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hypertension is common in hemolytic uremic syndrome ( hus ) , and often difficult to control in an acute stage . malignant hypertension associated with hus leads to reversible posterior encephalopathy syndrome , seizures , heart failure , and other adverse consequences which increase the morbidity and mortality of the disease . renin - mediated mechanism is believed to be the main factor responsible for hypertension seen in these cases . drugs that act by blocking renin - angiotensin axis ( ras ) are thus ideal for such cases , however , due to concern of progression of renal failure and lack of experience of these agents in children , these are not preferred or used commonly in acute stages . we hereby report two cases of hus with severe refractory malignant hypertension in which we targeted ras by using intravenous ( iv ) enalaprilat , oral aliskiren , and oral enalapril with quick and dramatic response of blood pressure ( bp ) . a 6-year - old male was admitted with a history of vomiting , fever since 2 weeks , hematuria and decreased urine output since 1 week . on evaluation by his local practitioner , he was found to have anemia ( hemoglobin [ hb ] 6.3 g / dl ) , thrombocytopenia ( platelet 72,000/mm ) , active urine sediment ( red blood cell [ rbc ] 4060/hpf , albumin 3 + ) , and azotemia ( blood urea 200 mg / dl , creatinine 4.2 mg / dl ) . he had an episode of seizure ( due to accelerated hypertension ) , hence was brought to our hospital for further management . on evaluation , he was hypertensive ( bp 150/100 mmhg ) with generalized edema , oliguria , and a normal systemic examination . investigations were suggestive of hus ( hb 4.8 g / dl , white blood cell [ wbc ] 11,190 cmm , platelet 1.84/mm , peripheral smear : schistocytes positive , reticulocyte count 6.8% , lactate dehydrogenase [ ldh ] 4300 u / l , direct coombs test and indirect coombs tests were negative , urea 67 mg / dl , creatinine 2.6 mg / dl ) . septic work up , dengue serology , and malarial antigen were negative , and he became afebrile on the 4 day of admission . his antinuclear antibodies ( ana ) and antineutrophil cytoplasmic antibody ( anca ) were negative . he was started on empiric antibiotics ( injection ceftriaxone ) and daily plasmapheresis for hus . detailed complement regulator assay showed very high anti - factor h antibody ( 41,000 iu ) . c3 , c4 , antigenic levels of factor h , factor i , factor b , and cd46 were normal [ table 1 ] . he was given a blood transfusion and initiated on hemodialysis and daily plasma exchanges in view of oligo - anuric acute kidney injury ( aki ) . complement assay in cases * for the child 's height percentile , the bp percentiles were : 90 percentile : 113/72 mmhg and 95 percentile 117/76 mmhg ( blood pressure references used were as per the fourth report ) . for arterial hypertension [ figure 1 ] , he was started on sustained release nifedepine , clonidine , and metoprolol and subsequently prazosin with a gradual increase in dosage . however , arterial bp remained persistently high ( > 99 centile ; up to 170/120 mmhg ) , and he developed blurring of vision , with abdominal pain and vomiting on the 3 day of admission necessitating need for iv nitroglycerine ( up to 5 mcg / kg / min ) and subsequently labetolol infusion ( up to 2 mg / kg / h ) for refractory hypertension . child had persistent arterial hypertension ( > 99 centile for his age ) , despite vigorous fluid removal in hemodialysis sessions . response to antihypertensive medications in case 1 oral enalapril and minoxidil were also added and dosage of other oral antihypertensives optimized to the maximal doses [ figure 1 ] but arterial bp remained high and was difficult to control . but since within 48 h of adding oral enalapril and oral minoxidil , child went into hypertensive emergency ( bp 180/120 mmhg ) , with hallucinations and visual blurring , iv enalaprilat was added . hypertension showed a significant improvement after addition of iv enalaprilat ( 10 g / kg / dose q 8 hourly ) on the 5 day of admission . there was a consistent fall of bp within hours of giving individual iv enalaprilat boluses ( average fall in mean bp 9.5 mmhg ) . arterial bp decreased to 110/78 mmhg ; patient became asymptomatic , and nitroglycerine and labetolol infusions were tapered off successfully . he developed neutropenia a week following enalaprilat therapy ( wbc 1500 cmm , 50% neutrophils ) , which could not be attributed to any other cause ; hence , it was stopped followed by a rebound in