodination of t4-t3 are used in a dose of propranolol 0.27 - 0.75 mg / kg 8 hourly. steroids act by inhibiting the peripheral de - iodination of t4-t3 and by compensating for hypermetabolism of endogenous steroids induced by t4 and t3. supportive treatment is very important to manage respiratory distress, fluid and electrolyte imbalance, temperature and high output heart failure. specific treatment of congestive heart failure by diuretics and digoxin may be necessary. oxygen therapy, non - invasive and invasive ventilation may be required. diarrhea and hyperthermia may occur and patient may need intravenous fluids and nursing in a temperature controlled environment. the work - up includes complete blood count, electrocardiography, x - ray chest, blood and urine cultures, echocardiogram and renal and liver function tests. monitoring also has to be done for side - effects of ptu and carbimazole such as leucopenia, liver dysfunction and thrombocytopenia.	fetal thyrotoxicosis is a rare disease occurring in 1 out of 70 pregnancies with grave 's disease or in 1 out of 4000 - 50,000 deliveries. the mortality is 12 - 20%, usually from heart failure, but other complications are tracheal compression, infections and thrombocytopenia. it results from transfer of thyroid stimulating immunoglobulins from mother to fetus through the placenta. this transplacental transfer begins around 20th week of pregnancy and reaches its maximum by 30th week. these autoantibodies bind to the fetal thyroid stimulating hormone (tsh) receptors and increase the secretion of the thyroid hormones. the mother has an active autoimmune thyroid disease or has been treated for it in the past. she may be absolutely euthyroid due to past treatment by drugs, surgery or radioiodine ablation, but still have active tsh receptor stimulating autoantibodies, which can cause fetal thyrotoxicosis. the other features of this disease are fetal tachycardia, fetal goiter and history of spontaneous abortions and findings of goiter, ascites, craniosyntosis, fetal growth retardation, maceration and hydrops at fetal autopsy. if untreated, this disease can result in intrauterine death. the treatment for this disease consists of giving carbimazole to the mother, which is transferred through the placenta to the fetus. the dose of carbimazole is titrated with the fetal heart rate. if the mother becomes hypothyroid due to carbimazole, thyroxine is added taking advantage of the fact that very little of thyroxine is transferred across the placenta. neonatal thyrotoxicosis patients are very sick and require emergency treatment. the goal of the treatment is to normalize thyroid functions as quickly as possible, to avoid iatrogenic hypothyroidism while providing management and supportive therapy for the infant 's specific signs and symptoms.
steroid 21-hydroxylase deficiency accounts for over 90% of cah cases and can have diverse manifestations : from the salt - wasting to the non - classical form due to a highly variable genetic mutation (1, 2). in the case of the simple virilizing or non - classical form, the symptoms related to the enzyme deficiency may be so insidiously progressive that it is not easy to identify the disease. impaired negative feedback by reduced cortisol causes an exaggerated rise of adrenocorticotrophic hormone (acth) levels, which stimulates the adrenal cortex to become hyperplastic, and sometimes nodular or tumorous (3 - 5). if we encounter a patient with adrenal incidentaloma(s), we may consider a variety of adrenal diseases, such as cushing 's syndrome or cah (6) ; however, the two conditions are mutually exclusive with regard to cortisol production. here a 41-yr - old man was referred to our emergency room from a local clinic on july 3, 2006, with the impression of acute viral hepatitis due to a remarkable elevation of liver enzymes. he had experienced generalized weakness, dark urine, and jaundice for a two week period. his previous history included community - acquired pneumonia and carpal tunnel syndrome, but neither diabetes mellitus nor hypertension. he complained of impotence, and his mother described precocious puberty and hirsutism beginning at the age of 7. his vital signs were a blood pressure of 115/92 mmhg, a regular pulse of 105/min, and a respiratory rate of 18/min. his height was 152.5 cm (95% suppression), which may suggest the hyperresponsiveness of the hypothalamus - pituitary - adrenal axis to dexamethasone. in search for conditions showing hypercortisolism, adrenal tumor and normal - to - increased responsiveness of the hypothalamus - pituitary - adrenal axis, we noticed that this patient has an extreme short stature (< 3th percentile) and precocious puberty which may suggest the diagnosis of the virilizing type of cah. in fact, hyperresponsiveness of the adrenal gland to acth or adrenal steroids is a characteristic feature of cah (10). we suspected that urine cortisol level in cah might be able to exceed upper normal limit on certain conditions if the adrenal gland that has become tumorous is chronically stimulated by acth (11, 12). gene mutation analysis further corroborated our diagnosis of cah, and this was similar to previous case heterozygote for a single mutation of cyp21 gene (13). i173n is one of the most frequent specific point mutations in the simple virilizing form of 21-hydroxylase deficiency, and the r357w mutation is also well - known in the cyp21 gene. these two mutations are generated by intergenic recombination between the cyp21 and cyp21p genes, a frequent cause of 21-hydroxylase deficiency (14 - 18). according to previous studies, the i173n mutation is specifically associated with the simple virilizing phenotype because of a partial defect in activity. the r357w mutation is known to completely abolish enzymatic activity and, in its homozygous form, is associated with the salt - wasting phenotype (2). disease severity in 21-hydroxylase deficiency is known to correlate with the less severely mutated allele. our patient exhibited a simple virilizing phenotype because he had i173n mutation with a partial enzymatic defect in agreement with previous data (15 - 18). in summary, we describe a case of cah with adrenal tumorous lesion and hypercortisoluria mimicking cushing 's syndrome. cortisol production in cah may exceed the upper limit of normal production under stressed conditions as shown in this case. unnecessary testing for differential diagnosis of cushing 's syndrome could be avoided if a detailed medical history was obtained from patient and 24-hr urine cortisol was measured in stable condition.	congenital adrenal hyperplasia (cah) is characterized by decreased adrenal hormone production due to enzymatic defects and subsequent rise of adrenocorticotrophic hormone that stimulates the adrenal cortex to become hyperplastic, and sometimes tumorous. as the pathophysiology is basically a defect in the biosynthesis of cortisol, one may not consider cah in patients with hypercortisolism. we report a case of a 41-yr - old man with a 4 cm - sized left adrenal tumorous lesion mimicking cushing 's syndrome who was diagnosed with cah. he had central obesity and acanthosis nigricans involving the axillae together with elevated 24-hr urine cortisol level, supporting the diagnosis of cushing 's syndrome. however, the 24-hr urine cortisol was suppressed by 95% with the low dose dexamethasone suppression test. cah was suspected based on the history of precocious puberty, short stature and a profound suppression of cortisol production by dexamethasone. cah was confirmed by a remarkably increased level of serum 17-hydroxyprogesterone level. gene mutation analysis revealed a compound heterozygote mutation of cyp21a2 (i173n and r357w).
vicinal azidoalcohols are precursors of aminoalcohols, which are well known as -blockers and present in various natural products and different bioactive compounds ; vicinal azidoalcohols not only constitute components of biologically active natural products but also serve as essential intermediates in the synthesis of amino sugar [13 ], carboxylic nucleosides, lactams, oxazolines, and aminoalcohols. ring - opening reactions of epoxides with nucleophiles are very useful approach in organic synthesis for the preparation of functionalized oxygenated compounds. epoxides are versatile intermediates in organic synthesis, and their reactions with a variety of reagents such as electrophiles, nucleophiles, acids, bases, reducing agents, and some oxidizing agents are widely studied. the reaction with nucleophiles such as oxygen compounds [810 ], nitrogen compounds (amine and derivatives of amines, azide, nitrate, and isocyanate), halides, and various carbon nucleophiles [11, the formation of basic oleochemical substances like fatty acids, fatty acid methyl esters (fames), fatty alcohols, fatty amines, and glycerols is by various chemical and enzymatic reactions. in the last decade, the oleochemical industry has been growing steadily due to increased global demand for more environment - friendly products. this is because of oleochemicals ' attractive traits such as being derived from renewable resources, nontoxic, and readily biodegradable. oils and fats of plant and animal origin offer possibilities of providing industries with a wealth of reaction products which will be of great value in the future. more than 90% of the hitherto published oleochemical reactions have been those occurring at the fatty acid carboxyl group while less than 10% have involved transformations of the alkyl chain. there has been a lot of interest in using vegetable oils as renewable raw materials for new industrial products. it is important to develop a range of relatively facile reactions on vegetable oils in order to facilitate their use. the reaction reported appears to be a useful reaction that has many potential applications for vegetable oils, especially the lesser known underutilized ones which are potentially cheap and readily available. this trend is very positive as it has the potential for considerably extending the range of compounds that may eventually be obtainable from oils and fats thereby leading to the growth in the use of fats as renewable raw materials. there is much interest in the literature in the reactions of these materials to produce cost - effective derivatives. some examples are epoxidised oil, soybean oil methyl ester (methyl soyate), maleated products, and derivatives of vegetable oils and soybean oil polymers [17, 18 ]. preparation of azido compounds from aliphatic and aromatic epoxides obtainable from petrochemical industries has been studied [1921 ]. such compounds are good starting blocks for organic molecules to be converted into nitrogen heterocycles by decomposition or addition reactions [22, 23 ]. though there have been reports on the preparation and some physical properties of azido compounds prepared from pure fatty acids and organic and polymeric substrates, there is however no depth of information on azido compounds prepared from the unsaturated systems in fatty acid methyl esters. such reaction should be feasible, and it forms the objective of this study using a lesser known underutilized seed oil of hura crepitans from nigeria. this present work aimed at introducing an azido group onto the fatty acid methyl esters of hura crepitans via epoxy cleavage in order to prepare vicinal amino hydroxyl methyl esters which are important in the surfactant industries. the mature seeds of hura crepitans were collected from the trees grown at the garden of the university of ibadan, ibadan, oyo state, nigeria. formic acid (100%) and hydrogen peroxide (30%) were purchased from merck, darmstadt, germany. further chemicals and all solvents used in this study were of analytical grade and were purchased from vwr international gmbh, darmstadt. the dried seeds of hura crepitans were extracted with n - hexane for 10 h using a soxhlet extractor. the extracted oil was analyzed for its iodine, saponification, and acid values employing methods described by the association of official analytical chemists. fatty acid methyl esters of the oil were prepared by refluxing the oil at 70c for 4 h in 2% sulphuric acid in methanol. the esters were extracted into ethyl acetate, washed free of acid, and passed over anhydrous sodium sulphate. the fatty acid composition was analyzed using an agilent 6890 n series gas chromatography equipped with fid detector on a split injector. a fused silica capillary column (db-225, 30 m 0.32 m i.d., j & w scientifics, usa) was used with the injector and detector temperature maintained at 230c and 250c, respectively. the oven temperature was programmed at 160c for 2 min and finally increased to 230c at 4c / min. the carrier gas was nitrogen at a flow rate of 1.5 ml / min. methyl esters were produced from the oil of hura crepitans using a two - step reaction system. the first step involved the use of 2% sulphuric acid in methanol and secondly transesterification reaction using koh as catalyst. the oil was first esterified using 2% sulphuric acid in methanol at 70c to convert the free fatty acid content to methyl esters. the esterification was carried out for 2 h, and the progress of the reaction was monitored using tlc to check the conversion of the free fatty acids to esters. the resultant product was extracted with ethyl acetate, washed with water until free of acid, passed over sodium sulphate, and concentrated using a rotary evaporator. the esterified oil was finally transesterified using 1% koh in methanol at 70c. the methyl esters formed were extracted with ethyl acetate, washed free of koh, dried over sodium sulphate, and concentrated in a rotary evaporator. the epoxidation was carried out in a 500 ml three - necked round - bottom flask equipped with a thermometer sensor and a magnetic stirrer. the methyl esters (0.0482 mol) and 100% formic acid (0.106 mol) were placed in the flask and cooled to a temperature of 15c while stirring. hydrogen peroxide (0.407 mol) this precaution was taken to prevent overheating of the system due to the exothermic nature of epoxidation reactions. the temperature was later raised to 70c and maintained at this temperature for 3 h. after the formation of epoxides, the mixture was cooled to room temperature, and the epoxidised methyl esters were extracted with ethyl acetate, washed with water until free of acid, and passed over sodium sulfate. the epoxidised methyl esters of hura crepitans (0.042 mol) were dissolved in 50 ml of dimethylformamide in a 250 ml three - necked round - bottom flask ; nh4cl (0.084 mol) was added to the solution while stirring as the mixture was gradually heated and maintained at 60c. after being heated and stirred for 10 min, nan3 (0.084 mol) the reaction continued until all the oxirane rings had disappeared as monitored by ftir and nmr. at the end of the reaction, the reaction mixture was allowed to cool and was later dissolved in diethyl ether in a separating funnel. the ftir spectra of oil, methyl esters, epoxidised methyl esters, and azidohydrin were recorded using avatar, 360 nicolet smart dura sample ftir. hnmr and cnmr spectra of oil, methyl esters, epoxidised methyl esters, and azidohydrin were obtained using a 500 and 75 mhz bruker avance iii biospin ag nmr spectrophotometer in cdcl3 containing some amount of tms as internal standard. the yield of oil from the seed of hura crepitans was 37.75 0.40% iodine value and acid values were 120.10 0.70 g iodine/100 g and 4.41 0.20%, respectively, while the saponification value was found to be 210.10 0.40 mg koh / g. the dominant fatty acid in the oil was c18:2 (52.8 0.10%) as presented in table 1. the iodine value sorts the oil in the range of cotton seed oil or corn oil (gossypium hirsutum and zea mays, resp.) [27, 28 ] while the saponification value is somewhat higher, like tamanu oil (calophyllum inophyllum). saponification values exceeding 200 are quite rare cases, which may be important for specific industrial applications. the ease of availability, economic viability and prospect, oil yield, and high amounts of unsaturated fatty acid (82.20 0.20%) were the major reasons for selecting hura crepitans seed oil for the synthesis of azidohydrin ; moreover, the double bonds are points at which different functional groups could be introduced into the oil. the hnmr spectra of the oil, methyl esters, epoxidised methyl esters, and azidohydrin are shown in figure 1. the peaks at around 0.88, 1.25, and 1.6 ppm were attributed to the terminal methyl protons, protons of the repeating methylene units, and protons of the methylene group to the carbonyl group, respectively [30, 31 ]. the signal at 2.0 ppm was assigned to allylic methylene protons while that at 2.7 ppm was assigned to the bisallylic methylene protons. the peak at 2.3 ppm was assigned to protons of the methylene group and to the carbonyl group while peak at 5.5 ppm was considered to be from the olefinic protons, confirming the presence of unsaturation in the oil and methyl esters as indicated by the ftir results. the signal appearing at around 4.14.3 ppm in the oil was assigned to the glyceridic methylene protons. an intense signal was also found at 3.7 ppm in the methyl esters which could be attributed to the protons of the methoxyl group of the esters. figure 2 presents the cnmr of the oil, methyl esters, epoxidised methyl esters, and azidohydrin. the signals at around 60.30, 62.10, and 69.20 in the cnmr spectra of the oil and methyl esters revealed the presence of glyceryl carbon atoms in the triglyceride molecules. disappearance of the glyceryl carbon signals in the oil spectrum and appearance of new signal at 51.60 (e) in the spectrum of the methyl esters could be accounted for as being due to methoxy carbon which is indicative of the formation of the methyl esters of fatty acids supporting the result of the hnmr (3.5 ppm). figure 3 shows the ftir result of the oil, methyl esters, epoxidised methyl esters, and azidohydrin. h stretching of c = c h in the oil and methyl esters of hura crepitans indicating the presence of unsaturated functional group. spectrum of the epoxidised methyl esters showed complete disappearance of the peak at 3003.10 cm after 3 h. the presence of peak at 834 cm in the epoxidised methyl esters suggested the formation of epoxides. this peak at 834 cm was due to the symmetric in - plane deformation of the epoxy group. also, the peak at 1250 cm may be attributed to the symmetric ring stretching of the epoxy group. in the hnmr, the signal of the epoxy protons was found at 2.9 ppm in the epoxidised methyl esters. the cnmr revealed signal at 5557 ppm (f) which accounted for the epoxy ring carbons of the methyl esters. the ftir, hnmr, and c - signal analysis confirmed the conversion of the epoxidised methyl esters to azidohydrin with a yield of 91.20%. the opening of the oxirane ring of the epoxidised methyl esters gave secondary hydroxyl group and vicinal azido group with band at around 3394 cm and 2101 cm, respectively, while the peak at 1737 cm was considered to be the carbonyl of ester group. in the hnmr spectra, the epoxides peak at 2.9 ppm disappeared, and the azidohydrin peak appeared at around 3.1 ppm. the signal of the secondary hydroxyl functional group was found at 3.4 ppm while the hydroxyl groups formed as a result of the opening of the epoxy ring were at 2.8 and 2.7 ppm. the c spectra revealed the carbinol peak at 73.25 and 73.30 ppm and the carbon bearing the azide resonates at 66.77 and 66.83 ppm. the azidohydrin prepared here can serve as starting material for the production of vicinal amino hydroxyl methyl esters, which is a class of compounds frequently used in the surfactant and cosmetic industries. in the present study oil was extracted from the seed of hura crepitans, which was analyzed and used in the synthesis of the corresponding azidohydrin. the oil of hura crepitans has an iodine value of 120.10 0.70 g iodine/100 g and a high saponification number of 210.10 0.40 mg koh / g with the dominant fatty acid being c18:2 (52.8 0.10%). the presented plant oil adds to the known renewable resources that can be used to make valuable products, and we anticipate that the azidohydrin synthesized will find applications in the formulation of high - performance chemical products.	the replacement of petrochemicals by oleochemical feedstocks in many industrial and domestic applications has resulted in an increase in demand for biobased products and as such recognizing and increasing the benefits of using renewable materials. in line with this, the oil extracted from the seed of hura crepitans was characterized by an iodine value of 120.10 0.70 g iodine/100 g and a saponification number of 210.10 0.40 mg koh / g with the dominant fatty acid being c18:2 (52.8 0.10%). the epoxidised fatty acid methyl esters prepared from the oil were used to synthesise the azidohydrin with a yield of 91.20%. the progress of the reaction was monitored and confirmed using ftir and nmr. this showed the seed oil of hura crepitans as a renewable resource that can be used to make valuable industrial and domestic products.