hypertension ( arterial bp 190/136 mmhg ) . he was not dialysis dependent at this stage with a good urine output , and serum creatinine had fallen to 0.6 mg / dl , and his hemodialysis catheter was removed . aliskiren ( 2 mg / kg / dose ) was then added with good response ( arterial bp decreased to 150/110 mmhg over 24 h , and 138/110 mmhg over 48 h ) ; however , it was withdrawn after 4 days due to hyperkalemia ( serum potassium 6.5 mmol / l ) . after improvement of neutopenia , oral enalapril was reintroduced along with telmisartan . bp control improved within 48 h on this angiotensin - converting enzyme inhibitor - angiotensin receptor blockade ( acei - arb ) combination ( bp < 90 percentile for age ) along with other oral antihypertensive agents . he received seven daily plasmapheresis sessions till active hemolysis subsided followed by alternate day sessions . he received prednisolone ( 1 mg / kg / day ) and iv immunoglobulin 2 g / day ( day 10 of admission ) , iv cyclophosphamide during his hospital stay . he received total six doses of iv cyclophosphamide followed by maintenance azathioprine . at his 1 year of follow - up , he is doing well , normotensive ( bp < 90 percentile for age ) and no proteinuria ( urine protein / creatinine ratio < 0.2 ) and a normal urine examination on angiotensin - converting enzyme ( ace ) and arb combination . a 7-month - old male was admitted with a history of vomiting , fever since 5 days and anuria since 2 days . , he was hypertensive ( bp 130/60 mmhg ) , with pallor , facial puffiness , and normal systemic examination . investigations were suggestive of atypical hus - microangiopathic hemolytic anemia with aki ( hb 7 g / dl , wbc 13,280/mm , platelets 100,000/mm , peripheral smear : schistocytes , elliptocytes , reticulocyte count 5.6% , ldh 4300 u / l ) and active urine sediment ( rbc 1020/hpf , albumin 2 + ) . the child had no evidence of pneumonia or sepsis , on clinical evaluation , and all cultures were sterile . the child had advanced azotemia ( urea 216 mg / dl , creatinine 7.2 mg / dl ) with severe hyperkalemia and metabolic acidosis . for hus with evidence of ongoing hemolysis and dialysis dependence , he was started on daily plasmapheresis . a detailed complement assay ( including c3 , c4 , antigenic levels of factor h , factor i , factor b , cd46 , and autoantibodies to factor h ) were normal [ table 1 ] . bp was observed to be high since admission and increased up to 160/110 mmhg on serial monitoring , although patient remained asymptomatic . for his arterial hypertension ( > 99 centile for age ) [ figure 2 ] , he was started on amlodipine and prazosin initially ; dosage was increased to the maximal dose , and clonidine and oral enalapril were added on the 3 and 4 day of admission , respectively . despite the addition of multiple antihypertensive agents and dosage optimization and aggressive ultrafiltration in hemodialysis sessions , arterial bp showed only marginal decrease and mean arterial bp remained high ( > 99 centile for age ; 100110 mmhg ) . intravenous enalaprilat ( 10 g / kg / dose q 8 hourly ) was added on day 6 of admission . the mean arterial bp improved ( 80 mmhg ) within 12 h of addition of enalaprilat . once arterial bp was controlled with no further increase , dose of oral enalapril was maximized , while tapering off iv enalaprilat and other antihypertensive medications were continued . response to antihypertensive medications in case 2 daily hemolytic parameters and renal function were monitored . he was discharged in 2 weeks time in a stable condition , with adequate urine output and bp controlled on oral antihypertensive therapy . at a follow - up of 1 year , currently the infant is doing well , with serum creatinine 0.7 mg / dl , urine protein / creatinine ratio 1.5 , and normal bp ( bp < 90 percentile for age ) on oral enalapril 0.4 mg / kg / day . a 6-year - old male was admitted with a history of vomiting , fever since 2 weeks , hematuria and decreased urine output since 1 week . on evaluation by his local practitioner , he was found to have anemia ( hemoglobin [ hb ] 6.3 g / dl ) , thrombocytopenia ( platelet 72,000/mm ) , active urine sediment ( red blood cell [ rbc ] 4060/hpf , albumin 3 + ) , and azotemia ( blood urea 200 mg / dl , creatinine 4.2 mg / dl ) . he had an episode of seizure ( due to accelerated hypertension ) , hence was brought to our hospital for further management . on evaluation , he was hypertensive ( bp 150/100 mmhg ) with generalized edema , oliguria , and a normal systemic examination . investigations were suggestive