as first defined by wilson (1), necrotizing fasciitis is a highly lethal infection of deep - seated subcutaneous tissue and superficial fascia. initial clinical characteristics usually include pain, swelling, and erythema, and as the disease progresses skin discoloration, bullae and necrosis are often observed. the mortality rate of necrotizing fasciitis ranges from 20% to 60% (2), and -hemolytic streptococci or mixed infections of both aerobic and anaerobic flora are the usual causes. streptococcus pneumoniae is a common pathogen implicated in community - acquired pneumonia, sinusitis, otitis media and meningitis., we describe a patient who presented with necrotizing fasciitis and meningitis from whom s. pneumoniae was isolated as the single pathogen. to the best of our knowledge, we reviewed all english language articles published from 1970 to 2010 through medline using the following keywords : " necrotizing fasciitis, " " soft tissue infection " and " streptococcus pneumoniae. " a 62-yr - old man was hospitalized in a tertiary medical center in march 2010 with a five day history of pain and swelling in the left lower leg and progressing swelling over the right hand. he had developed alcoholic liver cirrhosis 27 yr prior, and was being treated for his condition with furosemide. one year prior to presentation, he was also diagnosed with type 2 diabetes mellitus. on physical examination the patient was severely ill and had a temperature of 36.0. his blood pressure was 80/60 mmhg, and his pulse was 128 beats / min. signs indicative of compartment syndrome involving the right hand and left leg were present, and skin discoloration with bullae was observed over the lateral malleolar area of his left leg (fig. laboratory studies showed an elevated leukocyte count of 11,770/l and thrombocytopenia (platelet count, 44,000/l). there were signs of impaired coagulation, including a prolonged partial thromboplastin time of 50 sec (normal maximum, 36 sec) and an international normalized ratio of 1.6 (normal range, 0.90 - 1.20). the following values were also increased from normal levels : serum aspartate aminotransferase 82 u / l (normal range, 10 - 40 u / l) ; serum total bilirubin 2.63 mg / dl (normal range, 0.2 - 1.1 mg / dl) ; serum alkaline phosphatase 175 u / l (normal range, 50 - 128 u / l) ; creatinine 1.6 (normal range, 0.6 - 1.2 mg / dl) ; and creatine kinase 266 u / l (normal range, 60 - 220 u / l). serologic tests were negative for hiv, hepatitis b and hepatitis c. compartment syndrome was aggravated and the patient underwent surgical exploration of the left lower leg and the dorsum of right hand 9 hr after admission. intra - operative findings showed large areas of necrotic subcutaneous tissue and fascia but no pus formation (fig. microscopic histopathology of the sample taken from the necrotic area during debridement is shown fig. because of the patient 's history of eating sea squirts, we could not exclude the possibility of vibrio infection, and antibiotic therapy with dose - adjusted cefotaxime (2 g every 12 hr) and doxycycline (100 mg day 3, cultures of two blood samples and wound aspirates showed s. pneumoniae, so doxycycline was withdrawn. the e - test was used to determine the minimal inhibitory concentration (mic) of the antibiotic. antibiotic susceptibility testing of the isolate revealed mics to penicillin of 0.75 g / ml, cefotaxime 0.75 g / ml, and ceftriaxone 0.5 g / ml. serological analysis revealed that the s. pneumoniae isolate belonged to serotype 9 v. despite aggressive daily surgical debridement, the necrosis extended to the other leg. on hospital day 6, a change in the patient 's mental status occurred. no focal neurological deficits were noted. a lumbar puncture was performed, and the cerebrospinal fluid (csf) had a protein concentration of 66 mg / dl, a glucose level of 52 mg / dl, a csf - blood glucose ratio less than 0.25, 0 red blood cells/l, and 42 white blood cells/l (55% neutrophils and 18% lymphocytes). therapy with vancomycin (1 g every 12 hr) was added and the final result of csf culture was negative. dialysis was begun on day 7. on hospital day 8, the patient died of septic shock and multi - organ failure. necrotizing fasciitis is not so rare, but necrotizing fasciitis due to s. pneumonia is exceedingly rare : we found only 19 reported cases, including the present report (3 - 17). ages ranged from 21 - 83 yr, with an average of 50.1 yr (table 1). predisposing factors of necrotizing fasciitis are an age of more than 50 yr, diabetes mellitus, drug abuse, hypertension, vascular diseases and obesity (18). the majority of these pneumococcal necrotizing fasciitis patients had immunocompromising conditions, such as systemic lupus erythematosus (10, 14, 16), diabetes mellitus (4, 5, 17), chronic renal failure (8, 15), chronic liver disease (4, 12), intravenous drug use (13) and cardiovascular disease (4). we also found reports of two patients who had undergone intramuscular injection with nonsteroidal anti - inflammatory drugs (6). the present findings agree with those of previous studies (19) that the common risk factors for invasive pneumococcal infections are alcoholism, splenectomy, connective tissue disease, steroid use, diabetes mellitus and intravenous drug use. furthermore, these findings show that the immune system is an important defense against s. pneumoniae. four of the patients described in previous studies had no underlying disease (3, 7, 9, 11), but all patients had experienced some form of trauma before admission. therefore, small skin breaks due to blunt trauma are possible routes of inoculation, and any resulting hematoma might act as a nidus for the localization of s. pneumoniae infection (19). the salient feature of pneumococcal necrotizing fasciitis is its predilection for lower extremity infection (14 patients). the five other cases involved the neck, flank and upper extremities (3, 4, 8, 14, 16). s. pneumoniae was isolated from the cultures of blood and site aspirations in 13 cases, while in 3 cases it was found only in the blood (10, 12, 14), and in 3 cases only in the local aspirates (3, 5, 7). even though there were the possibilities of report bias, which means more fatal cases had been reported, ten of the 19 patients died of pneumococcal necrotizing fasciitis (mortality rate, 52.6%). all three patients who did not receive aggressive surgical debridement died (4, 9, 10), which underscores the importance of early diagnosis and prompt surgical intervention. the clinical characteristics of necrotizing fasciitis caused by group a -hemolytic streptococci and s. pneumoniae are similar. they all occur frequently in elderly patients and in patients who are immunocompromised or have other underlying conditions. in the present case, several antigenic molecular determinants such as streptococcus pyrogenic exotoxins a, b and c encoded by the spe loci have been associated with necrotizing fasciitis due to group a -hemolytic streptococcus. pneumococcal necrotizing fasciitis, on the other hand, causes an invasive infection through hyaluronidase and neuraminidase activity, which play roles in the migration of the organism through the fascial tissue (3). moreover, there no dna fragments corresponding to the spe gene were detected in three previously reported cases of pneum ococcal necrotizing fasciitis (4). current pneumococcal vaccines contain a mixture of the capsular polysaccharides from the more common serotypes and are effective against invasive disease (20). to assess whether certain serogroups of s. pneumoniae are preferentially associated with specific necrotizing fasciitis, we analyzed the pneumococcal cases we found in the literature. a total of 7 cases included information about serotyping (3, 4, 8, 11). serogroup 9 was the most often isolated (3 cases), while serogroup 5, 6a, 10a and 14 were also isolated from one patient each. not much is currently known about the effect of pneumococcal vaccines on necrotizing fasciitis, and our analysis suggests a possible approach to preventing it. pneumococcal vaccines may prevent not only common infections like pneumonia, but also necrotizing fasciitis in patients with predisposing factors as in the present case. we suggest that clinicians consider the use of pneumococcal vaccines for high risk patients. in summary, we describe a case of necrotizing fasciitis caused by s. pneumoniae. to the best of our knowledge, our case differs from the 18 other cases we reviewed in the literature in that our patient 's necrotizing fasciitis was followed by meningitis. host factors such as immunodeficiency or underlying diseases may affect the course and severity of pneumococcal necrotizing fasciitis. vaccination and early implementation of optimal therapies may reduce the mortality rate of pneumococcal necrotizing fasciitis.	necrotizing fasciitis is known to be a highly lethal infection of deep - seated subcutaneous tissue and superficial fascia. reports of necrotizing fasciitis due to streptococcus pneumoniae are exceedingly rare. we report a case of necrotizing fasciitis in a 62-yr - old man with liver cirrhosis and diabetes mellitus. he presented with painful swelling of left leg and right hand. on the day of admission, compartment syndrome was aggravated and the patient underwent surgical exploration. intra - operative findings revealed necrotizing fasciitis and cultures of two blood samples and wound aspirates showed s. pneumoniae. the patient died despite debridement and proper antimicrobial treatment. to the best of our knowledge, this is the first case of fatal necrotizing fasciitis with meningitis reported in korea. we also review and discuss the literature on pneumococcal necrotizing fasciitis.
eighty - five percent of salivary gland tumors of the parotid gland are pleomorphic adenomas. all demonstrate low recurrence risk of between 5 and 9.7%, but carry significant risks of facial nerve damage [1, 2 ]. effects of facial nerve damage can include facial asymmetry, synkinesis and frey 's syndrome that can impact an individual 's self - image, mental health and ability to communicate [1, 3 ]. brackmann score more than grade ii and 11.3% will demonstrate a score of grade iii or more 1 year after surgery. this may be a consequence of greater infiltration of the facial nerve or increased surgical difficulty due to disturbed anatomy [2, 4 ]. electrical stimulation (es) involves placing surface electrodes over the affected nerve to stimulate and recover its function. the stimulator generates an action potential in both nerves and muscle tissues that has been demonstrated in animal models to promote their structural recovery. we report our promising findings of two cases treated with es for facial weakness after revision surgery for pleomorphic adenoma. two subjects were referred to the national clinical functional electrical stimulation centre with significant facial palsy after revision pleomorphic adenoma surgery : subject a was a 35-year - old woman who had significant left facial weakness after surgery for her third recurrence of pleomorphic adenoma. she had previously been treated with enucleation at age 15, superficial left parotidectomy at age 30 and local resection using an intraoral approach at age 34. three months after surgery, her house brackmann score was grade iv with a sunnybrook score of 54 due to resting asymmetry and weakness particularly effecting the left side of the mouth. subject b was a 25-year - old man who had significant facial asymmetry after a right superficial parotidectomy for pleomorphic adenoma with repair of a communicating branch between the zygomatic and buccal facial nerve branches. previous treatments included removal of a presumed right sebaceous cyst at age 15 (histologically demonstrated pleomorphic adenoma) with facial nerve branch repair. his face was symmetrical at rest ; however, he had significant reduction of right - side mouth movement on smiling. stimulation was provided by a two - channel microprocessor - controlled stimulator (microstim2v2(ms2v2) [odstock medical limited ]) producing 300 s balanced monophasic pulses at 40 pps with an output of up to 120 ma. this was applied to both patients affected sides overlying the buccal branch of the facial nerve using pals plus electrodes (axelgaard). the unaffected side was stimulated initially to find motor points and estimate thresholds for contraction of the contralateral side. electrodes were placed as close as possible together to ensure that the current path was superficial. this was increased by 5 minutes per session per week until a target of 30 minutes twice a day was reached. subjects were reviewed at set up and 2, 6, 10, 18 and 26 weeks after. at reviews, digital photographs, facial measurements, house facial measurements were mapped during expressions using an engineer 's vernier caliper (table 1). comparison of measurements between the affected and unaffected sides of the face indicated the severity of asymmetry. table 1:measurements depicting distances (in millimeters) between corner of the mouth and various markers when performing expressions.distance (mm)subject asubject bpre - espost - espre - espost - esr sidel sider sidel sider sidel sider sidel sidelateral canthus corner of mouth when smiling6174676771676062lateral canthus corner of mouth when kissing7476797980827475corner of mouth ear lobe when smiling8393858584798484r = right ; measurements depicting distances (in millimeters) between corner of the mouth and various markers when performing expressions. figure 1:facial symmetry of subjects performing a smile before, during and after treatment with es. facial symmetry of subjects performing a smile before, during and after treatment with es. facial measurements initially demonstrated a large difference in the distance between the unaffected and affected side in both patients. both subjects showed a marked improvement in symmetry when smiling after treatment (table 1). her resting symmetry demonstrated drooping of the cheek and mouth on the affected side that resolved after treatment. initially there was only slight movement of mouth opening, snarling and lip puckering on the affected side that improved to almost complete movement. brackmann score improved from grade iv to ii. at the initiation of the study, he had no resting asymmetry. prior to stimulation, his ability to open his mouth, snarl and pucker his lip only showed slight movement, but recovered to complete movement in all domains except lip pucker (table 2). table 2:sunnybrook scores of both subjects before and after es treatment.sunnybrooksubject asubject bscore criteriapre - espost - espre - espost - essymmetry of voluntary movement forehead wrinkle5555 gentle eye closure4555 smile2425 snarl2425 lip pucker3424 total16221624 total 464886496resting symmetry eye shape0000 nasolabial fold1000 mouth1000 total2000 total 510000synkinesis with voluntary movement forehead wrinkle0000 gentle eye closure0000 smile0000 snarl0000 pucker0000total0000total composite score54886496composite score = symmetry of voluntary movement 4 ([resting facial symmetry 5 ] + synkinesis with voluntary movement) composite score = symmetry of voluntary movement 4 ([resting facial symmetry 5 ] + synkinesis with voluntary movement) neither patient suffered from frey 's syndrome or synkinesis, and both have sustained their facial symmetry for 3 and 15 months after treatment. interest in es began in the late 1950s focusing on facial paresis after bell 's palsy. brackmann scores of those treated for early bell 's palsy with es compared with no treatment. in contrast, tucany. noted the addition of es to physical therapy and corticosteroids significantly improved house assessed those with stable chronic facial palsies due to bell 's palsy (lasting on average 3.7 years) and surgical sacrifice (lasting on average 7.2 years) noting significant improvement in house unfortunately, there are many differences between studies such as stimulator settings and duration of treatment, which make comparison difficult. to our knowledge, no studies have evaluated es in patients after revision pleomorphic adenoma surgery. in our case reports, both subjects had unchanged severe facial palsy since revision pleomorphic adenoma excision for 1 and 3 months postoperatively. during treatment with es, these findings suggest that es may benefit patients with facial palsy after revision pleomorphic adenoma surgery. both subjects felt their facial palsy was stable prior to treatment ; however, it is possible that spontaneous recovery may have occurred. unfortunately, larger studies would be difficult to pursue due to a low incidence of cases. i.s. has a financial relationship with odstock medical ltd (oml), the company that manufactures and distributes the ms2v2 stimulator used for treatment. oml is majorly owned by salisbury nhs foundation trust and pays the trust a license fee for use of the patents.	surgery for pleomorphic adenoma recurrence presents a significant risk of facial nerve damage that can result in facial weakness effecting patients ability to communicate, mental health and self - image. we report two case studies that had marked facial weakness after resection of recurrent pleomorphic adenoma and their progress with electrical stimulation. subjects received electrical stimulation twice daily for 24 weeks during which photographs of expressions, facial measurements and sunnybrook scores were recorded. both subjects recovered good facial function demonstrating sunnybrook scores of 54 and 64 that improved to 88 and 96, respectively. neither subjects demonstrated adverse effects of treatment. we conclude that electrical stimulation is a safe treatment and may improve facial palsy in patients after resection of recurrent pleomorphic adenoma. larger studies would be difficult to pursue due to the low incidence of cases.
	mussel adhesion is mediated by foot proteins (mfp) rich in a catecholic amino acid, 3, 4-dihydroxyphenylalanine (dopa), capable of forming strong bidentate interactions with a variety of surfaces. a facile tendency toward auto - oxidation, however, often renders dopa unreliable for adhesion. mussels limit dopa oxidation during adhesive plaque formation by imposing an acidic, reducing regime based on thiol - rich mfp-6, which restores dopa by coupling the oxidation of thiols to dopaquinone reduction.
in the past decade or so, there have been many developments in minimally invasive glaucoma surgeries in the hope to minimize complications from traditional filtration surgeries while achieving a reasonable amount of intraocular pressure (iop) lowering. the trabectome is a surgical device approved for use by the food and drug administration in 2004. it uses an electrical probe placed through a 1.8 mm clear corneal incision to reestablish the outflow of aqueous by stripping the trabecular meshwork and the inner wall of the schlemm canal. the trabectome can be done as a stand - alone procedure or in combination with cataract extraction or other glaucoma surgeries. in a retrospective case series involving 1127 trabectome cases, the mean iop reduction was approximately 39% and antiglaucoma medication reduction was around 57% at 24 months. however, about 34.5% of the reported trabectome cases were combined with other procedures and not just the trabectome procedure alone. the patient demographics were also predominantly caucasian or hispanics, with only 3.5% of the patients being of asian ethnicity. the aim of this study was to investigate the safety and efficacy of using the trabectome as a stand - alone procedure in the treatment of pseudophakic eyes in chinese open - angle glaucoma (oag) patients. this was a prospective study conducted at a district hospital in hong kong special administrative region, china. oag patients requiring filtration surgery for iop control despite maximally tolerated antiglaucoma medications were recruited. the inclusion criteria included : consenting adults > 18 years of age ; open - angle configuration of grade 2 or above in 90 on gonioscopy (shaffer grading), pseudophakia, and evidence of glaucomatous optic neuropathy on optical coherence tomography or humphrey visual field. the exclusion criteria included subjects with only one functional eye and those with preexisting corneal pathologies or scars. the authors had full autonomy over the data analysis and write - up with no involvement by the funding source. the trabectome procedure : topical anesthesia with xylocaine gel 2% and intracameral lignocaine 2%.the operating microscope was tilted to 30 away from the surgeon and the patient 's head was turned in the opposite direction of the operating eye to maximize the visualization of the nasal angle.a 1.8 mm clear cornea incision was made temporally.placement of the trabectome tip into the schlemm canal under gonioscopic guidance.120 ablation was made with the trabectome to strip the inner wall of schlemm and trabecular meshwork.irrigation and aspiration was performed to washout any blood reflux.stromal hydration to close the corneal wound.intracameral cefuroxime 1 mg in 0.1 ml. topical anesthesia with xylocaine gel 2% and intracameral lignocaine 2%. the operating microscope was tilted to 30 away from the surgeon and the patient 's head was turned in the opposite direction of the operating eye to maximize the visualization of the nasal angle. 120 ablation was made with the trabectome to strip the inner wall of schlemm and trabecular meshwork. postoperative medications include pilocarpine 1% 4 times daily for 4 weeks, topical antibiotic 4 times daily, and topical steroids 4 times daily for 1 to 2 months postoperatively. patients were seen the day 1 postoperatively and at 1 week, 1 month, and every 3 months thereafter. at about 1 month postoperatively, antiglaucoma medications were stepped down in the following order : pilocarpine, prostaglandin analogs, topical carbonic anhydrase inhibitors, alpha - agonists, and then beta - blockers. medications were titrated according to the attending ophthalmologist 's discretion taking into consideration the severity of each patient 's disease and individual target iop 's, using a 25% iop reduction from initial iop as a minimum reference as per the findings from the early manifest glaucoma trial. the main outcome measure was the goldmann applanation - measured iop at the preoperative baseline and after the trabectome procedure (day 1, 1 week as well as 1, 3, and 6 months). secondary outcome measures included : complications from the trabectome procedure (intraoperative and postoperative) as well as the number of antiglaucoma medications at baseline as well as 3 and 6 months postoperatively. success was defined as iop 21 mm hg with or without topical antiglaucoma medications at 6 months. failure was defined as iop > 21 mm hg at 6 months or requiring secondary filtration surgery within 6 months postoperatively. normality testing of the data was performed using the dagostino & pearson omnibus normality test to confirm gaussian distribution. the following were compared using the repeated measures anova with tukey posttest : iop at baseline and day 1, 1 week, 1 month, 3 months, and 6 months postoperatively.number of antiglaucoma medications at baseline and 3 and 6 months postoperatively. iop at baseline and day 1, 1 week, 1 month, 3 months, and 6 months postoperatively. number of antiglaucoma medications at baseline and 3 and 6 months postoperatively. for iop and medication analyses, subjects that had secondary filtration procedure within 6 months of the trabectome were excluded from the analyses, as their results would not be solely contributed to the effects of the trabectome. for non - numerical va, the following denotations were used : finger count (fc) = 1.7 logmar, hand movement (hm) = 2.0 logmar, light perception (lp) = 2.3 logmar, and no light perception (nlp) = 3.0 logmar. normality testing of the data was performed using the dagostino & pearson omnibus normality test to confirm gaussian distribution. the following were compared using the repeated measures anova with tukey posttest : iop at baseline and day 1, 1 week, 1 month, 3 months, and 6 months postoperatively.number of antiglaucoma medications at baseline and 3 and 6 months postoperatively. iop at baseline and day 1, 1 week, 1 month, 3 months, and 6 months postoperatively. number of antiglaucoma medications at baseline and 3 and 6 months postoperatively. for iop and medication analyses, subjects that had secondary filtration procedure within 6 months of the trabectome were excluded from the analyses, as their results would not be solely contributed to the effects of the trabectome. snellen visual acuity (va) was converted to logmar units for statistical analysis. for non - numerical va, the following denotations were used : finger count (fc) = 1.7 logmar, hand movement (hm) = 2.0 logmar, light perception (lp) = 2.3 logmar, and no light perception (nlp) = 3.0 logmar. in 19 eyes of 19 chinese oag subjects, 26.3% were uveitic, 68.4% were primary open angle glaucoma, and 5.3% had a history of chronic angle - closure glaucoma with open - angle configuration after cataract extraction. the subject mean age was 67.5 14.4 years, with 4 females and 15 males. two subjects received secondary filtration surgery at 1 and 3 months after the trabectome surgery, respectively. these 2 cases were excluded from the analyses of iop and medications changes, as per study protocol but were included in the survival analysis. in 17 eyes of 17 remaining subjects, the mean iop before trabectome was 24.4 4.4 mm hg while on 3.9 0.8 to 2.8 types of antiglaucoma eye drops. both the iop and number of medications were significantly reduced at all time intervals following the trabectome procedure as compared to the preoperative baseline (all p 21 mm hg at 1 week after trabectome. three of which subsided with antiglaucoma medications and 2 required secondary filtration surgery as described above. mortality = iop > 21 mm hg or requiring secondary glaucoma filtration surgery. the trabectome has been a minimally invasive procedure that has been popularized in north america and europe for the past decade. its main advantages are that it only requires a small clear corneal incision that is smaller than that of a phacoemulsification wound, it does not disturb or rely on the conjunctiva, and it reestablishes aqueous outflow through the physiological channel with very little risk of postoperative hypotony due to the presence of the episcleral venous pressure. it is a relatively safe procedure with the most common postoperative complications being the need of a secondary trabeculectomy or other glaucoma surgeries and its failure does not affect the success of future trabeculectomies. in our study, we selectively performed the trabectome in pseudophakic eyes for 2 main reasons : to evaluate the stand - alone effect of the trabectome and because in chinese phakic eyes, even with open - angle configuration on gonsioscopy, there is often not enough space for adequate exposure of the trabecular meshwork to allow a safe access for angle surgery without damaging the surrounding structure like the iris. this was gathered from the authors previous experiences with similar angle surgeries like goniosynechialysis and also from the understanding that there may be higher risk of peripheral anterior synechiae formation, iridodialysis, or postoperative fibrosis leading to failure. our results of an iop reduction of 34.8% was comparable to the 25% to 38% reductions reported in the literature for trabectome as a stand - alone procedure in primary open - angle glaucoma (poag). the iop reduction can vary among studies based on whether or not the trabectome was combined with other procedures like phacoemulsification or other glaucoma filtration procedures, as these are surely to reduce the iop more ; thus, we have only quoted studies using the trabectome as a single procedure. furthermore, the duration of follow - up may also affect the level of iop reduction as the trabectome, just like another other glaucoma surgery, is not ever lasting. our follow - up duration was comparable to studies by pantcheva and kahook while in one of the longest follow - up studies by jordan, the iop reduction dropped to 25% at 40 months postoperatively. our medication reduction of 28.2% after a single session of trabectome also falls within the range as reported from the literature (2167%). the more variable range of medication reduction is related to both patient and surgeon factors including patient 's disease severity, individual target iop, protocol of individual institutions, and the discretion of the attending ophthalmologist. there are very few publications available in the literature reporting the effects of the trabectome for the chinese population. huang published an article in chinese in february 2015 reporting an iop reduction of 21.7%, medication reduction of 40.0% and a success rate of 85.0% at 12 months, which is comparable to our results. mizoguchi reported the results of the trabectome in a japanese population, demonstrating iop reductions of 23.0% and success rates of 51.2% at 2 years. in many of the patients, the surgical groove was no longer clearly visible at 1 month after surgery as it was covered by normal iris tissue (not peripheral anterior synechiae) although this finding did not correlate with iop rise. this is because in the chinese population, there tends to be a smaller anterior chamber area / volume so even in those with open angles, visibility of the angle structures may be limited by gonioscopy alone but during angle surgery, the influx of balanced salt solution helps to deepen the angle improving accessibility. one subject developed herpetic keratitis reactivation, which was unrelated to the procedure itself but arising from the postoperative steroid use. another subject had persistent hyphema requiring anterior chamber washout and on retrospect, keeping the initial iop higher at the end of the operation by reforming the anterior chamber or injecting an air bubble temponade may reduce the chance of bleeding in those high - risk subjects. hyphema is often due to the persistent intraoperative reflux of blood from the episcleral venous system after the sudden pressure lowering ; reflux has been reported to be as frequent as 78%. about 26.3% of our subjects had an iop spike > 21 mm hg at 1 week after trabectome which may be related to postoperative inflammation or an hyphened sensitivity to steroid response after removal of the inner wall of the schelmm canal ; most pressures went down after the temporary addition of antiglaucoma medications. the percentage of iop spike is much higher than the previously reported 5.8% in other studies and this may be related to the release of iris pigments due to the relatively shallower angle and more pigmented iris in our population. of the 2 subjects that required additional glaucoma filtration surgery for iop control, both had a history of uveitic glaucoma. while the trabectome has the theoretical advantage of being minimally invasive without the need of a peripheral iridotomy and has minimal risk of postoperative hypotony, making it a seemingly ideal surgery in uveitic glaucoma, our experience with uveitic patients is that the iop control from trabectome alone may be short - lived or inadequate. on the whole, another point of interest from our study is that 5% of our study subjects had a history of angle - closure configuration before phacoemulsification but at the time of trabectome, all had open - angle configuration suggesting that the procedure can still be useful in those with angle - closure as long as the angles are reopened with other modalities before trabectome. in a study by bussel, there was no significant difference in iop or medication reduction between those with shaffer angle grading 2 versus those with grading 3. our study was limited by the relative small sample size although there are very few studies in the literature reporting the effects of trabectome for the chinese glaucoma population. ideally, a larger sample size would also follow us to conduct subclasses analyses on the different subtypes of oag. furthermore, a longer follow - up duration will enable us to have a clearer picture on the long - term effects and sustainability of the trabectome. future studies involving randomization of treatment and comparison to other glaucoma surgeries can provide a head - to - head comparison to conventional treatments. lastly, the results of this study may not be generalizable to other populations that are significantly different to the one studied. in conclusion, the trabectome 's performance in the chinese oag population was comparable to the data reported in caucasian populations. the mean iop and medication requirement was reduced by about a third from the pretreatment baseline. the procedure was more effective in poag or even angle - closure glaucoma after angle reopening but less so in uveitic glaucoma ; the overall success rate was around 90% at 6 months.	abstractto investigate the clinical outcome of the trabectome in chinese open - angle glaucoma (oag).this prospective case series recruited pseudophakic glaucoma subjects with open - angle configuration. trabeculectomy ab interno was performed using the trabectome to 120 of the trabecular meshwork. intraocular pressure (iop) and medications were recorded preoperatively and every 3 months postoperatively. visual acuity was measured preoperatively and at 1 and 6 months postoperatively. one - way anova with tukey multiple comparison test were used to measure the pre and postoperative parameters.in 19 eyes of 19 chinese subjects, 26.3% were uveitic, 68.4% were primary open - angle glaucoma, and 5.3% had a history of chronic angle - closure glaucoma with open - angles after cataract extraction. the subjects mean age was 67.5 14.4 years, with 4 females and 15 males. two patients required secondary filtration procedure. at 6 months, the iop reduced by 34.8% (24.4 4.4 mm hg to 15.9 5.1 mm hg, p 21 mm hg, 1 had hyphema requiring washout, and 1 had reactivation of herpetic keratitis. the success rate at 6 months was 89.5%.trabectome achieved a modest reduction in iop and medications in the majority of pseudophakic chinese oag eyes.
peripheral artery disease (pad) affects 12%20% of americans 60 years of age and older.1,2 although balloon angioplasty (ba) is typically the first - line revascularization strategy, intermediate and long - term outcomes are uniformly poor for all but the simplest transatlantic inter - society consensus type a lesions, with rates of restenosis ranging from 40% to 60% at 1 year.3,4 american college of cardiology / american heart association guidelines currently recommend against primary stenting of femoropopliteal lesions (class iii indication).3 remarkably, atherectomy devices as well as stenting are indicated (class iib) as salvage devices to be used in the event of suboptimal ba results.3 the presence of calcified plaque in femoropopliteal lesions is common, and arterial wall calcium is associated with higher rates of procedural complications, including flow - limiting dissections which frequently require stent deployment to maintain vessel patency.5,6 further, stenting as salvage therapy in calcified segments after failed ba often results in stent underexpansion and malapposition, which increase the probability of subsequent stent fracture and/or restenosis.7,8 lastly, diffuse pattern in - stent restenosis (isr) has no approved treatment modality and is often a harbinger for multiple reinterventions or definitive bypass surgery. biomechanical stresses across the dynamic femoropopliteal segment further add to the complexity of realizing successful long - term clinical outcomes. lesion modification utilizing atherectomy prior to percutaneous transluminal angioplasty (pta) or stent implantation may be recommended as an alternative treatment option in patients with severely calcified lesions., st paul, mn, usa) is a minimally invasive catheter - based device that may be safely utilized for revascularization of occluded peripheral vessels.911 for example, the oas device has been used to effectively modify severely calcified infrapopliteal lesions prior to ba by changing vessel compliance, resulting in fewer dissections, less bailout stenting, and lower adjunctive balloon pressure to achieve the desired angioplasty result.12 a recent study evaluating modification of calcified lesions in above - the - knee, below - the - knee, and popliteal arteries utilizing the oas device found an elimination of bailout stenting, improved patency, and low reintervention rates.11 despite the recent advances in endovascular therapy, cost - effective methods of care have not been well defined. the direct and indirect costs of cardiovascular and stroke - related care in the us in 2008 were estimated to be us$448.5 billion and represented the largest disease costs to medicare.13 mean medicare expenditures in 2001 for pad were us$13,901 or more than double the average expenditure per enrollee (us$5,833).14 however, little is known about the actual costs associated with different pad interventions among patients with calcified lesions. understanding the economic impact of care for this patient population is vital. using data from the compliance 360 randomized pilot study, we conducted a cost - effectiveness analysis comparing oas with adjunctive ba vs ba alone. utilizing a threshold of us$50,000 per quality - adjusted life year (qaly), the objective of the study is to determine whether orbital atherectomy is a cost - effective alternative for treatment of pad. the study was performed from the third - party payer perspective and is intended for policymakers weighing decisions regarding technology adoption. compliance 360, a prospective study, was conducted to compare the performance of the oas followed by ba (oas+ba) vs ba alone at nine us centers. subjects with rutherford class 24 classification had lesions of $ 70% stenosis and fluoroscopically visible calcium and were equally randomized to each arm of the study after meeting all inclusion criteria and no exclusion criteria. the compliance 360 study design has been previously described.15 oas+ba and ba procedure charges, procedure time, length of stay (los), and stent and balloon utilization were collected for enrolled subjects. the following data were collected for the index procedures and associated hospitalizations from the subjects uniform billing statements (ub-04s) : hospital charges, site of service (inpatient or outpatient setting), and associated medical resource utilization (mru). los and procedural data (procedure time, number of lesions treated, stents utilized, and balloons utilized) were collected from the participating compliance 360 study sites. hospital costs were estimated from hospital charges using hospital - specific, procedure - specific cost - to - charge ratios (ccrs) obtained through the american hospital directory (http://www.ahd.com) and based on the hospital s most recent medicare cost report. all charges and costs are reported in 2010 us dollars. each patient s ub-04 charges for the entire procedure report were used to calculate the estimated costs based on reported inpatient diagnosis - related group (drg) billing code or hospital outpatient international classification of diseases, ninth revision (icd-9) diagnostic code. for inpatient procedures, the average among the three hospitals that specifically reported drgs 252, 253, and 254 was used to calculate the average ccr. for hospital outpatient procedures, the hospital - specific ccr was calculated from the reported average cost and charge listed for the icd-9 diagnostic codes from 440.2x ; ambulatory payment classification codes were not reported. a decision tree was constructed based on procedural success and outcomes through 1 year (figure 1). these endpoints are defined as patency, restenosis of the treated vessel (defined as reported target lesion revascularization [tlr ] or target vessel revascularization [tvr ]), and claudication with no subsequent treatment. since more subjects in the oas arm had multiple lesions requiring treatment, the decision tree and cost - effectiveness analysis compared the subset of subjects with only one lesion treated. primary and secondary clinical study results are presented alongside cost and qaly data to compare the index procedure costs and longer - term (1-year) comparative effectiveness of oas+ba vs ba alone. for this analysis, we defined procedural success as freedom from adjunctive stenting. procedural success and failure the average cost of reintervention for each arm was estimated based on the types of interventions performed (surgical bypass, stenting, or ba) and the hospital - specific ccrs for those interventions. restenosis requiring intervention evaluated by duplex ultrasound and claudication without intervention were both considered separately for each procedure arm. periprocedural and acute adverse events (pseudoaneurysm and embolic event) were included in procedure costs. an incremental cost - effectiveness ratio was calculated based on proportional cost and qaly for each outcome in oas+ba compared with ba alone. statistics included the number of observations (n), mean, standard deviation, and 95% confidence intervals. statistical analyses were conducted in sas version 9.3 (sas institute, cary, nc, usa). compliance 360, a prospective study, was conducted to compare the performance of the oas followed by ba (oas+ba) vs ba alone at nine us centers. subjects with rutherford class 24 classification had lesions of $ 70% stenosis and fluoroscopically visible calcium and were equally randomized to each arm of the study after meeting all inclusion criteria and no exclusion criteria. oas+ba and ba procedure charges, procedure time, length of stay (los), and stent and balloon utilization were collected for enrolled subjects. the following data were collected for the index procedures and associated hospitalizations from the subjects uniform billing statements (ub-04s) : hospital charges, site of service (inpatient or outpatient setting), and associated medical resource utilization (mru). los and procedural data (procedure time, number of lesions treated, stents utilized, and balloons utilized) were collected from the participating compliance 360 study sites. hospital costs were estimated from hospital charges using hospital - specific, procedure - specific cost - to - charge ratios (ccrs) obtained through the american hospital directory (http://www.ahd.com) and based on the hospital s most recent medicare cost report. all charges and costs are reported in 2010 us dollars. each patient s ub-04 charges for the entire procedure report were used to calculate the estimated costs based on reported inpatient diagnosis - related group (drg) billing code or hospital outpatient international classification of diseases, ninth revision (icd-9) diagnostic code. for inpatient procedures, the average among the three hospitals that specifically reported drgs 252, 253, and 254 was used to calculate the average ccr. for hospital outpatient procedures, the hospital - specific ccr was calculated from the reported average cost and charge listed for the icd-9 diagnostic codes from 440.2x ; ambulatory payment classification codes were not reported. a decision tree was constructed based on procedural success and outcomes through 1 year (figure 1). these endpoints are defined as patency, restenosis of the treated vessel (defined as reported target lesion revascularization [tlr ] or target vessel revascularization [tvr ]), and claudication with no subsequent treatment. since more subjects in the oas arm had multiple lesions requiring treatment, the decision tree and cost - effectiveness analysis compared the subset of subjects with only one lesion treated. primary and secondary clinical study results are presented alongside cost and qaly data to compare the index procedure costs and longer - term (1-year) comparative effectiveness of oas+ba vs ba alone. for this analysis, we defined procedural success as freedom from adjunctive stenting. the average cost of reintervention for each arm was estimated based on the types of interventions performed (surgical bypass, stenting, or ba) and the hospital - specific ccrs for those interventions. restenosis requiring intervention evaluated by duplex ultrasound and claudication without intervention were both considered separately for each procedure arm. periprocedural and acute adverse events (pseudoaneurysm and embolic event) were included in procedure costs. an incremental cost - effectiveness ratio was calculated based on proportional cost and qaly for each outcome in oas+ba compared with ba alone., statistics included the number of observations (n), mean, standard deviation, and 95% confidence intervals. statistical analyses were conducted in sas version 9.3 (sas institute, cary, nc, usa). twenty - five subjects with 38 lesions and 25 subjects with 27 lesions were randomized to receive oas+ba and ba alone, respectively. prevalence of diabetes was significantly higher (p<0.05) in the oas+ba arm (72%) than in the ba - alone arm (40%). the number of lesions per patient (1.5 vs 1.1, p<0.05) and number of lesions with single - vessel runoff (18 vs 3, p<0.05) were also significantly higher in the oas+ba arm, while mean percent stenosis was significantly lower (83.9% vs 92.8%, p=0.003). there were no other statistically significant differences in patient demographics, comorbidities, medical history, lesion characteristics, plaque morphology, or calcium scores. in the oas+ba study arm, 17 outpatient and eight inpatient procedures were performed, with two of the inpatient procedures performed on subjects with complications and/or comorbidities (drg code 253) and an additional two on subjects with major complications and/or comorbidities (drg code 254). in the ba arm, 19 procedures were performed on an outpatient basis, with the six remaining procedures resulting in an inpatient hospital stay. of the six inpatient procedures, two were for subjects with diagnostic codes indicating procedural complications and/or patient comorbidities. table 1 provides summary statistics regarding number of lesions treated as well as intra- and post - procedure mru as specified above. despite the statistically higher lesion counts, significantly fewer stents (0.1 vs 1.1 per person) were utilized during the oas+ba procedures, while mean balloon usage was comparable between treatment groups. mean hospital charges (us$51,755 vs us$39,922) and estimated hospital costs (us$15,100 vs us$11,016) were higher for oas+ba compared with ba alone, but the difference was not statistically significant. mean procedure time was significantly longer (96 vs 69 minutes, p<0.005) in the oas+ba treatment group, while the mean patient los was not significantly different for oas+ba (22.4 hours) vs ba alone (23.7 hours). a linear regression analysis was fit to determine whether there was an association of cost and number of lesions treated. because of the limited number of subjects with more than one lesion treated, two lesion cohorts were created : 1) one lesion treated and 2) more than one lesion treated. there was a statistically significant difference in cost between the lesion cohorts, with the lower costs being seen in those with only one lesion treated (p=0.0014). this effect also holds when evaluating the number of lesions as a continuous covariate / predictor in the linear regression model (p=0.0076). there were six oas and two ba subjects with multiple lesions. among subjects with one lesion treated (the majority of subjects) where lesion cohort is included as a predictor in the model, we saw less evidence that a difference in average cost existed between arms (adjusted p - value for study arm = 0.1545). a linear regression model was fit to see whether there was a differential effect on cost between study arms across lesion cohorts (one lesion vs more than one lesion). there was statistical evidence of a possible interaction effect (p=0.0360) ; thus, it is appropriate to report on subjects separately by lesion cohort. figure 2 shows the average costs by study arm and lesion cohort, along with 95% confidence intervals of the mean. with only two subjects in the ba - alone arm with more than one lesion, there are insufficient data to get a reasonable estimate of cost in that group of subjects. excluding those subjects with more than one lesion, we see that there is no evidence of a statistical difference in cost between study arms (p=0.4694). the proportion of index procedure success and endpoints for the single - lesion population are presented in figure 1. note that the percentage of subjects who were free from acute adjunctive stenting was 94.7% (18/19) and 17.4% (4/23) for oas+ba vs ba alone, respectively (p<0.0001). freedom from acute stenting, restenosis (due to tlr or tvr), or claudication at 1 year was 73.7% (14/19) and 8.7% (2/23) for oas+ba vs ba alone, respectively. the estimated mean index procedure costs (for the single - lesion population) by procedural success and failure were us$10,516 and us$12,030, respectively, for oas+ba and us$6,951 and us$9,860, respectively, for ba alone. subjects that required intervention to treat restenosis were assumed to have transitioned to lower qaly prior to treatment and experienced improved qol post - procedure. using these data, the mean costs for each outcome, and previously reported qaly, we approximated incremental cost of oas+ba vs ba alone at 1 year as us$549 and incremental qaly as 0.16. this results in an incremental cost - effectiveness ratio of us$3,441 (table 4), well below the us$50,000 threshold. the proportion of index procedure success and endpoints for the single - lesion population are presented in figure 1. note that the percentage of subjects who were free from acute adjunctive stenting was 94.7% (18/19) and 17.4% (4/23) for oas+ba vs ba alone, respectively (p<0.0001). freedom from acute stenting, restenosis (due to tlr or tvr), or claudication at 1 year was 73.7% (14/19) and 8.7% (2/23) for oas+ba vs ba alone, respectively. the estimated mean index procedure costs (for the single - lesion population) by procedural success and failure were us$10,516 and us$12,030, respectively, for oas+ba and us$6,951 and us$9,860, respectively, for ba alone. in addition, the cost to treat restenosis was higher in ba alone vs oas+ba (us$13,734 vs us$20,609) (table 2). subjects that required intervention to treat restenosis were assumed to have transitioned to lower qaly prior to treatment and experienced improved qol post - procedure. using these data, the mean costs for each outcome, and previously reported qaly, we approximated incremental cost of oas+ba vs ba alone at 1 year as us$549 and incremental qaly as 0.16. this results in an incremental cost - effectiveness ratio of us$3,441 (table 4), well below the us$50,000 threshold. for the subset of subjects with one lesion, compliance 360 reported higher (though not significantly) index procedure costs for oas+ba compared with ba alone. when effectiveness is measured in terms of restenosis and/or subsequent tlr / tvr, oas+ba and ba alone were comparable in terms of outcomes at 12 months. when freedom from acute adjunctive stenting was considered as an additional effectiveness measure, oas+ba compared with ba alone had superior cost - effectiveness, with an expected cost of us$3,441 per qaly gained to achieve an additional patient in whom acute adjunctive stenting was not required. a recent study comparing directional atherectomy using the silverhawk device (covidien, plymouth, mn, usa) to pta to treat femoropopliteal disease reported similar procedural supply costs and overall hospital costs for both treatments with no significant difference in complication rates between the two groups, concluding that the choice of interventional procedure should depend on the operator s preference, skill, and experience.16 safley reported that more stents were used in the pta group compared with the silverhawk group (1.50.8 vs 0.20.5, p<0.001), a similar rate to that in compliance 360.16 complications occurred at a somewhat higher rate in the silverhawk study than the present study. in both studies, differences in device costs (atherectomy vs balloon) were offset by the additional cost of stenting as salvage therapy. long - term health, imputed qol, and economic outcomes were not assessed by safley ;16 therefore, compliance 360 is the first atherectomy study to examine cost - effectiveness explicitly. although overall patency outcomes were similar between treatments at 1 year, the two treatment arms of the study essentially resulted in two distinct outcomes : isr vs rest - enosis without stenting. currently, isr remains an intractable problem with no approved and/or optimal treatments available, while oas+ba may offer more straightforward revascularization options using currently approved existing technologies. since the ba - alone group in compliance 360 led to primary stent placement in the majority of subjects (84%) compared with just 8% of subjects receiving atherectomy, a key outcome of this study is that future treatment options were preserved for the majority of subjects treated with atherectomy. preservation of treatment options translates into increased cost for reintervention as observed in the ba - alone group compared with the oas+ba group. although the number of reinterventions was small, the ba group experienced an average cost of nearly us$7,000 higher for those interventions. in addition, qaly and subsequent cost - effectiveness of oas+ba demonstrate the benefit of maintaining treatment options. this is consistent with previous findings that stenting leads to more stenting and that those subsequent procedures require more devices and lead to increased costs.17,18 while the 1-year restenosis rate for treating calcific occlusive femoropopliteal disease with atherectomy and ba was similar to ba with provisional stenting, the atherectomy - based approach may be more desirable given the high incidence of stenting in the angioplasty arm and the lack of a satisfactory treatment strategy for treating stent restenosis, especially diffuse pattern stent restenosis. the data from this study provide the opportunity to design future studies with the power necessary to draw more definitive conclusions. this study demonstrates that cost of the device alone should not be the deciding factor in selection of a vascular interventional treatment strategy but rather the overall cost of care, longer - term clinical outcomes, and consideration of the limitations and prognoses of salvage therapy. there are a number of limitations to the health economics analysis, including small sample size and limited cost data captured. the following data were not collected in the study : cost of the physician fee for the index procedure ; other post - hospital discharge mru data, including physician office visits and medications ; and patient - reported outcomes such as activities of daily living and qol. lastly, longer - term follow - up data and analysis is required to effectively analyze costs and benefits to the patient and society. additional studies are warranted to fully understand the effect orbital atherectomy has on health outcomes and economics. there are a number of limitations to the health economics analysis, including small sample size and limited cost data captured. the following data were not collected in the study : cost of the physician fee for the index procedure ; other post - hospital discharge mru data, including physician office visits and medications ; and patient - reported outcomes such as activities of daily living and qol. lastly, longer - term follow - up data and analysis is required to effectively analyze costs and benefits to the patient and society. additional studies are warranted to fully understand the effect orbital atherectomy has on health outcomes and economics. pad remains a common and costly disorder, and given an aging population and increasing prevalence of diabetes, the treatment of pad in these patients will have an expanding impact on clinical and economic resources. we found in this study that the index procedure costs and cost - effectiveness to 1 year were comparable for oas+ba vs ba alone. these results provide compelling short - term health and economic data supporting the use of atherectomy in treatment of calcified femoropopliteal lesions, a longstanding challenge for pad interventionalists. additional cost - effectiveness analysis of pad interventions is needed and should be included in all future peripheral vascular device trials.	introductionas cost considerations become increasingly critical when selecting optimal endovascular treatment strategies, a cost - benefit analysis was conducted comparing the diamondback 360 orbital atherectomy system (oas) (cardiovascular systems, inc., st paul, mn, usa) and balloon angioplasty (ba) vs ba alone for treatment of calcified femoropopliteal lesions.patients and methodsthe clinical outcomes from compliance 360, a prospective, multicenter, randomized study comparing oas+ba vs ba alone for treatment of calcified femoropopliteal lesions, were correlated with cost data and previously published quality of life data. site of service, hospital charges, and associated medical resource utilization were obtained from uniform billing statements for index treatments and associated revascularizations out to 1 year. hospital costs were estimated using hospital - specific, procedure - specific cost - to - charge ratios. length of stay and procedural data were collected from participating study sites.resultstwenty-five subjects with 38 lesions and 25 subjects with 27 lesions were randomized to the oas+ba and ba - alone groups, respectively. mean hospital charges (us$51,755 vs us$39,922) and estimated hospital costs (us$15,100 vs us$11,016) were higher for oas+ba compared with ba alone (not statistically significant). stent utilization was statistically significantly higher with ba - alone treatment for all subjects (1.1 vs 0.1, p=0.001) and in the subset of subjects with one lesion (1.0 vs 0.1, p<0.00001). there was a significant difference in cost for single - lesion versus multiple - lesion treatment. using costs and quality - adjusted life years (qalys) for the single - lesion cohort, the 1-year incremental cost of oas+ba vs ba alone was us$549, and incremental qaly was 0.16. this results in an incremental cost - effectiveness ratio of us$3,441, well below the us$50,000 threshold.conclusionone-year index procedure cost and cost - effectiveness were comparable for oas+ba vs ba alone. this study provides compelling cost - effectiveness data for using atherectomy for treatment of calcified femoropopliteal lesions, a longstanding challenge for peripheral artery disease interventionalists.