of hus ( hb 4.8 g / dl , white blood cell [ wbc ] 11,190 cmm , platelet 1.84/mm , peripheral smear : schistocytes positive , reticulocyte count 6.8% , lactate dehydrogenase [ ldh ] 4300 u / l , direct coombs test and indirect coombs tests were negative , urea 67 mg / dl , creatinine 2.6 mg / dl ) . septic work up , dengue serology , and malarial antigen were negative , and he became afebrile on the 4 day of admission . his antinuclear antibodies ( ana ) and antineutrophil cytoplasmic antibody ( anca ) were negative . he was started on empiric antibiotics ( injection ceftriaxone ) and daily plasmapheresis for hus . detailed complement regulator assay showed very high anti - factor h antibody ( 41,000 iu ) . c3 , c4 , antigenic levels of factor h , factor i , factor b , and cd46 were normal [ table 1 ] . he was given a blood transfusion and initiated on hemodialysis and daily plasma exchanges in view of oligo - anuric acute kidney injury ( aki ) . complement assay in cases * for the child 's height percentile , the bp percentiles were : 90 percentile : 113/72 mmhg and 95 percentile 117/76 mmhg ( blood pressure references used were as per the fourth report ) . for arterial hypertension [ figure 1 ] , he was started on sustained release nifedepine , clonidine , and metoprolol and subsequently prazosin with a gradual increase in dosage . however , arterial bp remained persistently high ( > 99 centile ; up to 170/120 mmhg ) , and he developed blurring of vision , with abdominal pain and vomiting on the 3 day of admission necessitating need for iv nitroglycerine ( up to 5 mcg / kg / min ) and subsequently labetolol infusion ( up to 2 mg / kg / h ) for refractory hypertension . child had persistent arterial hypertension ( > 99 centile for his age ) , despite vigorous fluid removal in hemodialysis sessions . response to antihypertensive medications in case 1 oral enalapril and minoxidil were also added and dosage of other oral antihypertensives optimized to the maximal doses [ figure 1 ] but arterial bp remained high and was difficult to control . but since within 48 h of adding oral enalapril and oral minoxidil , child went into hypertensive emergency ( bp 180/120 mmhg ) , with hallucinations and visual blurring , iv enalaprilat was added . hypertension showed a significant improvement after addition of iv enalaprilat ( 10 g / kg / dose q 8 hourly ) on the 5 day of admission . there was a consistent fall of bp within hours of giving individual iv enalaprilat boluses ( average fall in mean bp 9.5 mmhg ) . arterial bp decreased to 110/78 mmhg ; patient became asymptomatic , and nitroglycerine and labetolol infusions were tapered off successfully . he developed neutropenia a week following enalaprilat therapy ( wbc 1500 cmm , 50% neutrophils ) , which could not be attributed to any other cause ; hence , it was stopped followed by a rebound in hypertension ( arterial bp 190/136 mmhg ) . he was not dialysis dependent at this stage with a good urine output , and serum creatinine had fallen to 0.6 mg / dl , and his hemodialysis catheter was removed . aliskiren ( 2 mg / kg / dose ) was then added with good response ( arterial bp decreased to 150/110 mmhg over 24 h , and 138/110 mmhg over 48 h ) ; however , it was withdrawn after 4 days due to hyperkalemia ( serum potassium 6.5 mmol / l ) . after improvement of neutopenia , oral enalapril was reintroduced along with telmisartan . bp control improved within 48 h on this angiotensin - converting enzyme inhibitor - angiotensin receptor blockade ( acei - arb ) combination ( bp < 90 percentile for age ) along with other oral antihypertensive agents . he received seven daily plasmapheresis sessions till active hemolysis subsided followed by alternate day sessions . he received prednisolone ( 1 mg / kg / day ) and iv immunoglobulin 2 g / day ( day 10 of admission ) , iv cyclophosphamide during his hospital stay . he received total six doses of iv cyclophosphamide followed by maintenance azathioprine . at his 1 year of follow - up , he is doing well , normotensive ( bp < 90 percentile for age ) and no proteinuria ( urine protein / creatinine ratio < 0.2 ) and a normal urine examination on angiotensin - converting enzyme ( ace ) and arb combination . a 7-month - old male was admitted with a history of vomiting , fever since 5 days and anuria since 2 days . there was no history of diarrhea or dysentery in the past . on admission , he was hypertensive ( bp 130/60 mmhg ) , with pallor , facial puffiness , and normal systemic examination . investigations were suggestive of atypical hus - microangiopathic hemolytic anemia with aki ( hb 7 g / dl , wbc 13,280/mm , platelets 100,000/mm , peripheral smear : schistocytes , elliptocytes , reticulocyte count 5.6% , ldh 4300 u / l ) and active urine sediment ( rbc 1020/hpf , albumin 2 + ) . the child had no evidence of pneumonia or sepsis , on clinical evaluation , and all cultures were sterile . the child had advanced azotemia ( urea 216 mg / dl , creatinine 7.2 mg / dl ) with severe hyperkalemia and metabolic acidosis . for hus with evidence of ongoing hemolysis and dialysis dependence , he was started on daily plasmapheresis . a detailed complement assay ( including c3 , c4 , antigenic levels of factor h , factor i , factor b , cd46 , and autoantibodies to factor h ) were normal [ table 1 ] . bp was observed to be high since admission and increased up to 160/110 mmhg on serial monitoring , although patient remained asymptomatic . echocardiography and fundus were normal . for his arterial hypertension ( > 99 centile for age ) [ figure 2 ] , he was started on amlodipine and prazosin initially ; dosage was increased to the maximal dose , and clonidine and oral enalapril were added on the 3 and 4 day of admission , respectively . despite the addition of multiple antihypertensive agents and dosage optimization and aggressive ultrafiltration in hemodialysis sessions , arterial bp showed only marginal decrease and mean arterial bp remained high ( > 99 centile for age ; 100110 mmhg ) . intravenous enalaprilat ( 10 g / kg / dose q 8 hourly ) was added on day 6 of admission . the mean arterial bp improved ( 80 mmhg ) within 12 h of addition of enalaprilat . once arterial bp was controlled with no further increase , dose of oral enalapril was maximized , while tapering off iv enalaprilat and other antihypertensive medications were continued . response to antihypertensive medications in case 2 daily hemolytic parameters and renal function were monitored . he was discharged in 2 weeks time in a stable condition , with adequate urine output and bp controlled on oral antihypertensive therapy . at a follow - up of 1 year , currently the infant is doing well , with serum creatinine 0.7 mg / dl , urine protein / creatinine ratio 1.5 , and normal bp ( bp < 90 percentile for age ) on oral enalapril 0.4 mg / kg / day . the extent of renal microangiopathic involvement appears to be responsible for the development of hypertension and renal failure in atypical hus . renal ischemia is triggered which leads to maximal activation of ras resulting in accelerated hypertension which is often very severe and resistant to antihypertensive therapy . local ras activation is believed to be an important key factor in the thrombotic microangiopathy in hus leading to intractable hypertension . this has been demonstrated to occur via enhanced tissue factor expression on glomerular endothelial cells which is enhanced by angiotension ii . on the other hand two studies have shown elevated plasma renin levels in children with hus , irrespective of systemic hypertension . severe hypertension that ensues is a clinician 's nightmare as it is hard to control despite use of multiple drug combinations and careful drug titration . in extreme cases , bilateral nephrectomy is ultimately required as a life - saving measure for achieving control of bp , thus again underlining the pivotal role of hyperreninemia in the development of hypertension in hus . although there is plenty of evidence in favor of renin - mediated mechanism in the pathogenesis of hypertension in hus , in practice there is hesitation to use ras inhibitors or their combinations in the acute stage for hypertension . this is due to fear of worsening of renal failure and lack of pediatric experience with newer ras inhibitors . oral aceis ( enalapril , captopril ) have been shown to be renoprotective and of benefit for long - term bp control and reduction in proteinuria in patients with persistent disease . however , they are not preferred in the acute stage of disease , used with great caution and generally initiated after improvement of renal function . moreover , aceis including enalaprilat are seldom used in hypertensive emergencies due to concerns regarding slow onset of action and variable effectiveness , especially in children . we used iv enalaprilat in both patients for acute hypertension who showed significant and consistent decline in bp . it helped in successful reversal of hypertensive urgency in the older child whose bp remained high and was extremely difficult to control despite multiple drugs including iv nitroglycerine and