mycetoma is a chronic granulomatous disease affecting mainly the feet, which are more prone to trauma, and hence more likely to get infected, as compared to other organs in the body such as the lower legs, hands, head, neck, chest, shoulders and arms. although mainly a disease of the tropics, patients residing in temperate regions may also be affected. a noninvasive and early diagnosis may be possible with usg and mri.[358 ] a 50-year - old male from tamil nadu presented with a soft tissue swelling of his ankle and foot. mri showed extensive soft tissue edema in the foot and ankle region with tibial and talar involvement [figure 1a ] and multiple small cystic areas with central hypointensity, suggestive of the dot - in - circle sign [figure 1a, c, d ]. sagittal short - tau inversion recovery (stir) mri image (a) shows osteolytic areas in the tibia (grey arrow) and talus (white arrowhead) ; dot - in - circle lesions (white arrows) are seen in the soft tissue. sagittal ct scan (b) shows osteolytic areas (black arrowhead) and periosteal reaction (white arrow) in the tibia. axial t2w mri image through the sole of the foot (c) and sagittal t2w mri image (d) show multiple t2-bright round lesions (white arrow) with a central dot (white arrowheads), seen clearly in some lesions and faintly in others usg image shows hypoechoic lesions with hyperechoic centers (white arrows) mycetoma or madura foot is a chronic granulomatous infection of the dermis and epidermis caused by the bacteria actinomyces (actinomycetoma) or by true fungi (eumycetoma). it was first described in the indian district of madura in 1846, hence the eponym madura foot. endemic in africa, mexico and india, it is also found in central and south america and the middle and the far east. eumycetoma is more common in areas with scarce rainfall and actinomycetoma in areas of abundant rainfall. the infecting organism is presumed to be directly inoculated after penetration of the skin with a sharp object, e.g., a thorn. patients present with painless subcutaneous nodules and fistulae, from which a purulent exudate may be discharged. the process is usually indolent but with a potential for abscess formation, draining sinus tracts, osteomyelitis, and fistula formation, with severe deformity and disability ensuing if treatment is not provided. although antifungal medication is successful in almost 90% of cases, lesions not arising in the foot or due to fungus tend to have a worse prognosis and require surgery. histologically, the lesion consists of grains of fungal hyphae or bacteria in microabscesses within a granulomatous fibrous - tissue reaction. gram stain, gomori methenamine silver, periodic acid - schiff and lactophenol blue stains are useful to differentiate actinomycetoma and eumycetoma. early laboratory diagnosis, before the appearance of the sinuses and grains, is difficult. though biopsy (with demonstration of the characteristic features) or staining and microbiological culture of the discharge from the lesion usually gives the definitive diagnosis, both are time - consuming procedures and diagnosis may be difficult to achieve, especially with fastidious organisms. radiographs may be normal, demonstrate soft tissue enlargement, bone sclerosis, bone cavities, periosteal reaction, bone expansion, extrinsic cortical scalloping, fanning of the rays or osteoporosis. the bones are almost always attacked from the outside, in contrast to bacterial osteomyelitis. a few radiographic bone changes have been described that help distinguish between actinomycetoma and eumycetoma. eumycotic lesions tend to form a few cavities in bone that are 1 cm in diameter, while actinomycetes often form smaller but more numerous cavities, leading to a moth - eaten appearance. initial reports of the mri findings of mycetoma described lesions with low signal on t1w and t2w images, which were assumed to be due to susceptibility from the metabolic products of the grains. the dot - in - circle sign, seen as tiny hypointense foci within the hyperintense spherical lesions, was initially described by sarris., in 2003 on t2w, stir, and t1w fat - saturated gadolinium - enhanced images. correlating the mri and histological findings, they suggested that the high - signal areas seen on mri represented inflammatory granulomata, the low - intensity tissue seen surrounding these lesions represented the fibrous matrix, and the small central hypointense foci within the granulomata represented the fungal balls or grains. it was also later reported in 2007, 2009 and 2010. the last (2010) case was misdiagnosed as a soft tissue hemangioma on mri due to the presence of serpiginous enhancing masses with the dot - in - circle sign (the dots were mistaken for phleboliths). who demonstrated on in vitro imaging of the mycetoma lesions that the hyper - reflective echoes corresponded to the grains ; eumycetoma grains produce sharp hyperechoiec foci, while actinomycetomas produce fine hyperechoiec foci that commonly settle at the bottom of the rounded lesions. the usg dot - in - circle sign is similar to the mri sign, with multiple round hypoechoiec lesions containing hyperechoiec foci. sarris. had predicted that with the increasing availability of mri in the metropolitan centers of countries where mycetoma is endemic, the sensitivity and specificity of this sign could be determined. this case from tamil nadu, the land of the madura foot (madura being a district in the indian state of tamil nadu), reiterates the specificity of the dot - in - circle sign.	mycetoma is a chronic granulomatous disease that is more common in tropical than in temperate regions. early diagnosis is important due to the therapeutic implications. although biopsy and microbiological culture provide definitive diagnosis, they are time - consuming procedures and may not be able to provide a definite diagnosis in cases of fastidious organisms. the dot - in - circle sign has recently been proposed as a highly specific magnetic resonance imaging (mri) and ultrasonography (usg) sign of mycetoma, which may allow a noninvasive as well as early diagnosis. we present a case of histologically proven mycetoma that demonstrated this sign.
there have been several reports on arthroscopically assisted removal of the bullet imbedded in hip joint in the literature. similarly, in this case, a bullet lodged in acetabulum was extracted with arthroscopic technique. what makes this case unique in the literature is that the bullet removed from the acetabulum traversed the femoral neck. male patient aged 32 years with a low - velocity gunshot wound was referred to the emergency room on august 28, 2012. the projectile was lodged in acetabular side of the hip joint transversing through the femoral neck. two years after surgery, the patient had groin pain and underwent a safe dislocation for femoral chondral injury. in the last follow - up in the second post - operative year, hip arthroscopy is a minimally invasive and proper procedure for removal of foreign materials such as a bullet in the hip joint. arthrotomy can be reserved for further complications such as chondral injury as in this case. low - velocity gunshot wound has been encountered with increasing frequency. foreign materials in the soft tissue caused by gunshot wound can be managed with antibiotic treatment and superficial debridement. however, it is suggested that foreign materials sucked into joint be removed in an attempt to prevent joint arthropathy, cartilage damage, and septic arthritis. not long ago, arthrotomy was a technique used to remove intra - articular foreign materials. at present, arthroscopic methods for the removal of intra - articular foreign material are described for the knee, hip, ankle, and shoulder. we are unaware of any report describing removal of a bullet imbedded in acetabulum passing through the femoral neck. this makes our report unique when compared with the other reports on arthroscopically assisted bullet removal. a male patient aged 32 years with a low - velocity gunshot wound was referred to the emergency room on august 28, 2012. wound debridement, tetanus prophylaxis, and antibiotic treatment were initially administered for the management of the wound. physical examination showed that entry wound was placed 1 cm lateral to femoral artery, 10 cm distal and 5 cm medial to spina iliaca anterior superior (fig. entry wound, x - ray view of the bullet and its computed tomography cross - section within the hip joint. the relation of the projectile lodged in acetabulum with the joint and its path through the femoral neck was seen at radiography and computed tomography (ct) scanning (fig. hip arthroscopy was performed using the supine position with the extremity in traction on a fracture table with general anesthesia. traction weight of 30 kg was applied to the left hip joint of the patient. the projectile was found to be subchondrally located in posterior superior acetabulum, relating with the joint (fig. exit point of the projectile was seen on the femoral side and chondral damage was treated with debridement. the surrounding of the projectile lodged in the acetabular side was enlarged with a probe for the removal of it. computed tomography cross - section showing the path traversed by the bullet, arthroscopic view of the bullet, the removal of the bullet. following the debridement of the chondral damage occurred in the femoral and acetabular sides and the application of microfractures for the appropriate areas, the joint was washed out and the procedure was terminated. limited mobilization with double crutches was permitted for 6 weeks. then, full weight bearing was initiated. in the 2 year of follow - up period, the patient had full range of motion but had hip pain, especially with weight bearing. magnetic resonance images (mris) were taken and the path traversed by the projectile through the femoral neck and 4 cm 2 cm chondral defect on the femoral head was apparent (fig. he was put on surgery table in lateral decubitis position with general anesthesia, and safe surgical dislocation of the hip was applied (fig. acellular collagen scaffold (cares-1s arthro kinetics, esslingen, germany) was implanted on chondral defect area and fixed with fibrin glue (fig. passive motions were allowed after 2 days and weight bearing was prohibited for 6 weeks. 1 year after second surgery, the patient had full range of motion and had no hip pain. gunshot wounds to the hip joint account for 2% of all extremity gunshot wounds and 4% of lower - extremity gunshot wounds [4, 5 ]. considering this frequency, it is a low possibility for the bullet to be imbedded in acetabulum, passing through the femoral neck. it is a unique case and the pathway of the bullet is really interesting. to the best of our knowledge, there are no published reports describing the arthroscopic removal of a bullet imbedded in acetabulum, passing through the femoral neck. when left in the joint, the bullet may cause complications such as deep infection, lead intoxication, synovitis, and traumatic arthritis in the long - term [1, 2, 6 ]. thus, the projectile imbedded in the hip joint is required to be removed and the joint to be irrigated. the location of the bullet needs to be determined with ct scanning in the pre - operative period and surgical operation should be planned accordingly. in the report by delaney., they opted for safe surgical dislocation to retrieve a bullet from the femoral head since fragmentation at the femoral head was observed from ct image and the bullet was located posterior to the femoral head. an experienced surgeon is able to access a satisfying view over the hip joint with arthroscopically assisted technique. compared to arthrotomy, this technique which is less invasive allows the patient to return daily activities more rapidly. in our case, having established the location of the projectile with the help of pre - operative radiography and ct we opted for hip arthroscopy. arthroscopic technique for the extraction of the bullet from the hip joint was applied for a number of cases in the literature [9, 10, 11 ]. in the case report by singleton., it was noted that a gunshot wound entered the abdomen, traversed the rectum, and ended up in the weight - bearing dome of acetabulum, and the bullet was extracted with an arthroscopically assisted technique. they noted that this procedure can be performed safely, quickly and with minimal complications. in our report cory and ruch reported on the arthroscopic removal of a 0.44 caliber bullet from the femoral head using debridement of the articular surface. in our case, in contrast with arthrotomy, hip arthroscopy prevents blood loss, causes less osteonecrosis of femoral head, and yields better cosmetic appearance with shorter rehabilitation time [4, 12 ]. however, infection was the most expected complication in the cases reported in the literature since an accompanying abdomen wound existed. osteonecrosis of the femoral head affected by the projectile and hip joint arthrosis were potential complications in our case. mri, taken in the second post - operative year, revealed that chondral defect of the femoral head was observed. after 1 year of arthrotomy, the patient had full range of motion and had no hip joint pain. arthroscopically assisted technique allows minimally invasive access to hip joint for the retrieval of foreign material lodged in minimizing possible complications. arthrotomy can be reserved to deal with further complications such as chondral problems as this case. we suggest using arthroscopic techniques for initial bullet removal, and open surgery may be reserved to handle further complications as chondral injury like in this case. at the same time, we can conclude that acellular collagen scaffolds may be useful for chondral injuries caused by bullets. it is suggested to remove foreign materials sucked into joint to prevent joint arthropathy, cartilage damage, and septic arthritis.	introduction : there have been several reports on arthroscopically assisted removal of the bullet imbedded in hip joint in the literature. similarly, in this case, a bullet lodged in acetabulum was extracted with arthroscopic technique. what makes this case unique in the literature is that the bullet removed from the acetabulum traversed the femoral neck.case report : male patient aged 32 years with a low - velocity gunshot wound was referred to the emergency room on august 28, 2012. the projectile was lodged in acetabular side of the hip joint transversing through the femoral neck. a hip arthroscopy was performed for bullet removal. two years after surgery, the patient had groin pain and underwent a safe dislocation for femoral chondral injury. in the last follow - up in the second post - operative year, the patient had no clinical complaint.conclusion:hip arthroscopy is a minimally invasive and proper procedure for removal of foreign materials such as a bullet in the hip joint. arthrotomy can be reserved for further complications such as chondral injury as in this case.
this study was a randomised, placebo - controlled, parallel - group, phase ii, orphan drug study of anti - esbl igy for the eradication of esbl - producing k. pneumoniae and e. coli in faecal carriers. the study was approved by the regional ethical committee (dnr 2011/170/1) and the medical products agency in sweden (eudract 2009 - 011446). study flow chart of the screening and randomisation process, treatment, and follow - up for the participants in the study. two hundred forty - seven patients colonised or infected with ctx - m - producing k. pneumonia during the hospital outbreak during 2005 to 2007 were registered in an internal database at the department of microbiology, uppsala university hospital, and formed the base for this study. to increase the inclusion rate, patients found to be colonised or infected with esbl - producing k. pneumonia or e. coli at uppsala university hospital between 2008 and 2013, and falun hospital between 2012 and 2013 were also added. the screening procedure and typing of esbl - producing strains were carried out as previously described (6). esbl - producing k. pneumonia and e. coli isolates were reported to the research team. written information and contact data individuals who did not reply spontaneously were contacted by phone at least two times on separate occasions. after informed consent, patients were screened for colonisation with esbl - producing k. pneumonia or e. coli, and, if positive, randomised to the study drug or placebo at a 1:1 ratio. briefly, igy antibodies against esbl - producing k. pneumoniae and e. coli were developed as follows : recombinant variants of the two fimbrial k. pneumoniae subunits mrkd and fimh were developed by detailed instructions in the investigator 's brochure. a combination of recombinant proteins was used for immunisation of one group of hens and a combination of esbl k. pneumoniae and e. coli bacteria was used for the immunisation of another group of hens. egg yolk antibodies are actively transferred from the hen to the egg yolk where they are found in high concentration (15). equal amounts of eggs from the two immunisation groups were used for production of the active drug. eggs from immunised hens were visually inspected and defective eggs were excluded from further processing. the remaining eggs were then washed in 70% ethanol and dried before the egg yolk was separated and weighed : the egg yolk was diluted with purified water (16) ; the lipoproteins were sedimented ; and the supernatant decanted, filtered, and finally dispensed in high - density polyethylene bottles with a polypropylene screw cap (70 ml in each) and labelled. the specificity of the anti - esbl igy antibodies against the two antigen pairs, that is, the fimbrial proteins (mrkd and fimh) and esbl - producing e. coli / k. there was a high specific binding of anti - esbl igy in igy batches, whereas the placebo batches showed very low activity. thus, an exact lower limit for the acceptance criterion has not been established, but samples tested with elisa from the igy batches showed 11.615.1 bnu / ml (limit, > 5 bio unit / ml), regardless of storage conditions (i.e. 5 bio unit / ml), regardless of storage conditions (i.e. 4 (21), indicating that igy activity remains after passage through the ventricle. as an extra protection against acidity, sodium hydrogen bicarbonate was used. at the time of the initial idea of this study, we were in the midst of a turbulent hospital care situation, that is, we were treating a large number of patients infected or colonised with esbl - producing k. pneumoniae. the hospital 's structure, care process, and economic status were all put under tremendous stress. the timeline for the development of this study was severely delayed because of different manufacturer and regulatory regulations. thus, we propose that further studies on igy chicken antibodies should be done in a preclinical setting to better understand the mechanisms of action. human studies of this kind may be performed as a long - term investigation on discovered carriers of esbl genes in faeces. such an ongoing study process may be suitable to intensify in an outbreak situation. for now, increasing the inclusion rate to a reasonable level in such a study should be done in medium- to high - prevalent regions. in summary, the present study of anti - esbl igy for the eradication of esbl k. pneumoniae and e. coli in faecal carriers was prematurely discontinued because of high mortality and other factors resulting in a low inclusion rate. spontaneous eradication of esbl - producing bacteria was frequently observed in recruited participants, which is consistent with previous reports. the authors have not received any funding or benefits from industry or elsewhere to conduct this study.	backgroundextended - spectrum beta - lactamase (esbl)-producing enterobacteriaceae is an emerging therapeutic challenge, especially in the treatment of urinary tract infections. following an outbreak of ctx - m-15 klebsiella pneumoniae in uppsala, sweden, an orphan drug trial on igy chicken antibodies was undertaken in an attempt to eradicate faecal carriage of esbl - producing k. pneumoniae and escherichia coli.methodshens were immunised with epitopes from freeze - dried, whole - cell bacteria (esbl - producing k. pneumoniae and e. coli) and recombinant proteins of two k. pneumoniae fimbriae subunits (fimh and mrkd). the egg yolks were processed according to good manufacturing practice and the product was stored at20c until used. using an internal database from the outbreak and the regular laboratory database, faecal carriers were identified and recruited from may 2005 to december 2013. the participants were randomised in a placebo - controlled 1:1 manner.resultsfrom 749 eligible patients, 327 (44%) had deceased, and only 91 (12%) were recruited and signed the informed consent. in the initial screening performed using the polymerase chain reaction, 24 participants were esbl positive and subsequently randomised and treated with either the study drug or a placebo. the study was powered for 124 participants. because of a very high dropout rate, the study was prematurely terminated. from the outbreak cohort (n=247), only eight patients were screened, and only one was positive with the outbreak strain in faeces.conclusionsthe present study design, using igy chicken antibodies for the eradication of esbl - producing k. pneumonia and e. coli, was ineffective in reaching its goal due to high mortality and other factors resulting in a low inclusion rate. spontaneous eradication of esbl - producing bacteria was frequently observed in recruited participants, which is consistent with previous reports.
g - protein coupled receptors (gpcrs) with seven transmembrane helices are the major membrane proteins that play the important interface role for signaling to the inner cell. an external ligand stimulus to a gpcr induces the coupling with g - proteins (gi / o, gq/11, gs and g12/13) followed by different kinds of signal transduction. since about half (1) of all drugs distributed throughout the world are designed to control these mechanisms, gpcrs are important targets in the development of effective drugs. from the viewpoint of drug design, it will be of utmost importance to screen a drug for its ability to effectively control the activation of a specific g - protein, by monitoring the stimulation by different ligands. in general, it is quite difficult to develop such a high - throughput experimental system ; however, g - protein activity prediction made using bioinformatics techniques contributes to the design of an effective experimental system. g - protein binding selectivity when both the gpcr sequence and ligand information are submitted. the established way to predict protein function is to classify proteins into functional groups whose members are linked by sequence similarity using a conventional sequence search method such as blast (2) and fasta (3). however, in the case of gpcrs, the function - similarity relationship is unclear. for example, (i) some homologous gpcr pairs with the same ligands bind to different kinds of g - protein ; (ii) those pairs that bind to the same type of g - protein bind to a different ligand ; and furthermore (iii) some gpcr pairs bind to both the same ligand and the same g - protein even though they show sequence similarity of 85% on average). based on this study, we developed a griffin web server () that can predict g - protein coupling specificity using the svm and hmm methods. the svm process is suitable for predicting g - protein coupling selectivity for the class a gpcr family. it is well known that the class a gpcr family is huge, as it is the major family, and its large - scale diversity makes it difficult to predict its coupling g - proteins using only sequence similarity information. therefore, we first applied the svm method using characteristic quantities extracted from the ligand information and gpcr structure. the hmm method is suitable for predicting the coupling selectivity of g - proteins with gpcrs belonging to opsins and olfactory receptors (in class a), class b, c, frizzled and smoothened families. although it is still unclear what kind of g - protein binds to frizzled and smoothened gpcrs, these two families can be used as a filter to classify other gpcrs. for these families, the g - protein prediction is easier because sequence similarity information (described in terms of the hmm) directly correlates with functional annotation of the binding g - protein type. the svm and hmm calculations were performed using the libsvm (14) and hmmer (15) software packages, respectively. the detailed parameters and thresholds used are described below. for class a gpcrs, amino acid sequences were obtained from the swissprot and trembl databases. these gpcr sequences include both ligand and g - protein information written in tips (16) and gpcrdb (17). the number of class a gpcr sequences selected as training data for svm classification is 132 (gi / o binding type : 61 sequences ; gq/11 binding type : 47 sequences ; gs binding type : 24 sequences). and in this work, gpcrs which are coupled with multiple g - proteins and the g12/13 g - protein family are not considered because there are not sufficient data to construct a prediction system. the redundancy of these sequences was evaluated by analyzing clusters formed under sequence similarity set to decrease from 100% to 30% in steps of 1% using blastclust from the blast software package (2). one cluster consisted of two gpcrs (swissprot ids pkr1_human and pkr2_human) and appeared at 87% sequence identity, and the other clusters were not detected until the sequence identity reached 68%. though pkr1_human and pkr2_human show strong sequence similarity, as described above, they bind to different ligands and, therefore, both sequences should be used as training datasets. for this reason, 132 sequences are used in this work without a process of elimination of redundancy. for opsins and olfactory receptors, classes b, c, frizzled and smoothened families, sequences were obtained from the swissprot and trembl databases as well as the above - mentioned 132 class a gpcrs. class c gpcr sequences can be classified into two types, gi / o binding type and gq/11 binding type ; therefore, this family is separately collected into two groups. the numbers of gpcrs are 170, 394, 34, 20, 9, 40 and 5 for opsins, olfactory receptors, class b, class c for gi / o specific, class c for gq/11 specific, frizzled and smoothened families, respectively. to develop the program, we assumed that the ligand, gpcr and g - protein form a complex, and that therefore the interactions among this complex are all essential factors for activating g - protein bindings. from this viewpoint, structural characteristics should be extracted comprehensively from the ligand, extracellular loops, intracellular loops and transmembrane domain of gpcrs, although the tertiary positions of some characteristics are distant from the g - protein binding site. to calculate these parameters, the boundaries of the transmembrane helix and loop regions of gpcr sequences were determined from multiple alignments of known class a families with bovine rhodopsin as a three - dimensional structure template (pdb i d : 1f88) using clustal w (18). in addition to the above parameters, two bit scores are calculated from gpcr sequences. one is obtained when a query gpcr sequence is searched against the hmm profile (peptide profile) constructed from multiple alignments of gpcr groups binding to a peptide ligand. the other is calculated by the hmm profile (amine profile) of a gpcr group which is bound to a small amine ligand. each gpcr in these two groups was obtained from swissprot : 439 and 243 for the peptide ligand type and the small amine ligand type, respectively. svm classifies these vector representations (feature vectors) of gpcrs using a multidimensional hyperplane called the kernel function. since the svm is a classifier used to divide data into two groups, classifications such as (gs binding type and others), (gq/11 binding type and others) and (gi / o binding type and others) are performed. in order to calculate the accuracy in discriminating each g - protein type (gi / o, gq/11 and gs for the training dataset containing gi / o, gq/11 and gs binding gpcrs), svm training was performed by changing the combination of the feature vector elements, kernel functions (linear, polynomial, rbf and sigmoid formula) with parameters c and, which determine the shape of kernel function. the variable ranges of the parameters c, and cross - validation fold are from 2 to 2, from 2 to 2, and from 2 to 5, respectively. the best combination of feature vector elements and kernel functions is determined when the product of sensitivity and specificity shows the highest value of accuracy for evaluating g - protein coupling prediction. as indicated in table 2, the discrimination of the gs binding type is the most successful, with the following five feature vector elements : (i) the third intracellular loop length, (ii) the c - terminal loop length, (iii) the total number of arginines and lysines in the c - terminal region of the intracellular loop, (iv) the existence of proline at the position corresponding to the 170th residue on rhodopsin, and (v) the bit score of the amine profile. however, under the same condition, gi / o and gq/11 types can not be classified with high accuracy. thus, in order to predict gi / o or gq/11 from the two proteins with high accuracy, svm training was performed again. the best performance results for gi / o and gq/11 classifications are shown in table 3. the highest sensitivity and specificity for classifying as gi / o type or gq/11 type were achieved when five parameters [(i), (ii), (v) and two additional parameters : (vi) the bit score of the peptide profile and (vii) the ligand molecular weight ] were used. hmm profiles were made from each member of the opsins, olfactory receptors, classes b, c, frizzled and smoothened families using the hmmer program (15) (class c hmm profiles were made separately from two groups which bind to gi / o or gq/11). to verify the reliability of each profile used for prediction, all gpcrs were first picked up from gpcrdb to add as false data for each family. each family was divided into four subgroups and 4-fold cross - validation tests were executed to verify the reliability of hmm profiles (that is, three - fourths of the subgroups are used as training datasets and the remaining fourth are used as test data). as a result, for each hmm profile, we determined the safe threshold score to discriminate subfamilies with the highest sensitivity and specificity (table 4). as shown in table 4, these results suggest that for each family, sequence information (described in terms of the hmm) corresponds to specific g - protein type : opsins bind to gt, olfactory receptors bind to golf, most of the class b family binds to gs, and the class c family binds to gi / o or gq/11. the type of g - protein binding to the frizzled and smoothened families is still unclear ; therefore, these gpcrs can be classified as unknown g - protein type. thus, an hmm profile search can directly link the g - protein information, and these profiles are useful filters in the classification of the class a gpcrs and others. g - protein coupling selectivity is shown as the flowchart in figure 1. as input data, this system requires the sequence of query gpcr and ligand molecular weight, which are converted to feature vector elements. at the first stage, a query sequence is searched against the hmm profiles of the opsins and olfactory receptor subfamilies, classes b, c, frizzled and smoothened families by the hmmer program (15) with high accuracy, as shown in table 4. if the computed hmm profile score is larger than the threshold of a certain subfamily (see table 4), the query sequence can immediately link to the g - protein information, and griffin stops the prediction process. however, if the query does not meet the above conditions (i.e. all profile scores are less than each corresponding family threshold), griffin continues the processing to the second stage, which uses the svm with feature vectors which are converted from sequence and ligand molecular weight. since the prediction of gs from other g - proteins and gi / o or gq/11 in these two proteins requires different parameter sets and conditions to achieve the best performance, we constructed the following hierarchical system. first, this system determines whether the query sequence is binding to gs by using five parameters and the rbf function. if the query is predicted to be of the gs binding type (with 95.2% specificity and 83.3% sensitivity, table 2), this result is displayed and griffin stops the prediction process. if it is predicted not to be of the gs binding type, griffin changes the process to predict gi / o or gq/11 coupling selectivity by using the other five parameters and the polynomial function, with high sensitivity and specificity as shown in table 3. after applying this hierarchical system to known sequences through 10 000 rounds of 4-fold cross - validation, the average discrimination sensitivities and specificities were 87% and 88% for gi / o, 85% and 84% for gq/11, and 85% and 89% for gs, respectively. in previous studies, three methods (1113) is based on the naive bayes model and it predicted the g - protein with 72% sensitivity from 55 gpcrs (11). mller. indicated > 90% specificity with 3040% sensitivity using pattern extraction (12). succeeded in reducing the error rate of prediction to 85%. hmm profiles by sreekumar. indicated higher performance of prediction compared with our method. however, it is difficult to compare their performance with our method because the gpcr sequences used in their dataset and their evaluation methodology are different from ours. most importantly, our method is the first one to predict g - protein types by inputting both ligand molecular weight and gpcr sequence, and this prediction processing is available on the useful web server griffin. for class a gpcrs, amino acid sequences were obtained from the swissprot and trembl databases. these gpcr sequences include both ligand and g - protein information written in tips (16) and gpcrdb (17). the number of class a gpcr sequences selected as training data for svm classification is 132 (gi / o binding type : 61 sequences ; gq/11 binding type : 47 sequences ; gs binding type : 24 sequences). and in this work, gpcrs which are coupled with multiple g - proteins and the g12/13 g - protein family are not considered because there are not sufficient data to construct a prediction system. the redundancy of these sequences was evaluated by analyzing clusters formed under sequence similarity set to decrease from 100% to 30% in steps of 1% using blastclust from the blast software package (2). one cluster consisted of two gpcrs (swissprot ids pkr1_human and pkr2_human) and appeared at 87% sequence identity, and the other clusters were not detected until the sequence identity reached 68%. though pkr1_human and pkr2_human show strong sequence similarity, as described above, they bind to different ligands and, therefore, both sequences should be used as training datasets. for this reason, 132 sequences are used in this work without a process of elimination of redundancy. for opsins and olfactory receptors, classes b, c, frizzled and smoothened families, sequences were obtained from the swissprot and trembl databases as well as the above - mentioned 132 class a gpcrs. class c gpcr sequences can be classified into two types, gi / o binding type and gq/11 binding type ; therefore, this family is separately collected into two groups. the numbers of gpcrs are 170, 394, 34, 20, 9, 40 and 5 for opsins, olfactory receptors, class b, class c for gi / o specific, class c for gq/11 specific, frizzled and smoothened families, respectively. to develop the program, we assumed that the ligand, gpcr and g - protein form a complex, and that therefore the interactions among this complex are all essential factors for activating g - protein bindings. from this viewpoint, structural characteristics should be extracted comprehensively from the ligand, extracellular loops, intracellular loops and transmembrane domain of gpcrs, although the tertiary positions of some characteristics are distant from the g - protein binding site. to calculate these parameters, the boundaries of the transmembrane helix and loop regions of gpcr sequences were determined from multiple alignments of known class a families with bovine rhodopsin as a three - dimensional structure template (pdb i d : 1f88) using clustal w (18). in addition to the above parameters, two bit scores are calculated from gpcr sequences. one is obtained when a query gpcr sequence is searched against the hmm profile (peptide profile) constructed from multiple alignments of gpcr groups binding to a peptide ligand. the other is calculated by the hmm profile (amine profile) of a gpcr group which is bound to a small amine ligand. each gpcr in these two groups was obtained from swissprot : 439 and 243 for the peptide ligand type and the small amine ligand type, respectively. svm classifies these vector representations (feature vectors) of gpcrs using a multidimensional hyperplane called the kernel function. since the svm is a classifier used to divide data into two groups, classifications such as (gs binding type and others), (gq/11 binding type and others) and (gi / o binding type and others) are performed. in order to calculate the accuracy in discriminating each g - protein type (gi / o, gq/11 and gs for the training dataset containing gi / o, gq/11 and gs binding gpcrs), svm training was performed by changing the combination of the feature vector elements, kernel functions (linear, polynomial, rbf and sigmoid formula) with parameters c and, which determine the shape of kernel function. the variable ranges of the parameters c, and cross - validation fold are from 2 to 2, from 2 to 2, and from 2 to 5, respectively. the best combination of feature vector elements and kernel functions is determined when the product of sensitivity and specificity shows the highest value of accuracy for evaluating g - protein coupling prediction. as indicated in table 2, the discrimination of the gs binding type is the most successful, with the following five feature vector elements : (i) the third intracellular loop length, (ii) the c - terminal loop length, (iii) the total number of arginines and lysines in the c - terminal region of the intracellular loop, (iv) the existence of proline at the position corresponding to the 170th residue on rhodopsin, and (v) the bit score of the amine profile. however, under the same condition, gi / o and gq/11 types can not be classified with high accuracy. thus, in order to predict gi / o or gq/11 from the two proteins with high accuracy, svm training was performed again. the best performance results for gi / o and gq/11 classifications are shown in table 3. the highest sensitivity and specificity for classifying as gi / o type or gq/11 type were achieved when five parameters [(i), (ii), (v) and two additional parameters : (vi) the bit score of the peptide profile and (vii) the ligand molecular weight ] were used. hmm profiles were made from each member of the opsins, olfactory receptors, classes b, c, frizzled and smoothened families using the hmmer program (15) (class c hmm profiles were made separately from two groups which bind to gi / o or gq/11). to verify the reliability of each profile used for prediction, all gpcrs were first picked up from gpcrdb to add as false data for each family. each family was divided into four subgroups and 4-fold cross - validation tests were executed to verify the reliability of hmm profiles (that is, three - fourths of the subgroups are used as training datasets and the remaining fourth are used as test data). as a result, for each hmm profile, we determined the safe threshold score to discriminate subfamilies with the highest sensitivity and specificity (table 4). as shown in table 4, these results suggest that for each family, sequence information (described in terms of the hmm) corresponds to specific g - protein type : opsins bind to gt, olfactory receptors bind to golf, most of the class b family binds to gs, and the class c family binds to gi / o or gq/11. the type of g - protein binding to the frizzled and smoothened families is still unclear ; therefore, these gpcrs can be classified as unknown g - protein type. thus, an hmm profile search can directly link the g - protein information, and these profiles are useful filters in the classification of the class a gpcrs and others. g - protein coupling selectivity is shown as the flowchart in figure 1. as input data, this system requires the sequence of query gpcr and ligand molecular weight, which are converted to feature vector elements. at the first stage, a query sequence is searched against the hmm profiles of the opsins and olfactory receptor subfamilies, classes b, c, frizzled and smoothened families by the hmmer program (15) with high accuracy, as shown in table 4. if the computed hmm profile score is larger than the threshold of a certain subfamily (see table 4), the query sequence can immediately link to the g - protein information, and griffin stops the prediction process. however, if the query does not meet the above conditions (i.e. all profile scores are less than each corresponding family threshold), griffin continues the processing to the second stage, which uses the svm with feature vectors which are converted from sequence and ligand molecular weight. since the prediction of gs from other g - proteins and gi / o or gq/11 in these two proteins requires different parameter sets and conditions to achieve the best performance, we constructed the following hierarchical system. first, this system determines whether the query sequence is binding to gs by using five parameters and the rbf function. if the query is predicted to be of the gs binding type (with 95.2% specificity and 83.3% sensitivity, table 2), this result is displayed and griffin stops the prediction process. if it is predicted not to be of the gs binding type, griffin changes the process to predict gi / o or gq/11 coupling selectivity by using the other five parameters and the polynomial function, with high sensitivity and specificity as shown in table 3. after applying this hierarchical system to known sequences through 10 000 rounds of 4-fold cross - validation, the average discrimination sensitivities and specificities were 87% and 88% for gi / o, 85% and 84% for gq/11, and 85% and 89% for gs, respectively. in previous studies, three methods (1113) were developed in order to predict g - protein binding selectivity. the method of cao. is based on the naive bayes model and it predicted the g - protein with 72% sensitivity from 55 gpcrs (11). mller. indicated > 90% specificity with 3040% sensitivity using pattern extraction (12). succeeded in reducing the error rate of prediction to 85%. hmm profiles by sreekumar. indicated higher performance of prediction compared with our method. however, it is difficult to compare their performance with our method because the gpcr sequences used in their dataset and their evaluation methodology are different from ours. most importantly, our method is the first one to predict g - protein types by inputting both ligand molecular weight and gpcr sequence, and this prediction processing is available on the useful web server griffin. figure 2a shows the home page of the griffin website (). on this home page, there are small and large text boxes for entering a sequence name and an amino acid sequence, respectively. the three small text boxes at the bottom of the page are for entering the range of ligand molecular weight (onset, termination and differential values) from left to right. with this function, by changing the range of ligand molecular weight, the user can perform the computational experiment to monitor g - protein binding for orphan receptors whose ligands are still unknown. of course, the user can also predict the type of g - protein by entering only one value for ligand molecular weight. if molecular weight calculator is clicked, it navigates to a page where the user can calculate the molecular weight of a chemical compound when the chemical equation is entered in the text box and the the pubchem website, which contains chemical compound information, is also available via a link at the top of the page. when the predict button is clicked, the griffin system navigates to the results page (figure 2b). when the user enters a range of ligand molecular weight, and if this range matches a certain g - protein type, the results are displayed with each line representing a predicted g - protein type. for example, figure 2b shows the result when a wide molecular range (from 100 to 30 000 in steps of 100) is entered ; this query sequence changes between the coupling g - proteins gi / o and gq/11. the query sequence and user - defined name are displayed in the fasta format, with transmembrane regions colored in red, when the query gpcr belongs to the class a family. in addition, feature vector elements and their scores, which are calculated in the process of prediction, are displayed in a table. we believe that griffin will contribute to the research into functional assignment of orphan gpcrs and to the design of experimental systems for screening effective drugs. g - protein coupling selectivity. (a) the top of the griffin website, where the gpcr sequence and ligand molecular weight can be entered. (b) the result page of a griffin calculation, where the predicted g - proteins of the user - defined sequence are indicated together with physicochemical parameters used in the svm or hmm calculation. the prediction accuracy of svm part performed with 132 gpcrs the prediction accuracy of svm part performed with 108 gpcrs the prediction accuracy of hmm part performed with 4-fold cross - validation	we describe a novel system, griffin (g - protein and receptor interaction feature finding instrument), that predicts g - protein coupled receptor (gpcr) and g - protein coupling selectivity based on a support vector machine (svm) and a hidden markov model (hmm) with high sensitivity and specificity. based on our assumption that whole structural segments of ligands, gpcrs and g - proteins are essential to determine gpcr and g - protein coupling, various quantitative features were selected for ligands, gpcrs and g - protein complex structures, and those parameters that are the most effective in selecting g - protein type were used as feature vectors in the svm. the main part of griffin includes a hierarchical svm classifier using the feature vectors, which is useful for class a gpcrs, the major family. for the opsins and olfactory subfamilies of class a and other minor families (classes b, c, frizzled and smoothened), the binding g - protein is predicted with high accuracy using the hmm. applying this system to known gpcr sequences, each binding g - protein is predicted with high sensitivity and specificity (> 85% on average). griffin () is freely available and allows users to easily execute this reliable prediction of g - proteins.