labetolol . the onset of action begins in 15 min , but the peak effect may take 14 h. the duration of action is usually 46 h. the half life of the drug is usually 11.1 h in infants and children . there is one case series reporting the neonatal use of enalaprilat that reported that doses even at the lower end of what was used in this cohort may lead to significant , prolonged hypotension and oliguric acute renal failure . if it is used in the newborn or children , it should be used with caution with ongoing monitoring of bp , serum potassium , and renal function . the adverse effects of enalaprilat are hyperkalemia , hypotension , cough , diarrhea , angioedema , and leucopenia ( agranulocytosis ) . we encountered neutropenia in one of our patients a week after initiation of iv enalaprilat which reversed quickly on drug withdrawal . however , severe rebound hypertension also occurred on stopping enalaprilat , while other drugs were continued , which reiterates its effectiveness in the management of the hypertensive urgency . studied the bp response to iv enalaprilat in 35 patients with hypertensive crisis , and found that the extent of systolic and diastolic bp reduction correlated well with the pretreatment plasma renin and angiotensin ii levels . thus , it appears that the status of ras determines the efficacy of iv enalaprilat , and hence it was successful in both our patients with hus - induced hypertension . therapeutic enalaprilat levels can probably be achieved with 1/4 total cumulative dose of enalapril , administered as 6 hourly enalaprilat : recommended pediatric dosing is 510 mcg / kg / dose . we used a dose of 10 mcg / kg / dose q 8 hourly in both of our cases based on the previous report . we used aliskiren in the older child which too was very effective in lowering bp . aliskiren is the first direct renin inhibitor available in an oral form approved for adults by the us food and drug administration in 2007 . as renin is the first and rate - limiting step in angiotensin ii synthesis in ras , its direct inhibition is theoretically more advantageous as compared to ace inhibition / arb . as it is a nonpeptide molecule , it has better bioavailability and a long half - life and can , therefore , lower bp effectively . once administered orally , the effect of the drug peaks in 13 h , achieves its steady state in 57 days and has a half - life of 40 h. aliskiren produces dose - dependent bp reduction and its potency has been shown to be equivalent or better than aceis , arbs in various trials . moreover , when administered as a combination with acei , it helps to block an increase in plasma renin activity induced by acei monotherapy . combination therapy ( aliskiren + acei ) has been demonstrated to have greater bp - lowering potential as compared to either alone . dual ras blockade however must be cautiously monitored as there is a higher chance of adverse effects . a case series of children with chronic kidney disease receiving combination aliskiren / acei showed > 45% proteinuria reduction , however side effects in the form of hyperkalemia , worsening of renal function , and hypotension were seen . mild hyperkalemia was seen in our patient but was asymptomatic , and potassium normalized quickly on stopping the drug . other adverse effects including nausea , angioedema , diarrhea , abdominal pain , and headache may be seen with aliskiren but are usually mild and not dose related . adult studies have shown aliskiren to be a safe and effective antihypertensive drug ; however , its use in children has been restricted so far due to the paucity of data . a recent prospective , randomized controlled in children between ages 6 and 17 years concluded that aliskiren in once daily doses of 2 mg / kg or 6 mg / kg was well tolerated and safe . we used a dose of 2 mg / kg in case 1 . in the above two patients , we used two unconventional drugs : aliskiren and iv enalaprilat , both of which were very quick and effective in controlling high bp refractory to multiple antihypertensive medications and aggressive ultrafiltration during dialysis sessions . the limitation of the report is small number , multiple antihypertensives in these patients , and simultaneous use of plasma exchanges and immunosuppression in anti - factor h antibody positive case 1 to help resolution of illness , which might make interpretation difficult . these appear to be promising alternatives in the treatment of severe atypical hus - induced hypertension and hypertensive emergency , and there is a need to have more trials targeting renin in these cases .