international humanitarian law affords special protection to medical property and personnel whose mission is to save lives and provide health care for civilians and combatants alike. presented below are examples of the afghanistan analyses where, as a result of war situations, people are most vulnerable. discussed are the minimum protection and standards applicable to such situations specified by the international humanitarian law. its rules and provisions obligate fighting parties to take all necessary measures to protect and respect medical missions in all circumstances. as contemporary armed conflicts become more common, the lack of respect for the signs of the red cross and red crescent, under which medical services operate, is noticeable. as a result of the treachery and abuse of pictographic representations of the red cross and red crescent, it has reached a point where the people and objects entitled to be protected have become as much of a target as military units. despite the undeniable recognition of these protective symbols, the parties involved in the conflict can not always be certain whether the signs of the red cross or the red crescent are being used by people with authorization. this situation is widespread in the conflict in afghanistan, where the usage of these symbols is inconsistent with international conventions, and has led to numerous events where medical vehicles marked with the red cross or the red crescent symbols, together with their employees, are often targets of terrorist attacks. additionally, similar reactions have been encountered by the units of the international committee of the red cross (icrc) when providing humanitarian help to citizens of afghanistan. this is considered a violation of the geneva conventions, which prohibit attacks on people and objects bearing the emblem of protection. however, it automatically raises the problem of obtaining and/or guaranteeing that the marked unit is actually an ambulance or a humanitarian mission, not a branch of the armed forces of the enemy. this situation applies to both verifying the credibility of the mark and its protection, and protecting the rights established in order to save human life and health. all of these issues influence the work of the icrc, but mainly they affect the neediest, who are involved in situations of armed conflict and are waiting for help from humanitarian organizations and emergency medical services. the international humanitarian law of armed conflict emerged in the nineteenth century. the first piece of legislation regulating this issue was signed on the 22nd of august 1864 : the convention for the amelioration of the condition of the wounded and sick in armies in the field. the convention not only introduced an important international obligation to comply with humanitarian principles, but it correspondingly created the basis for contemporary humanitarian law. moreover, it formulated standards aimed at protecting victims of armed conflict particularly wounded soldiers. the convention reaffirmed the neutrality of medical services and obliged others to respect their actions. the convention defined the rules pertaining to the usage of and the reverence that should be shown to the red cross emblem on a white background. a valuable feature of the convention was its multilateral nature, which made it possible for all interested countries to accede to it. in 1906, the convention was replaced by a new agreement, which was also signed in geneva. that new convention extended the scope of protection regarding wounded and injured soldiers by introducing the principle of keeping records of victims of armed conflicts and established a system for exchanging information about those people. experiences gained in further conflicts caused the undertaking of additional work on the development of international humanitarian law. by 1949, many different kinds of declarations and conventions came into effect that developed issues outlined in the geneva convention of 1906. afterwards, the hague conventions on laws and customs of war on land and the adaptation to maritime warfare of principles of geneva convention of 1864 were signed in 1899. in 1907, then, in 1925, the geneva protocol was signed, banning the usage of asphyxiating, poisonous gas or similar substances and bacteriological methods in war. another two geneva conventions were introduced in 1929 to amend the geneva convention of 1906 and the geneva convention on the treatment of war prisoners. at the moment, international humanitarian law and the activities of the icrc during armed conflict are based on four geneva conventions of 1949, as well as on the additional protocols for these conventions. combined, these constitute a system of international law that protects victims of armed conflicts. one of the basic principles of this system is that medical personnel and military civilians are entitled to special protection from attacks, and another is that the activities of medical personnel can not be prohibited or violated. medical personnel, in accordance with article 24 of the geneva convention, should only be exclusively engaged in the search for, or the collection, transport or treatment of the wounded or sick, or in the prevention of disease, staff exclusively engaged in the administration of medical units and establishments, as well as chaplains attached to the armed forces, and shall be respected and protected in all circumstances. geneva convention ii grants protection to medical personnel, as well as to personnel of hospital ships. according to article 36 of geneva convention ii, these personnel shall be respected and protected ; they may not be captured during the time they are in the service of the hospital ship, whether or not there are wounded and sick on board. article 37 constitutes that if religious, medical, and hospital personnel fall into the hands of the enemy, they must be respected and protected ; they may continue to carry out their duties as long as necessary for the care of the wounded and sick. they shall afterwards be sent back as soon as the commander - in - chief, under whose authority they are operating, considers it practicable. the first additional protocol to the geneva convention defines that civilian medical personnel shall be respected and protected and, if needed, all available help shall be afforded to civilian medical personnel in an area where civilian medical services are disrupted due to combat activity. the occupying power shall afford civilian medical personnel in occupied territories any assistance required in order to enable them to perform, to the best of their ability, their humanitarian functions. when they are performing those functions, the occupying power can not compel these personnel to give priority to the treatment of any person, except on medical grounds. the personnel can not be compelled to carry out tasks that are not compatible with their humanitarian mission. civilian medical personnel shall have access to any place where their services are essential, subject to such supervisory and safety measures as the relevant party to the conflict may deem necessary. the provisions of the conventions and of this protocol concerning the protection and identification of medical personnel shall apply equally to such persons. according to article 16 regarding the general protection of medical duties, under no circumstances shall any person be punished for carrying out medical activities compatible with medical ethics, regardless of the person benefiting from them. persons engaged in medical activities shall not be compelled to perform acts or to carry out work contrary to the rules of medical ethics or to other medical rules designed for the benefit of the wounded and sick, or to the provisions of the conventions or of this protocol, or to refrain from performing acts or from carrying out work required by those rules and provisions. no person engaged in medical activities shall be compelled to give to anyone belonging either to an adverse party, or to his own party (except as required by the law of the latter party) any information concerning the wounded and sick who are (or who have been) under his care, if such information would in his opinion prove harmful to the patients concerned or to their families. article 38 of protocol i, additional to the geneva conventions, prohibits the unauthorized use of the red cross image and other emblems, signs, and signals recognized internationally. article 9 of protocol ii, also additional to the geneva conventions, explores the issue of protection regarding medical personnel. furthermore, according to this article, all possible help must be provided to enable medical personnel to fulfill their functions. medical personnel should also not be forced to perform tasks that are impossible to reconcile with their humanitarian mission. moreover, they should not be asked to give priority to any person for reasons other for than medical purposes. article 10 of protocol i, additional to the geneva conventions, establishes general rules concerning the protection of medical duties. due to the general protection of medical duties, under no circumstances shall any person be punished for having carried out medical activities that are compatible with medical ethics, regardless of the person benefiting from them. persons engaged in medical activities shall neither be compelled to perform acts or to carry out work contrary to nor be compelled to refrain from acts required by the rules of medical ethics or other rules designed for the benefit of the wounded and sick, or this protocol. the professional obligations of persons engaged in medical activities regarding information which they may acquire concerning the wounded and sick under their care shall, subject to national law, be respected. subject to national law, no person engaged in medical activities may be penalized in any way for refusing or failing to give information concerning the wounded and sick who are, or who have been, in their care. article 11 treats the issue of protection of medical units and transports, which shall be respected and protected at all times and shall not be the objects of attack. the protection to which medical units and transports are entitled shall not cease unless they are used to commit hostile acts outside of their humanitarian function. protection may, however, cease only after a warning has been given, setting whenever appropriate a reasonable time limit, and after such a warning has remained unheeded. nowadays, the geneva conventions apply in all cases where hostilities are ongoing, regardless of whether the war is declared or not. in addition to war, the geneva conventions are concerned with occupation, even if there is no armed resistance. furthermore, the conventions apply to all states, including situations where one of the countries involved in the conflict may not be a party to the convention. in accordance with articles 47, 48, 127, and 144 of geneva conventions i, ii, iii, and iv, respectively, it is a legal obligation of countries to spread knowledge of these conventions and protocols. since 1949, the geneva conventions have been extended by a number of other regulations, conventions, agreements, and protocols, which have significantly influenced the current shape of international humanitarian law. contemporary humanitarian law is defined as the norms of conduct devoted to humanity, human dignity, life, and health ; the norms adopted jointly by the countries and adopted widely in international law ; legal standards that take the form of conventions and other international agreements, as well as those included by custom ; norms of international law applicable throughout the world in times of peace, war, and other circumstances ; and a set of rules of international law, enacted and adopted in order to provide assistance and care for human beings, in particular the geneva conventions of 12 august 1949 for the protection of war victims and the additional protocols of 1977 to those conventions. moreover, the red cross is a promoter of humanitarian law. in order to facilitate the dissemination of knowledge regarding international humanitarian law, the icrc has developed a set of standards that constitutes the essence of international humanitarian law. these norms, which are customary in nature, state that nonparticipants or people that are not able to partake in a fight are entitled to respect for their lives and their physical and mental integrity. such individuals must always be protected and treated humanely, without any discussion ; it is forbidden to kill or inflict wounds on an enemy who surrenders or can no longer take part in a fight ; wounded and sick must be gathered and taken into the care of the party in the conflict who they are collected by. additionally, medical staff and medical devices, vehicles, and equipment are also subject to this protection ; apprehended combatants and civilians that are under the power of the opposing party are entitled to respect for their lives, dignity, personal rights, and their political, religious, and other opinions ; everyone must adhere to the basic judicial guarantees, and no one can be held responsible for an act they did not commit. no one shall be subjected to physical or mental torture or corporal punishment or any other cruel or degrading treatment ; parties to the conflict and members of their armed forces do not have the unlimited right to choose methods and means of warfare. it is forbidden to use weapons or methods of warfare that may cause unnecessary losses or excessive suffering ; parties to the conflict shall at all times distinguish between the civilian population and combatants in order to spare the civilian population and their property. the civilian population as a whole and individual civilians shall not be the objects of attack. it is designed to provide assistance and protection to all people and to reduce the suffering caused by war. moreover, humanitarian law provisions regulate relations with the enemy, the management of war prisoners, and the rights of residents of a territory occupied by a foreign country. the preamble to the protocol i (additional to the geneva conventions) contains the following statement : () reaffirming further that the provisions of the geneva conventions of 12 august 1949 and of this protocol must be fully applied in all circumstances to all persons who are protected by those instruments, without any adverse distinction based on the nature or origin of the armed conflict or on the causes espoused by or attributed to the parties to the conflict (). human rights are in no way concerned with the methods used in military operations (for example, the methods of use of weapons). their purpose is to protect individuals ; to facilitate the development and strengthening of the human being in opposition to a government. only in exceptional circumstances and in specific cases (as described in acts of international and national law) is it possible to ignore some of its provisions. in international regulations dealing with human rights issues, there have been provisions that authorize the state to suspend these rights in a situation threatening its existence. this applies in particular to the right to live, the prohibition of torture and inhuman behavior, the prohibition of slavery and servitude, as well as the principle of the legality and nonretroactivity of the law. most of the rights included in article 4 of the international covenant on civil and political rights may be repealed in the case of armed conflict. conversely, the following rights can never be repealed : prohibition of death penalty rulings except in court proceedings and some limitations to this penalty ; prohibition of torture and inhuman or degrading treatment ; prohibition of slavery and servitude ; prohibition of retroactivity of new or stricter rules of substantive criminal law ; the right to own a legal personality forever ; the rights to freedom of thought, conscience, and religion. according to the icrc : () the mechanisms controlling obeying human rights are very diverse. in many cases, relevant institutions are responsible to determine whether a breach of the law has taken place or not. for example, the european court of human rights may, after completion of the procedure in a particular case, state that the authorities of the country violated the european convention on human rights. afterwards, the authorities have the obligation to implement the necessary measures to ensure that the internal situation comply with the standards set out in the convention. all in all, mechanisms responsible for the implementation of human rights are essentially designed to allow compensation for the harm suffered. additionally, human rights applicable in the context of armed conflict are complemented by international humanitarian law. for that reason, both systems aim to ensure the protection of the human being, but through the use of other means and in other circumstances. the war in afghanistan was recognized as an international armed conflict in the beginning. consequently, in the course of the conflict, the geneva conventions and additional protocols have applied. humanitarian law has been applied to the parties of the conflict, but has also provided protection to people and groups who have not participated in the conflict or who have ceased to take part in it. in accordance with the additional protocol provisions by special care, the following are also covered : wounded and ill soldiers in terrestrial conflicts, as well as members of the medical services of the armed forces ; wounded, ill, or shipwrecked soldiers in the war at sea, as well as members of the naval medical service ; prisoners of war ; and the civilian population, such as foreign civilians who are present on territory belonging to the parties in the conflict, including refugees, civilians in occupied areas, arrested and interned civilians, medical and religious personnel, and civil defense units. many of the standards contained in the additional protocols to the geneva conventions referring to international conflicts are treated as a norm of customary law applicable in all armed conflicts. this is important because additional protocol i discusses the issue of protecting civilians and the measures necessary to shield them from the effects of hostilities. according to the nuremberg tribunal : the law of war is not only contained in treaties, but also in the habits and customs, which gradually have gained general recognition, as well as the general principles of justice applied by jurists and military courts. as a result, the law is not static, but is adapted by continual embellishments to the needs of a changing world. however, in many cases the treaty merely expresses and defines existing legal principles in more detail. in the international humanitarian law of armed conflict,, each high contracting party shall allow the free passage of all consignments of medical and hospital stores and objects necessary for religious worship intended only for civilians of another high contracting party, even if the latter is its adversary. it shall likewise permit the free passage of all consignments of essential foodstuffs, clothing, and tonics intended for children under fifteen, expectant mothers, and maternity cases. to the fullest extent of the means available to it, the occupying power has the duty to ensure the food and medical supplies of the population ; it should, in particular, bring in the necessary foodstuffs, medical stores, and other articles if the resources of the occupied territory are inadequate. if the whole or part of the population of an occupied territory is inadequately supplied, the occupying power shall agree to relief schemes on behalf of the said population, and shall facilitate them by all of the means at its disposal. in all cases, the duration of the period during which a protected person accused of an offence is under arrest awaiting trial or punishment shall be deducted from any period of imprisonment awarded. protected persons shall not be arrested, prosecuted, or convicted by the occupying power for acts committed or for opinions expressed before the occupation, or during a temporary interruption thereof, with the exception of breaches of the laws and customs of war. nationals of the occupying power who sought refuge in the territory of the occupied state before the outbreak of hostilities shall not be arrested, prosecuted, convicted, or deported from the occupied territory, except for offences committed after the outbreak of hostilities, or for offences under common law committed before the outbreak of hostilities which, according to the law of the occupied state, would have justified extradition in peacetime. at this stage, the military encounter in afghanistan should be classified as a non - international armed conflict due to the fact that it takes place between afghan guerrillas, which are not the government s armed forces and nato troops. moreover, it is noteworthy that the conditions for the implementation of additional protocol ii are stricter than the conditions needed for the application of article 3 of the geneva conventions. furthermore, in such situations, humanitarian law applies to the armed forces both regular and irregular that are involved in the conflict, and it protects any person or class of people who do not participate in hostilities or have ceased to take part in them (i.e., wounded and sick combatants, individuals deprived of their liberty as a result of the conflict, civilians, and medical and religious personnel). on the other hand, during a non - international armed conflict, humanitarian law provides material aid to victims of the struggle. in article 18 of additional protocol ii, it is stated that : if the civilian population is suffering undue hardship owing to a lack of the supplies essential for its survival, such as food - stuffs and medical supplies, relief actions for the civilian population which are of an exclusively humanitarian and impartial nature and which are conducted without any adverse distinction shall be undertaken subject to the consent of the high contracting party concerned. furthermore, the conflict in afghanistan is not only a war against the taliban but a combination of several minor conflicts that involve in addition to an international coalition gathered under the banner of the isaf all sorts of entities (both state and private), international terrorist organizations, and criminals. this would also include different tribes, mercenaries, religious and ideological leaders, and intelligence services that have broken away from the control of the state. therefore, ending the conflict in afghanistan is an extremely difficult task, and probably one that will prove impossible to achieve for a long time. additionally, in the case of a new war, it is very important that international humanitarian law is not toothless and is fully applied. all in all, in the history of armed conflict there have been many examples of neglect and failure to comply with international humanitarian law, relating to members of the armed forces, medical personnel, humanitarian workers (icrc), as well as civilians. this is because of a lack of respect for the signs of the red cross and red crescent under which the medical professionals operate, the attitudes of governments that ignore the action of international organizations and institutions on the grounds that this action constitutes interference in their internal affairs (such as the armed conflict in darfur, the western province of sudan), and the occurrence of a new kinds of armed conflict, including so - called unstructured conflicts (french destructursthere is no clear division between the warring parties) and fights between armed forces and terrorists in circumstances of large - scale terrorist activity (such as the russian in addition, modern international humanitarian law provides protection to devices (means of transport, hospitals) and medical staff. consequently, in situations of international armed conflict, the geneva conventions and additional protocol i should be implemented. in this type of conflict, humanitarian law is intended to primarily protect the parties of the conflict, as well as any individuals or any group of people who do not participate in the conflict or have ceased to take part in it. in the case of a non - international armed conflict, article 3 common to the four geneva conventions and additional protocol ii is applied. nevertheless, in line with contemporary international humanitarian law in armed conflicts (especially non - international conflicts, in which there is no clear division between the warring parties) where the application of military force is legally and morally justified, there are certain measures that can not be executed. the validity of the fight against terrorism, the scourge of the twenty - first century does not justify the use of certain forms of violence, especially against civilians. for that reason, attacking civilians, including medical personnel, is a violation of the geneva conventions ; it is a war crime and a crime against humanity. thus, all countries that have ratified the geneva conventions and additional protocols are obliged to follow the rules of war outlined in them and to ensure their dissemination during peacetime. educating societies in the field of international humanitarian law may help to prevent attacks on medical facilities and personnel, as well as significantly improve the fate of the victims of armed conflict.	introductioninternational humanitarian law affords special protection to medical property and personnel whose mission is to save lives and provide health care for civilians and combatants alike.discussionthis paper presents the legal aspects of medical - personnel protection in armed conflicts. presented below are examples of the afghanistan analyses where, as a result of war situations, people are most vulnerable. discussed are the minimum protection and standards applicable to such situations specified by the international humanitarian law.conclusionits rules and provisions obligate fighting parties to take all necessary measures to protect and respect medical missions in all circumstances.
photorhabdus (family : enterobacteriaceae) with their natural vectors, the entomopathogenic nematodes (epn) (phylum : nematoda ; order : rhabditida ; family : heterorhabditidae), have emerged as important biological control agents of insect pests, and are capable of production and delivery of diverse compounds to influence host biology,,. raw sequencing reads, and whole - genome shotgun assemblies for three p. luminescens strains have been deposited at ddbj / embl / genbank under the accession numbers provided in table 1. total raw reads, sequenced base - pairs, n50 value of wgs assembly and snps identified from p. luminescens nbaii h75hrpl105, p. luminescens nbaii hipl101, and p. luminescens nbaii hbpl105 have been summarized in table 2. are symbiotic bacteria associated with soil - born heterorhabditis and steinernema species of entomopathogenic nematodes. bacterial cultures were established from isolation in these natural hosts, then cultured in lab hosts galleria mellonella. finally, pure monoxenic cultures were then grown in lb media. purity of these isolations was checked with 16s rrna gene sequences, before they were used for whole genome sequencing. isolated genomic dna was then used for sequencing and library preparation using the illumina miseq platform (at chromous biotech ltd., bengaluru, 560692, karnataka, india) with paired - end libraries generated for each of the three bacterial genomes. reads were processed, analyzed and trimmed according to fastqc to remove illumina adapter sequences. trimmed reads were assembled into contigs to capture whole - genome shotgun sequences (wgs) using de novo and reference - guided methods using clcbio genomics workbench v. 7.5. luminescens strains were mapped to the reference genome of p. luminescens laumondii strain tt01 (ncbi accession nc_005126.1, for reference - guided genome assemblies) using global alignment, and trimmed where base - call confidence was less than 95%. sequence variants (snps, multiple nucleotide polymorphisms and indels) were identified against the reference genome of p. luminescens subsp. laumondii tt01 reference genome (ncbi accession nc_005126.1) in clcbio genomics workbench using the following parameters : minimum variant coverage50, minimum variant count9, minimum variant frequency50%, minimum quality score neighborhood radius13, minimum variant quality score30, and minimum neighborhood quality score25, p. luminescens strain nbaii h75hrpl105, p. luminescens strain nbaii hipl101, and, p. luminescens strain nbaii plhb105 isolated from the entomopathogenic nematodes, heterorhabditis species strain nbaii h75hr, heterorhabditis indica strain nbaiihi101 and heterorhabditis bacteriophora strain nbaii hb105, respectively.	we report here draft whole genome sequences of three novel strains of photorhabdus luminescens of 5.25.3 mbps in size, and with a g + c content of 42.5% (each). symbiotic -proteobacteria belonging to the genera, photorhabdus (family : enterobacteriaceae) with their natural vectors, the entomopathogenic nematodes (epn) (phylum : nematoda ; order : rhabditida ; family : heterorhabditidae), have emerged as important biological control agents of insect pests, and are capable of production and delivery of diverse compounds to influence host biology [1 ], [2 ], [3 ]. analysis of these genomes is expected to provide enhanced insight into mechanisms of virulence, insecticidal toxin genetic diversity, antibiotic resistance and monoxenicity. the nucleotide sequence information for the three strains nbaii plhb105, nbaii hipl101 and nbaii h75hrpl105 has been deposited in ncbi nucleotide database and is accessible via azab00000000, jthj00000000 and jxur00000000 accession numbers respectively.
mathematical models can explain elegantly what might appear as bewilderingly complex variations in species abundances. seminal work starting in the early 20 century [2 - 4 ] has, in fact, become so familiar to population biologists and beyond that today we are hardly surprised to see complex oscillatory patterns or complex dependencies of population dynamics on a myriad of environmental and demographic factors. many of these phenomena can straightforwardly be explained in terms of relatively simple population dynamics models. the success of these models has also meant that ecological ideas are coming to pervade the analysis of other interacting systems, including cancer, stem cells, and even the banking system, all of which are characterized by the interactions between different entities that affect the overall dynamics of the system and its stability. simple models are beguiling and shape our intuition and allow us to explain trends in data. in many important scenarios, however, different factors come together with sometimes complex patterns resulting from their interplay. thus, understanding realistic systems subject to a multitude of internal and external factors is hard. this is further complicated in situations where models are used to make predictions or assess different types of interventions in silico prior to their implementation in, for example, conservation biology. these challenges are not unique to theoretical ecology, of course, and recent years have seen concerted efforts to tackle the so - called inverse problem : estimating parameters of a model from data ; choosing from among a set of plausible candidate models the model that is best able to explain the data ; or inferring mechanistic or statistical dependencies between the different state variables making up a system in an ecological case, this would, for example, be the species considered in the model. below, we are considering population dynamical models where a vector, x, containing n species, x1,x2,,xn describes the abundances of species in the ecosystem. these are assumed to change as a result of interactions among the species (and potentially external factors) according to some rate laws, dxdt = f(x;),(1) where, with slight abuse of notations, we will also implicitly allow for stochastic dynamics. the community matrix of the ecological system (1) is, of course, given by a=f(x;)x, which captures the ecological relationships among the species. finally, the (vector - valued) parameter denotes the typically unknown demographic and system parameters (for example, birth, death, and migration rates) as well as parameters characterizing the interactions between and within species. below, we will discuss methods that allow us to infer the parameters,, and choose between different potential models (for example, f1,f2,,fk). the statistical toolset that we will discuss, centered primarily around abc, complements traditional mathematical approaches that have been used in theoretical population biology to great effect since the 1950s. but the aim here is rather than to focus on general mathematical laws governing the behavior and fate of natural populations to make models as specific to a given problem, to identify the key factors driving an ecosystem 's dynamics, or to make predictions about the future of an ecosystem. model selection the process of comparing the ability of different models to explain some data is continuing to attract the attention of statisticians and domain experts in different scientific disciplines. but for many challenging real - world problems, conventional statistical approaches become computationally too cumbersome very quickly. this class of problems includes many stochastic processes, highly structured populations, and those where different types of data need to be considered. often, it is still straightforward to establish simulation models in general, real - world problems tend to defy purely analytical approaches but conventional statistical approaches become computationally too expensive. arguably, many of the most contentious problems in population biology (or science in general) fall into this category of problems. a model abstracts from reality what are known or believed to be the essential features of a real natural (or technological or social) system. this fact alone has in the past added to some controversies : as all models are wrong, it is necessary to identify the best model that captures and allows us to quantitatively and qualitatively understand the dynamics of the real system. thus, we need statistical tools that allow us to deal with complex systems, many of which are expected to stretch conventional statistics. here, we develop a viable alternative that maintains most if not all of the advantages of the bayesian inferential apparatus but can be extended to problems defying conventional statistics.,dn }, we need to infer the parameters,, from the data. the likelihood is defined as the probability of obtaining some data d given a parameter value ', l()=pr(d\|)(2) this is the central quantity in likelihood inference ; crucially, the likelihood contains all the information about the parameter that can be extracted from the data d. in bayesian inference, it, together with the prior distribution of, pr(), strikes a balance between what is or can be known about the parameter prior to having seen the data, and the information contained in the data, to give rise to the posterior distribution, pr(\|d)=pr(d\|)pr()pr(d).(3) here, pr(d) denotes the evidence. it is often thought of as a normalization constant but does in fact contain information about the ability of a model to describe the data. obtaining the posterior distribution, or a sample from it, is computationally demanding. in general, computing the evidence pr(d), which is typically a high - dimensional integral, is complicated. sometimes, the focus, therefore, may shift from consideration of the whole (posterior) distribution to the maximum (mode) of the posterior distribution ; this maximum a posteriori estimate is the bayesian equivalent to the maximum likelihood estimate. so that the additional information contained in the distribution markov chain monte carlo (mcmc) methods have become the main workhorses of computational bayesian statistics and have allowed us to generate samples from the posterior distribution. recent years have witnessed increased interest in these and related methods such as population and sequential monte carlo techniques but even the most sophisticated approaches reach their limits when the number of parameters or the complexity of the model increases. the first problem, the so - called curse of dimensionality, is shared by all statistical inference procedures. for example, we may ask ourselves whether there are simpler versions, ma(a), of the model that we are considering, m0(), that would, despite the simplification or coarse - graining (by simplification we typically mean that the dimension of the parameter vector is smaller in the simplified model, that is, \|a\|<\|\|), allow us to draw meaningful, verifiable (or falsifiable) mechanistic insights from the available data. in principle, this might appear to exacerbate the statistical problem, for we would have to find computationally affordable and sufficiently discriminatory ways of deciding if and when a simpler model ma(a) is a good approximation to the original model, m0(). first, however, we discuss abc methods, which form an alternative approach to tackling statistically challenging problems in a bayesian framework and which have become a popular alternative to conventional (or exact) bayesian inference in many applications, especially in evolutionary, population, and systems biology. in abc, we stay as close as possible to the model of interest but instead forgo evaluation of the likelihood in favor of a comparison between simulated and real data. for many systems, the likelihood becomes computationally intractable, either because of the model complexity or the detailed nature of the data. the principal underlying insight of abc is that we can consider pr(d\|)=lim0pr([d, d])\|),(4) where d is data obtained by simulating from our model with parameter, [x, x ' ] is a distance function that can be chosen flexibly to suit the problem at hand, and is a tolerance threshold that reflects the desired accuracy of our inference. the essential problem is that for any complicated problem, it is impossible to obtain the precise dataset, d, by simulating from the model d, even if we know the true parameter (we ignore the artificial problem of deterministic dynamics with no observational noise). by increasing the threshold, our inference becomes more approximate, but the chance of obtaining a simulated dataset for which [d, d]< increases. the comparison of real and simulated data is particularly straightforward for ecological time - series data, for example. here, d might take the form of vectors of population abundances, xt, for n species collected at t=1,, the euclidean (or any other vector) norm provides a suitable distance. generalization to compartmental or spatio - temporal models (or both) is straightforward : if we can simulate data efficiently, we can appeal to the bayesian inference formalism via abc (keeping in mind the nature of the approximation and the tolerance threshold). instead of comparing the data this has been one of the main advantages as well as sources of contention for abc inference. we call a statistic, s of the data, sufficient if and only if pr(d\|,s)=pr(d\|s). in this case, we can replace the data by the sufficient statistic without any loss of information about the parameter,. the attraction of using sufficient summary statistics lies in the fact that their dimension, ds, is typically much smaller than the dimension of the data itself, dd (in the above example of n species sampled at m time points, dd = mn) ; that is, ds dd. especially in population genetics, which has inspired the rise of abc methods since the late 1990s, the use of summary statistics has been popular (see, for example, [24 - 28 ]). with the use of summary statistics, the likelihood can be written as pr(d\|)=lim0pr([s(d),s(d)])\|),(5) with potentially an appropriate change in the distance function and. although equation 5 works very well for parameter inference if s is sufficient, it is important to note that sufficient statistics are few and far between for any real - world problem. unfortunately, abc requires appropriate sufficient statistics (or comparisons of the data directly, as in the case of time - series problems). there have been attempts to generate collections of statistics that together fulfil sufficiency properties [28 - 33 ], but these are computationally expensive in their own right. so far, we have implicitly considered abc in a simple rejection framework : (a) we sample a parameter from a suitable prior, (b) we simulate the model for the parameter, and then (c) we compare the simulated and the real data (or their respective summary) statistics and accept a parameter as a draw from the abc posterior if the distance is below some threshold. steps (a) to (c) are repeated until a sufficiently large number of parameter values have been accepted. the posterior in this case is represented as a sum over indicator functions, prabc(\|d)=i=1n1(i), where either [d, di] or [s(d),s(di)], depending on whether the data or sufficient summaries are used in the inference process. this framework is as simple as it is impractical : like all rejection samplers, it is limited to small problems involving less than a handful of parameters. it has been possible to construct abc - mcmc samplers, but the real workhorses of most abc approaches to real - world problems are based on sequential monte carlo (smc) approaches ; abc - smc has become a very popular field of research (arguably inspiring more detailed analysis also in exact smc samplers), and recent developments are allowing us to tackle larger and more complicated systems. the most widely used flavor of abc - smc proceeds by constructing a set of intermediate distributions that start from the prior and increasingly resemble the posterior. to do so, a sequence of decreasing thresholds, 12 k (with k=), is defined and the sequence of distributions is constructed by sampling parameter vectors from the previous distribution (or the prior in the first step), perturbing them by using some perturbation kernel function, and accepting those parameter vectors for which the distance between real and simulated data falls below the threshold k. choice of the thresholds and the nature of the perturbation kernels determine the computational efficiency and runtime of the inference, but both can be tuned to speed up the process and tackle larger problems. so far, we have assumed that we have a single model that describes our system of interest. the bayesian framework readily provides us with credible intervals for parameters, but it is also possible to assign probabilities for different models to be the correct model, conditional on the available data and the set of competing models, mim={m1,,mk}. in the likelihood framework, the comparison of general (that is, non - nested) models is made possible only through the use of information criteria ; in the bayesian framework, the posterior probability of a model is given analogously to equation 3 by pr(mi\|d)=pr(d\|mi)pr(mi)pr(d).(6) which is also known as the marginal likelihood of model mi. in principle, bayesian model selection allows us to compare any number of arbitrary models. an additional advantage is that the selection via the marginal likelihood, equation 6, automatically strikes a balance between the ability of models to reproduce or explain the observed data, the complexity of the model, and the robustness of the inference. equation 6 can be interpreted in the abc framework, and abc model selection has been an area of great interest and activity [40 - 45 ]. although model selection is indeed straightforward if experimental and simulated data are compared directly, it has been shown that model selection becomes unreliable when summary statistics instead of the data are compared : summary statistics are sufficient for model selection for only a very restricted set of problems. constructing sets of statistics that are sufficient for model selection (they must be sufficient for every model considered and across the models ; this is an area of active research), while possible in principle, is computationally enormously demanding. in many ecological problems, however, we deal with spatio - temporal time - series data, for which model selection is possible. our aim in such cases if no single model emerges from such a comparison, then we need to investigate those models that have comparably high marginal likelihoods. simulations from the respective model posteriors can then be used, for example, to develop more discriminatory experimental designs that allow us to further distinguish among these models. abc methods were borne out of a need to tackle problems that defy conventional statistical methodologies. it has become clear, however, that whenever suitable bayesian alternatives that do deal with the proper likelihood are available, abc becomes computationally too expensive. the reason for this is primarily the fact that the representation of the posterior (as a weighted sum over dirac functions) is not very efficient. in parallel to their role in computationally demanding applications, abc techniques have, more recently, also attracted attention as an inferential framework in their own right. from this, interesting new approaches to deal with real - world problems may well emerge. in conclusion, abc - based methods are best suited to those problems for which other likelihood - based (or exact) bayesian inference procedures do not yet exist. many stochastic and highly structured spatio - temporal problems in ecology, epidemiology, and evolutionary genetics clearly fall into this category. the recent developments discussed above mean that abc has become a viable new way of tackling computationally demanding parameter inference problems. given a model as long as we can simulate it abc gives us a handle to evaluate approximate posterior distributions, which then can be further evaluated. sensitivity and robustness analyses, but also predictions of future behavior or the likely effects of any interventions or perturbations, can be analyzed by simulating the model with parameters sampled from the posterior. there is enormous scope for basing the exploration of, for example, policy or conservation measures on the available data in this way. abc has, for example, been used in experimental design and in synthetic biology to generate designs of molecular pathways that exhibit certain types of behavior. in such cases, we replace the observed data, d, by a representation of the desired behavior (such as the desired abundance of a species). then the inference procedure is used to identify the scenario for which we are most likely to observe this outcome. such predictions then reflect the best available evidence in light of the data and the model. as an aside, it is worth keeping in mind that the technical challenges of statistical inference and modeling can often be minor compared with the difficulties in communicating the results to policy makers or the general public. many of the most pressing problems in ecology have become highly emotive topics as they nearly always involve a conflict between parties that have very different priorities (see, for example,). in many complicated situations, the nuance and cautiousness that accompany how we present, however, abc, with its explicit focus on simulation, may even have an advantage, as the underlying rationale is so straightforwardly explained and easy to understand.	mathematical models have been central to ecology for nearly a century. simple models of population dynamics have allowed us to understand fundamental aspects underlying the dynamics and stability of ecological systems. what has remained a challenge, however, is to meaningfully interpret experimental or observational data in light of mathematical models. here, we review recent developments, notably in the growing field of approximate bayesian computation (abc), that allow us to calibrate mathematical models against available data. estimating the population demographic parameters from data remains a formidable statistical challenge. here, we attempt to give a flavor and overview of abc and its applications in population biology and ecology and eschew a detailed technical discussion in favor of a general discussion of the advantages and potential pitfalls this framework offers to population biologists.
living cells emerged as open systems, exchanging matter and energy with their surrounding, extracting and channelling energy to maintain themselves in a dynamic steady state distant from equilibrium. one of the properties that distinguish living organisms is, among others, a high degree of chemical complexity and molecular organization, defined functions for each of their components and regulated interactions among them. for this reason, chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. however, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. aging, in this chemical context, could be viewed as an entropic process, the result of chemical side - reactions that chronically and cumulatively degrade the function of biological systems. death, the endpoint in the kinetic process of aging, represents the threshold of damage sufficient to compromise the normal function of an essential physiological subsystem or its ability to survive a challenge to that function. although aging seems to be a multicausal process (other causal factors involved in the aging process are, e.g., membrane unsaturation, maillard reaction, proteasome, autophagy), it is likely due to a relatively small number of main causes with major effects. this would make it possible to study the main endogenous mechanisms of aging. reactive oxygen species (ros), especially those of mitochondrial origin, are most likely among those main causes [911 ]. mitochondria are a main source of ros in healthy aerobic tissues and are the only known extra - nuclear cellular organelles in animal cells that contain their own dna (mtdna). mitochondrial ros generation occurs continuously throughout life at a animal species - specific rate independent of the rate of mitochondrial oxygen consumption but related to the longevity of each species and, consequently, with their rate of aging [11, 13 ]. in spite of the existence of many kinds of cellular antioxidants, as adaptive response to ros generation, there is always a certain steady - state level of oxidative damage to macromolecules even in healthy unstressed animals [14, 15 ]. ros can modify mtdna directly at the sugar - phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, including the most commonly measured 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodg), as well as single and double strand breaks and dna mutations [16, 17 ] (figure 1). reactive carbonyl compounds derived from carbohydrates and lipid oxidation reactions can also react at the exocyclic amino groups of deoxyguanosine, deoxyadenosine, and deoxycytosine to form various alkylated products [18, 19 ] (figure 1). some common carbonyl compounds that can potentially cause mtdna damage are malondialdehyde, acrolein and 4-hydroxynonenal, among others. the most common adducts arising from enals are exocyclic adducts such as malondialdehyde - deoxyguanosine (m1 g). many investigations have found that tissue steady - state levels of 8-oxodg show a moderate increase during aging in brain, heart, or liver nuclear (ndna) or mitochondrial dna (mtdna) in rodents and humans [2023 ], while such increases were not detected in other cases [2325 ]. while 8-oxodg is normally present both in ndna and in mtdna, its level is several folds higher in mtdna than in ndna [12, 23, 26 ]. this has been attributed to various reasons like the lack of protective histones and polyamines or to a lower repair of mtdna in relation to ndna. however, while mtdna lacks some forms of dna repair, its capacity to repair 8-oxodg seems to be similar to that of ndna. thus, the most probable reason for the higher level of 8-oxodg in mtdna than in ndna is its closeness to the main ros generator of healthy cells, the inner mitochondrial membrane (figure 1). other factors, like the lack of introns in mtdna, can amplify the detrimental consequences of the relatively higher level of oxidative damage of mtdna. the genetic information is tightly packed on the mtdna molecule and, therefore, most mutational changes on this genome should have injurious effects. the observed increases in oxidative damage to mtdna with aging likely reflect the net flux of oxidative damage resulting from the balance between prooxidant sources and antioxidant / detoxifying / repair systems that would contribute to age - related deterioration at all levels of the biological organization, possibly due to its capacity to generate mtdna mutations. the mitochondrial genome principally suffers from two types of mutations : point mutations, which are changes of one or a few nucleotides ; and large deletions, which involve the removal of large portions of the genome (from a few hundred base pairs to almost the entire genome). the exact sources of mtdna mutations are matter of debate, but might be similar to those causing nuclear dna mutations, that is, errors during dna damage processing or spontaneous polymerase errors [28, 29 ]. inside mitochondrial respiratory chain, electrons from reduced substrates are passed from complexes i and ii of the electron transport chain through complexes iii and iv to oxygen, forming water and causing protons to be pumped across the mitochondrial inner membrane. the proton motive force set up by proton pumping drives protons back through the atp synthase in the inner membrane, forming atp from adp and phosphate. in this context, a major side reaction is that electrons may leak from the respiratory chain and react with oxygen to form the free radical superoxide. superoxide anion, the product of a one - electron reduction of oxygen, is the precursor of most ros and a mediator in oxidative chain reactions. there is, however, a lack of stoichiometric coupling of ros production to oxygen consumption. it is well known that mitochondrial ros (mtros) generation occurs at complex i and at complex iii (reviewed in [11, 14 ]). concerning the electron transport component responsible for mtros generation within complex i, flavin mononucleotide, ubisemiquinone species, or iron - sulphur clusters have been proposed (reviewed in [3135 ]). interestingly two of these, the flavin and all the fes clusters, are located in the hydrophilic complex i domain facing the mitochondrial matrix, which would facilitate close proximity, or even the contact between mtdna and the ros generator (see figure 1). in contrast, complex iii produces ros directed to the cytosolic side of the inner membrane, although recent studies suggest that part of the production could also occur towards the matrix side. the following can be included (reviewed in [14, 33, 37 ]) : (i) to adapt the amount of the respiratory complex / es responsible for ros generation. thus, a decrease in the amount of complex i protein will lead to a decreased rate of ros generation ; (ii) to adjust the amount of uncoupling proteins. the mitochondrial superoxide production is very sensitive to the proton motive force, so it can be strongly decreased by mild uncoupling. in this scenario, an ancestral function of uncoupling proteins has been proposed : to cause mild uncoupling and so diminish mitochondrial superoxide production, hence protecting against oxidative damage ; (iii) to regulate the degree of electronic reduction of these generators : the higher their degree of reduction, the higher will be their rate of ros production ; and (iv) to modify by enzymatic and nonenzymatic pathways such as s - nitrosation, acetylation, and glutathionylation specific peptides of the ros generators. these studies found that the rate of mtros production is lower in the tissues of long - lived than in those of short - lived animal species [11, 12, 14 ]. early studies, however, only included animal species following the rate of living theorythe inverse relationship between longevity and metabolic rate-, so the results obtained could also be interpreted as a correlate of that phenomenon. species with short longevity could show high mitochondrial ros production simply because their rates of mitochondrial oxygen consumption are higher. fortunately, comparative physiology offers more possibilities to test the hypothesis that mitochondrial ros production is involved in the rate of aging and, consequently, in determining longevity. three groups of homoeothermic vertebrates have an extraordinarily high longevity in relation to their body size and metabolic rate : birds, bats, and primates. birds can be a strong test because if a low rate of ros production contributes to slowing of the aging rate, the mitochondria of birds should show a low rate of ros generation in spite of the high rate of oxygen consumption of these animals. thus, in spite of their high rates of whole body o2 consumption, parakeets, canaries, and pigeons (with longevities of 21, 24, and 35 years, resp.) have lower rates of mtros generation than mice and rats (3.5 and 4 years of longevity, resp.), even though their metabolic rates and body sizes are of a similar magnitude. the difference in ros production between these species occurred at only one of the electron transport complexes : complex i. the low rate of mtros production of birds, in spite of their high rates of oxygen consumption, is possible because (a) the percent of total electron flow in the respiratory chain directed to ros production (the % free radical leak, % frl) is lower, and (b) the total amount of mitochondrial complex i is also lower [38, 39 ]. this means that their respiratory chain transports electrons more efficiently avoiding univalent electron leaks to oxygen upstream of cytochrome oxidase. all the comparative investigations performed in mammals and birds show that mitochondrial ros generation is lower in long- than in short - lived animals. interestingly, this finding is even more significant after correction for effects of body size and phylogeny. furthermore, other interspecies comparisons between rodents with widely different longevities but similar body size (see), or between rodents and bats, also showed that the longest - lived species had lowest mitochondrial ros production and leak. recent studies have also found much lower rates of ros generation in human (primates and especially humans also live much longer than expected for their body size and metabolic rate) than in rat brain mitochondria. if mitochondrial oxygen radical production controls aging rate, the rate of ros production of animals with different metabolic rates and body sizes but similar longevity should be equal. this is exactly what happened in another interspecies comparison when mitochondrial ros production of mouse (longevity, 3.5 yrs) and rat (longevity, 4 yrs) were compared [43, 44 ]. the longevity of these species are similar (3.5 and 4 years), whereas metabolic rate is threefold higher and body size is around 15 fold lower in the mouse than in the rat. the relevance of ros production in determining longevity has also been recently reinforced by using drosophila as experimental model. in this study, transgenic strains of drosophila were created that express yeast ndi1 (in yeast, the single - subunit nadh dehydrogenase ndi1 serves as a nonproton - translocating alternative enzyme that replaces complex i, bringing about the reoxidation of intramitochondrial nadh) ubiquitously. ndi1 expression mitigated the aging - associated decline in respiratory capacity and the accompanying increase in mitochondrial ros production, and resulted in decreased accumulation of markers of oxidative damage in aged flies, resulting in an increased median, mean, and maximum longevity. these results support a central role of mitochondrial complex i in influencing longevity via oxidative stress. in accordance with the low mitochondrial free radical production of long - lived animal species, there is an adaptive response concerning endogenous cellular antioxidant systems (reviewed in [14, 32 ]). thus, long - lived vertebrates, including mammals, constitutively have lower (instead of higher) tissue levels of antioxidant enzymes and low molecular weight endogenous antioxidants, as well as repair systems, than short - lived ones. that characteristic can thus explain why endogenous tissue antioxidants correlate negatively with longevity across species : long - lived animals have constitutively low levels of antioxidants because they produce ros at a low rate. if long - lived animals had high rates of ros production together with their very low levels of endogenous antioxidants, their tissue cells would not be able to maintain oxidative stress homeostasis. decreasing mitochondrial ros production instead of increasing antioxidants or repair systems makes sense when considered from the point of view of evolution of longevity among species. it would be very inefficient to generate large amounts of ros and, afterwards, try to intercept them before they reach cellular components like dna, or even worse, try to repair dna after heavily damaging it. this makes even more sense taking into account the high energetic cost of continuously maintaining high levels of antioxidant and repair molecules in cells. interestingly, there is a close proximity or even contact between main sources of ros at the inner mitochondrial membrane and mtdna. so, lowering the rate of mtros generation near mtdna decreases its damage much more efficiently and at much lower cost. in agreement with the adaptive response that represents antioxidant defences, and with the ability of all kinds of animals (short- or long - lived) to transitorily induce or repress these protective molecules when necessary in the needed amounts, the outcomes of two experimental paradigms deserve also special mention (reviewed in [11, 48, 49 ]) : (i) experimentally increasing tissue antioxidants through dietary supplementation, pharmacological induction, or transgenic techniques sometimes moderately increases mean longevity (life expectancy) but does not change maximum longevity ; and (ii) animals in which genes coding for particular antioxidant enzymes are knocked out can show different pathologies but their rates of aging do not seem to be affected. it is proposed that these findings are probably due to the activation of negative feedback mechanisms in order to maintain the redox balance, which is cell and species specific. it is this low endogenous rate of generation of highly damaging substances that can importantly contribute to a slower rate of accumulation of dna damage and mutations, and then to a longer life, in long - lived species. mitochondrial dna oxidation is one of the natural consequences of aerobic life. among different kinds of molecular damage caused by ros, that to mtdna is important for longevity because it can lead to irreversible loss or alteration of its coded information, which will be especially deleterious in postmitotic tissues. determinant mechanisms of the steady - state level of mtdna damage include (i) because long - lived animal species have low rates of mtros generation, this should be reflected in the steady - state level of oxidative damage and the accumulation of somatic mutations in their mtdna ; (ii) mtdna is located very close to or even in contact with the site / s of mitochondrial ros production (see figure 1) ; and (iii) the structural susceptibility of the mtdna molecule to damage. the susceptibility of mtdna to damage could be related to the simplest property of a dna sequence, the proportion of a, c, g, and t in the mtdna molecule. in a recent study, analyzing the light strand mtdna sequence of 94 animals species (including invertebrates, birds, and mammals), it was found that short - lived species have higher a and t abundances and lower c abundances than the long - lived species, while g is almost uniformly low in all species. the light strand was given this name because it is unusually deficient in g, and data from this study shows that this characteristic low g abundance carries through to all of these species. a conclusion is that there is some strong evolutionary pressure to keep the g abundance of the light strand low. the standard hypothesis is that the asymmetric mode of replication of the mtdna molecule causes an increase in the g to a transition mutations on the light strand, depleting it of g nucleotides. in addition to this finding, it was verified that this pattern of nucleotides affects a dna sequence property, the free energy (a physical property of the double - stranded dna molecule related to the binding energy between the two dna strands ; the more negative the free energy is, the less likely is the spontaneous separation of the two strands through thermal fluctuations, conferring to the mtdna a greater structural stability and lesser susceptibility to damage). thus, the longer the maximum longevity of a species, the lower is the free energy of mtdna. considering that of the four nucleobases, guanine has the lowest oxidation potential and is thus generally most easily oxidized, it is proposed that these mtdna base sequence pattern and derived properties represent an evolutionary adaptation of long - lived species to increase mtdna resistance to endogenous damage by chemical side - reactions and so to decrease the susceptibility of mtdna to damage and mutation. a similar evolutionary adaptation has been demonstrated for proteins (by the methionine content) and membrane unsaturation (by the polyunsaturated fatty acids content) in long - lived species [5, 14 ]. in agreement with their high resistance to endogenous damage and low rates of mitochondrial ros production, long - lived animal species have lower steady - state levels of 8-oxodg in their mtdna [51, 52 ] and lower rates of urinary excretion of 8-oxo-7,8-dihydroguanine than short - lived ones. long - lived birds also usually show lower oxidative damage to mtdna in their tissues than short - lived rodents of similar body size. it has been also described that the rate of accumulation of mtdna mutations with age is also much slower in humans than in mice. in this line, in a recent study, analyzing the lineage - specific mitochondrial mutation rate across 1,696 mammalian species and comparing it with the nuclear rate, is reported a selected decrease of substitution rate in long - lived species, in agreement with the evidence for a causal role of mtdna mutations in aging (reviewed in), suggesting that natural selection tends to decrease the mitochondrial mutation rate in long - lived species. in addition, oxidative damage is around 59 fold higher in mtdna than in ndna in the brain and heart of all the 11 mammalian and bird species studied [51, 54 ], a difference similar to that generally observed for spontaneous mutations when comparing both dnas. in order to maintain mitochondrial genomic integrity and to avoid the gradual accumulation of oxidative dna base lesions, mtdna damage is repaired by a number of mechanisms (for review see). in general, oxidized mtdna bases are removed by base excision repair (ber) mechanisms [59, 60 ] and although these correcting mechanisms show high fidelity, dna lesions can still accumulate with age. dna polymerase enzymes (as component of the ber machinery) have a proofreading error - correcting capacity, and when mitochondrial dna polymerase proofreading activity was eliminated in mice there was a 35 fold increase in mitochondrial mutations and a decreased longevity in such mice [61, 62 ]. in support for a relevant role of mtdna mutations in the rate of aging, in a recent study it has been demonstrated that mutations in mtdna profoundly affect the assembly of fully functional etc complexes, leading to mitochondrial dysfunction and ultimately, to the activation of a mitochondrial - mediated apoptotic program, as mechanism underlying the accelerated aging process of this experimental model. all that might suggest that the flux rates of both ros attack on and repair of dna are much higher in the mtdna of short - lived than in that of long - lived animals, and are also much higher in the mtdna than in the ndna of all species. in addition, the higher rate of mtros production and mtdna susceptibility to oxidative damage of short - lived animals may be an important cause of their much faster rate of accumulation of mtdna mutations during aging [55, 65 ]. thus, it could be caused in part by the long - life progressive accumulation of nonrepairable macromolecular damage. this occurs at the level of the main information - containing macromolecule of postmitotic cells, the dna. as mentioned above, it is known that somatic mtdna mutations can accumulate with age reaching very high levels in postmitotic tissues [66, 67 ], and some studies in mutant mice suggest that increasing the level of mtdna mutations increases the aging rate [6163 ]. it has been argued that the high copy number of mtdna molecules per cell is contradictory with a possible role of mtdna in aging, since loss of some mtdna molecules would not be deleterious to the cell because the remaining wild type ones would be enough for the continuation of cell function. however, it is known that clonal expansion of mutated mtdna molecules can bring them near to homoplasmy in old tissues. furthermore, the reliability theory of aging points out that aging appears in complex systems thanks to the presence of redundancy of vital system components. such redundancy also occurs at subcellular level in the case of mtdna which is present in multiple copies per cell. if a single mtdna molecule would exist per postmitotic cell instead of many, its deletion could easily result in cell death. according to gavrilov and gavrilova (2006), some cases of the so - called negliglible senescence in simple organisms would not be cases of very slow aging as it is frequently assumed. instead, they would be the simple result of a lack of redundancy in their vital component parts. they would just die when they are seriously damaged for the first time, and thus they could not tell us anything interesting about slow aging and longevity in mammals. this concept of reliability and aging due to high redundancy could be also applied to mtdna. cells containing large amounts of mutated mtdna molecules seem to survive many years or even decades in human postmitotic tissues, and their wrong functioning likely contributes to the age - related deterioration of tissue functions. large accumulations of mtdna mutations have been recently reported in substantia nigra [67, 70 ] as well as in human skeletal muscle in which 31% of the fibers of old individuals showed electron transport abnormalities and the abnormal fibers showed accumulations of deleted mtdna molecules to detrimental (> 90% of the total mtdna) levels [71, 72 ]. on the other hand, ros have mutagenic capacity, and the rate of accumulation of dna mutations is known to be higher when the cellular po2 is increased, suggesting a role for ros in their formation. mtros generation can be among the causes of such accumulation in vivo and thus of aging. also agreeing with this possibility, both 8-oxodg [51, 74 ] and somatic mutations are present in mtdna at levels around 10 folds higher than in nuclear dna (ndna). since it is now known that the capacity of the mitochondria to repair 8-oxodg is similar or even higher (instead of lower) than that of the nucleus, the difference in steady - state oxidative dna damage between both compartments should be mainly due to the much higher capacity of the mitochondria for continuous ros generation, not to differences in 8-oxodg repair, and possibly to the closeness or even the contact between mtdna and the inner mitochondrial membrane [7678 ]. the comparative studies described above are strongly suggestive of a causal relationship between mtros production and longevity via mtdna damage. but correlation does not necessarily mean that a cause - effect relationship exists, and experimental studies are needed for confirmation. dietary restriction (dr) is the best described experimental intervention that slows down aging and increases longevity in different animal species such as c. elegans, drosophila, rodents (mice and rats), and monkeys. the effect of dr on mtros production has been studied intensively in rodents and especially in rats. these investigations, usually applying 40% dr, consistently demonstrated that long - term dietary restriction (between 1 year of dr and life - long dr) significantly decreases the rate of mtros generation in rat tissues including heart, brain, skeletal muscle, liver, and kidney. it was also found that 6 - 7 weeks of dietary restriction are enough to decrease mtros production and 8-oxodg in mtdna and ndna in rat liver. it was also found that the decrease in mtros generation in dr rats specifically occurs at complex i in all the organs studied so far (heart, liver, and brain), along with a decrease in the amount of the mtros generators, and that it takes place together with lack of changes in mitochondrial o2 consumption and with a decrease in % frl, indicating that the mitochondria of dr animals are more efficient in avoiding ros production per unit electron flow [8284 ]. these characteristics were also observed in long - lived animal species when compared to those of short - lived ones. the lack of changes in mitochondrial o2 consumption also agreed with the lack of variations in basal metabolic rate in dr [85, 86 ]. in addition, the decrease in mtros generation observed in dr rats was accompanied by significant decreases in 8-oxodg levels in mtdna alone, or in mtdna and ndna, depending on the tissue studied [82, 84, 87 ]. on the other hand, the repair of 8-oxodg in mtdna through the mitochondrial base excision repair pathway does not increase and even decreases during dr. thus, the decrease in 8-oxodg steady - state level in mtdna during dr must be due to a decrease in 8-oxodg formation. this is further supported by the fact that the three implicated parameters, mtros generation, steady state 8-oxodg levels in mtdna, and 8-oxodg repair through mitochondrial base excision repair decrease by a similar quantitative extent during 40% dr : around 3040%. this agrees with the previously proposed model indicating that there is a decrease in the flow of oxidative damage through the mtdna in dr, similar to what occurs in long - lived compared to short - lived species. in other words, mitochondrial 8-oxodg repair, like tissue antioxidants in long - lived animals, is lower (instead of higher) in tissues of dr rodents because their rate of mtros production is lower than in those fed ad libitum. neither 8-oxodg repair nor endogenous antioxidants should be continuously maintained at levels higher than required because that would be energetically costly. thus, they can be transitorily increased at moments of higher than normal oxidative damage to quickly return to their basal levels when the situation is normalized. for instance, the repair of 8-oxodg is maximally induced in the rat kidney after only 6 hours of exposure to potassium bromate. the lower steady - state oxidative damage in the mtdna of long - lived and dr animals is much better obtained by decreasing the rate of mtros generation than by increasing antioxidants and repair because this is much less costly and much more efficient and simple [11, 33 ]. in summary, lowering the rate of mtros production seems to be a highly conserved mechanism developed during the evolution to be used both within (dr) and between species. it allows both long - lived species and dr animals to decrease steady - state oxidative damage to mtdna, and likely the rate of accumulation of mtdna mutations and the aging rate. in this scenario, it is proposed that natural selection tends to decrease the mitochondrial ros generation, the oxidative damage to mtdna and the mitochondrial mutation rate in long - lived species, in agreement with the mitochondrial oxidative stress theory of aging.	chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. however, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. mitochondria are a main source of reactive oxygen species (ros) and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own dna (mtdna). ros can modify mtdna directly at the sugar - phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and dna mutations. in this scenario, natural selection tends to decrease the mitochondrial ros generation, the oxidative damage to mtdna, and the mitochondrial mutation rate in long - lived species, in agreement with the mitochondrial oxidative stress theory of aging.
sickle cell (sc) disease is endemic in african subcontinent but is also common in parts of orissa ans andhra pradesh in india. sc disease patients have repeated sickling episodes leading to avascular necrosis of the femoral head. sc disease leading to endarteritis induces skeletal changes in the form of osteitis, sclerosis of the femoral canal and osteonecrosis of femoral head.1 all these make surgery difficult and prolonged. total hip replacement in cases of osteonecrosis of the hip secondary to sc disease poses a considerable challenge to the treating orthopedic surgeon. the soft spongy bone in sicklers is at risk of fracture and bleeds a lot. there is increased risk of infection, sc crisis and increased complication rate in these patients.2345 this study highlights the preoperative, intraoperative and postoperative hurdles encountered in performing a total hip replacement in patients with sc hemoglobinopathy and the short term outcome using cementless implants. thirty nine patients with sc disease, with osteonecrosis of the femoral head, operated between 2007 and 2011 were included in this study. nineteen patients were homozygous for sc (hemoglobin ss), 15 had hemoglobin (hb) s / c and rest were hemoglobin s / beta thalassemia. bilateral cementless total hip replacement was performed in 11 patients (22 hips) and the rest had unilateral involvement (28 hips). only one hip was operated at a time with an interval of 57 days between the surgeries. all patients were classified according to steinberg staging developed by the university of pennsylvania system.6 twenty six were stage v, 9 were stage iv and 4 were stage iii avascular necrosis hip. this was done by giving aggressive preoperative transfusions or using plasmapheresis and exchange transfusion [figure 1].7 intraoperative medullary canal sclerosis [figure 2a ] was cleared using a high speed burr (50% of patients). the operating room temperature was maintained at 22c and the patient was kept hydrated and warm using a bair hugger blanket to prevent hypothermia. all patients were operated using cementless implants (accolade stem, hydroxyapatite - coated acetabular cup, 28 mm/36 mm cocr head and polyethylene liner, stryker howmedica, uk). suction drains were placed in all patients. clinical photograph showing the patient undergoing exchange transfusion using the plasmapheresis machine (a) anteroposterior radiograph of the pelvis with both hips showing the sclerosis in the proximal femora and narrow medullary canals. left femur shows a broken screw left from previous surgery (b) postoperative anteroposterior radiograph of the pelvis with both hips showing cementless implants postoperatively, intravenous fluids were given at the rate of 120 ml / hour to maintain adequate hydration. humidified oxygen was given at 5 l / min for 4872 h. o2 saturation monitoring was carried up to 7 days postoperatively and maintained at 97%. postoperative haemoglobin levels were maintained at > 9 gm%.8 patients were mobilized toe touch weight bearing with the help of a walker next day. the spongy bone in sicklers is so soft that the stability of the cementless implant (bone in growth) may be jeopardized leading to early loosening. so as a precaution, the patients were only allowed partial toe touch weight bearing from first postoperative day and full weight bearing was started at 6 weeks [figure 4 ]. patients were followed clinically and radiologically at 2 weeks, 1 month, 3 months and then yearly [figures 2b and 3 ]. preoperative and postoperative modified harris hip score was evaluated.7 (a) preoperative anteroposterior view of the pelvis with both hips showing advanced stage osteonecrosis of femoral heads in a 22 year old male (b) anteroposterior radiograph of the pelvis with both hips after tha on the left side (c) anteroposterior radiograph of both hips at 2 years followup after tha on both sides with no signs of aseptic loosening (a) preoperative (b) postoperative radiograph of thirteen years girl who underwent total hip replacement due to avn hip secondary to sicking (c) clinical photograph of same girl showing toe touch walking the average operating time was 96 min (range 88148 min). the average blood loss in patients was 880 ml (range 6501200 ml). the average intraoperative blood (leukodepleted packed red blood cells) transfused were 2.3 units (range 25 units) to maintain a hemoglobin level of 810 gm% and dilute the hbs load. all the patients showed an improvement in harris hip score which improved from average 42 points preoperatively to average 92 points at latest followup. intraoperatively, one patient had a periprosthetic fracture for which stainless steel cerclage wiring was done. subsidence was not noticed in any of the cases till latest followup. there were no early or late dislocations. no heterotopic ossification was seen in any of the cases. there were no cases of sciatic nerve palsy. the incidence of avascular necrosis in sicklers has been reported to be between 10% and 30%.910 avascular necrosis of femoral head has been reported more commonly in sc disease (29%) and 19% in adults with ss disease.111213 preoperatively, average 45 units of leukodepleted packed red cells should be arranged in each case to attain a hemoglobin level of 810 gm%. this reduces the chances of development of postoperative acute sc crisis.3 all patients should be evaluated for antibodies in the blood as these patients have high chances of antibody levels due to repeated transfusions. these antibodies it is recommended to decrease the preoperative hbs level to 7 years and found no difference in aseptic implant survivorship (95% vs. 97%). they believed that the outcome of tha in sc patients can be improved by optimizing medical management and use of cementless prosthetic devices.22 we have used cementless implants in our series and have found them to shorten surgical time, are bone conserving and have a predictable outcome at mid term. high rates of aseptic loosening in tha in sicklers have been reported in the literature.32324 hernigou. in their review of 312 hip arthroplasties performed in 244 patients with sc disease at mean followup of 13 years reported an aseptic loosening rate of 8%20 cups and 5%16 stems. they also mentioned that the risk of aseptic loosening was less than that reported in the literature.8 although it is prematute to speak of loosening at 2 years of followup. we have had no aseptic loosening in our series and agree that loosening rates have decreased with the use of cementless implants. further followup is warranted to judge the future of total hip arthroplasty using cementless implants in patients with sc disease. management in sicklers require a multidisciplinary approach involving the anesthetist, hematologist and the orthopedic surgeon. the hematologists need to reduce the sc load preoperatively by exchange transfusion and build hemoglobin to > 9 gm%, the anesthetist need to keep the patient warm and hydrated to reduce sickling and the orthopedic surgeon needs to be meticulous and quick in technique and achieve adequate hemostasis during surgery. during the postoperative period, care needs to be taken to maintain hemoglobin > 9 gm%, reduce sc load by aggressive transfusions, hydration and oxygenation, to avoid sc crisis. contrary to previous reports, tha in sicklers now has a predictable outcome, less complications and failure rates especially with the use of cementless implants and better patient management in the pre, intra and postoperative periods.	background : sickle cell (sc) disease leading to endarteritis induces skeletal changes in the form of osteitis, sclerosis of femoral canal and osteonecrosis of the femoral head. all these make total hip arthroplasty (tha) difficult and prolonged. there is increased risk of infection, sc crisis and increased complication rate. our paper aims to highlight preoperative, intraoperative and postoperative hurdles encountered in performing tha in sicklers and the short term outcome using cementless implants.materials and methods : thirty - nine patients with sc disease, who had osteonecrosis of the femoral head, were operated between 2007 and 2011. the mean age of patients was 22 years (range 1349 years). there were twenty eight females and 11 males. bilateral cementless total hip replacement (thr) was performed in 11 patients (22 hips) and in the rest unilateral (28 hips). preoperative and postoperative modified harris hip score was evaluated. the average followup was 3.8 years (range 2 - 6 years).results : the average operating time was 96 min (range 88148 min). the average blood loss was 880 ml (range 6501200 ml). the average intraoperative blood transfused was 2.3 units (range 25 units). all patients showed an improvement in harris hip score from 42 points preoperatively to 92 points at latest followup. intraoperatively, one patient had a periprosthetic fracture. six patients developed acute sc crisis and were managed in intensive care unit. three patients developed wound hematoma. three patients developed limb length discrepancy less than 1 cm. none had early or late dislocations, infection, heterotopic ossification, sciatic nerve palsy and aseptic loosening.conclusion:tha in sicklers involves considerable challenge for the orthopedic surgeon. management requires a multidisciplinary approach involving the anesthetist, hematologist and the orthopedic surgeon. contrary to previous reports, tha in sicklers now has a predictable outcome especially with the use of cementless implants.
lipoma of the abdominal cavity, a benign neoplasm of mature fat cells usually presents as an asymptomatic abdominal mass or progressive abdominal distention. lipomas are sometimes detected incidentally as an intraperitoneal radiolucent fat density mass on a ct scan. extrahepatic, fat - containing masses of the abdomen and pelvis represent a broad spectrum of congenital, metabolic, inflammatory, traumatic, degenerative, and neoplastic processes. we present a rare case of giant retroperitoneal lipoma in an infant, mimicking hirschprung 's disease, and presenting as an emergency. at laparotomy, a six - month - old baby girl presented to our accident and emergency department with progressive abdominal distention and recurrent constipation, with episodes lasting four to nine days, for all of the four - month duration, and significant wasting. there was no history of delayed passage of the meconeum, no vomiting, and no fever. the child was in respiratory distress, the abdomen was grossly distended, tensed, and nontender, but with uniform dullness to percussion [figure 1 ]. a plain x - ray of the abdomen revealed a soft tissue mass displacing and compressing the bowel loops, with the dome of the diaphragm at the level of the fourth rib anteriorly. an ultrasound (us) examination of the abdomen showed a huge heterogeneously hyperechoic mass occupying the whole abdomen, with the bowel loops displaced toward the periphery. thin fibrous septations were also noted within the lesion. on doppler scanning of the abdomen the vessels the patient was optimized and the nasogastric tube (ngt) drained, with scanty, clear effluent and no reduction of the abdominal girth. an emergency laparotomy was performed, which revealed a huge yellowish tumor arising from the retroperitoneal space occupying the whole abdomen [figure 2 ]. by careful dissection the tumor was removed, it measured 26 19 13 cm and weighed 1.7 kg [figure 3 ]. gross abdominal distention due to retroperitoneal lipoma retroperitoneal lipoma being delivered at the laparotomy the lipoma weighing 1.7 kg retroperitoneal lipoma is an unusual entity that is most often found in adults between 40 and 60 years of age and rarely occurs in the first decade of life. retroperitoneal benign lipomas are extremely rare and represent about 2.9% of all primary retroperitoneal tumors and about 80% of them are malignant neoplasms. in a series of 190 retroperitoneal tumors in infants and children till 1979, only 12 cases of retroperitoneal lipoma in children, diagnosed in the first decade of life, were reported in the literature. clinically, these lipomas produce few symptoms and therefore tend to become large before being discovered. the only report of malignant degeneration was by kretschmer, who removed a large lipofibrosarcoma from a two - yearr - old girl. lipoma of the abdominal cavity usually presents as an asymptomatic abdominal mass or progressive abdominal distention. lipomas are sometimes detected incidentally as an intraperitoneal radiolucent fat density mass, on a ct scan. two cases of histologically proven giant retroperitoneal lipomas, evaluated by ct and sonography, appear to be similar on ct, but they exhibit different echographic patterns sonographically. weather the lipoma is congenital in infants is uncertain, as abdominal masses are not seen at birth in most cases, but the presence of a huge abdominal mass in early infancy, in this patient, indicates that this retroperitoneal lipoma is most probably congenital the long - term behavior of retroperitoneal lipoma in children is not well - defined as compared to adults, due to the inadequate number of cases, therefore, a long - term follow - up is essential in children. retroperitoneal lipoma is rare in children, more especially in infancy ; the emergency presentation was due to pressure symptoms. the characteristic behavior in children is yet to be defined ; therefore a long - term follow - up is recommended.	retroperitoneal lipomas have remained the essentially rare tumors seen in clinical practice. the tumors are rarer in children, with very few reported cases in surgical literature worldwide. we are reporting the case of a six - month - old child who presented with a giant retroperitoneal lipoma that was successfully managed by complete excision. there has been no recurrence noticed during follow - up.
minimally invasive approaches have been the actual goal for neurosurgery. in line with this philosophy besides the esthetic benefit of the minimally invasive approach, there is the possibility to minimalize the operative time and length of hospital stay. at an emergency setting, a 47-year - old patient was admitted with a glasgow coma scale (gcs) of 13 points and remained neurologically stable until surgery, which he was submitted after 72 h of trauma. although he was neurologically stable, the location of the hematoma implicated in the risk of brain stem compression with normal intracranial pressure. it would be necessary to observe for a long period to be sure that there were no more risks for the hematoma expansion. brain computed tomography (ct) scan showed a temporal hematoma with 30 ml of volume, extending from the temporal posterior region to the anterior compartment of the right media fossa. the volume and the location of the hematoma implicate in a high risk of brain stein compression. the brain ct scan was reconstructed using the program osirix, as shown in fig. 1, so that it was possible to establish accurate cranial landmarks to guide the burr hole position. figure 1:osirix ct reconstruction. was put in the proper position, guided by the 3d imaging, we were able to perform a single small incision and a single burr hole at the posterior aspect of the hematoma. the neuroendoscope (storz hopkinsforward oblique telescope 30) was positioned in a 45 direction, headed from the posterior limit of the hematoma to the media fossa floor, in a way that the whole hematoma could be visualized. with the neuroendoscope in place then, both the suctor and the neuroendoscope are progressively inserted through the burr hole, following the natural corridor provided by the hematoma evacuation. if some level of hemorrhage emerges, irrigation with 0.9% saline is enough to stop bleeding. if bleeding persists, pieces of surgicel can be left in place. a small piece of gelfoam is placed over the burr hole, and the wound is closed in layers. the comparison between the pre- and postoperative ct scans is shown in fig. 2. in the actual scenario of minimally invasive techniques, the endoscopic method has been at high spot, especially for intracerebral hematomas. in contrast, for hemorrhagic strokes several authors, with or without complementary methods, have described the technique. in 2010 the author used a 3d reconstructed ct scan to guide the entry point, depth of penetrating path and surgical trajectory. the average hematoma evacuation rate was 82%. using stereotactic guidance described a hematoma evacuation inserting the endoscopy through a metal tube positioned by stereotaxic. the holder assured that the guide tube did not move, providing safety to the procedure. other papers [35 ] confirm that the method is safe with lower mortality compared with the conventional method to treat spontaneous hematomas, and those do not extend the operative time. in traumatic brain injuries, these features may decrease the operative time, which is crucial for a safer procedure. when considering posterior fossa, it is marked the time gained with the endoscopic technique (64.5 versus 230.6 min), as described by yamamoto t.. considering these advantages, two groups applied the endoscopic technique for acute subdural hematomas drainage [7, 8 ] describing one case of non - traumatic and other traumatic large acute subdural hematomas. the technical note presented in this report expresses the tendency of minimally invasive surgeries, focused on lower operative time and lower blood loss, besides the esthetic aspect considered as another advantage. endoscopic surgery, usually described for spontaneous hematomas, may expend its perspectives considering the reduced time of surgery, blood loss and safety, and may also be considered for traumatic brain injuries. this case report is the first to describe the endoscopic technique applied to traumatic intracranial hematoma. also, comparing it to the other tools described before to guide the procedure, the surgical planning reported here has the great advantage that does not require any cost and is also simple to use. our group has already described the use of the open access osirix software for planning craniotomy or burr hole in the emergency setting with a detailed description on how to use the tools provided by the software, which surely does not postpone the procedure. nevertheless, its use to assist an endoscopic surgery was not described in our previous article. this first described technique could be widely spread considering its cost and all the possible morbidities that could be applied, gathering both safety and effectiveness for minimally invasive procedures.	the endoscopic technique has been described as a minimally invasive method for spontaneous hematoma evacuation, as a safe and effective treatment. nevertheless, to our knowledge, there is no description of a technical report of traumatic intracerebral hematoma removal using the neuroendoscope. a 47-year - old man was admitted sustaining 13 points in glasgow coma scale with brain computed tomography (ct) scan showing a temporal contusion. guided by a 3d reconstructed ct, using the program osirix, the posterior limit of the hematoma was identified. a burr hole was placed at the posterior temporal region, and we used the neuroendoscope to assist the hematoma evacuation. the postoperative tomography showed adequate hematoma removal. he was discharged from hospital 48 h after surgery. two weeks later, he was conscious and oriented temporally. this endoscopic - assisted technique can provide safe removal of traumatic hematomas of the temporal lobe.

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