hypertension is common in hemolytic uremic syndrome ( hus ) and often difficult to control . local renin - angiotensin activation is believed to be an important part of thrombotic microangiopathy , leading to a vicious cycle of progressive renal injury and intractable hypertension . this has been demonstrated in vitro via enhanced tissue factor expression on glomerular endothelial cells which is enhanced by angiotensin ii . we report two pediatric cases of atypical hus with severe refractory malignant hypertension , in which we targeted the renin - angiotensin system by using intravenous ( iv ) enalaprilat , oral aliskiren , and oral enalapril with quick and dramatic response of blood pressure . both drugs , aliskiren and iv enalaprilat , were effective in controlling hypertension refractory to multiple antihypertensive medications . these appear to be promising alternatives in the treatment of severe atypical hus - induced hypertension and hypertensive emergency .

Introduction Case Reports Case 1 Case 2 Discussion Financial support and sponsorship Conflicts of interest

in 2009 , a 52-year - old woman presented with a single lesion on her nose , which started as a papule , referred to sedighe tahereh clinic , isfahan , iran . the lesion had existed for a period of 14 months and was slowly increasing in size , enlarging to a plaque . the diagnosis of leishmaniasis was confirmed with a positive smear of the lesion showing leishmania bodies about 1 year before . all five members of her family had had a history of proven leishmaniasis . in the past medical history , the patient was a renal failure case since 11 years before and received a renal transplant 4 years after the diagnosis of renal failure . she was receiving oral mycophenolate mofetil ( 2 g daily ) and cyclosporine ( 100 mg daily ) . a 33 cm indurated ulcer with elevated borders was present on the tip of her nose ( figure 1 ) . her therapeutic plan was intralesional glucantime injection ( approximately 1 ml of 1.5 g vial per week , intralesional injection ) . after completing a therapeutic course of 20 sessions receiving intralesional glucantime injections , she was considered as glucantime therapy resistant . the occurrence of malignant neoplasms in sites of scars is an infrequent but well - known phenomenon.5 although the coexistence of cutaneous leishmaniasis and bcc may have been coincidental , some studies suggest that an association between these two entities does exist.6 leishmaniasis can directly or indirectly alter the diagnosis and course of different malignancies.7 there are reports of bcc in chronic leg ulcers.8 cases of bcc developing in a leishmania scar have also been documented,9 but to our knowledge , cases of both leishmaniasis and bcc in the same site and the same lesion are rare.10 however , in this case , solid organ transplantation and long term immuno suppressive therapy should be considered as risk factors for malignancy . advances in effective immuno suppression after organ transplantation have led to increased risk of malignancies , particularly skin cancers11 including squamous cell carcinoma , basal bcc and malignant melanoma.12 thus , malignancies should be considered in the differential diagnosis of leishmaniasis lesions difficult to treat . the possible role of cutaneous leishmaniasis , as a predisposing factor for skin cancer , should also be kept in mind . aa was the main therapeutic physician and helped write the manuscript . i m and pk contributed in writing the manuscript .

leishmaniasis is a protozoan infection due to organisms of the genus leishmania . the differential diagnosis of cutaneous leishmaniasis includes arthropod bites , basal cell carcinoma ( bcc ) and other malignancies . bcc is the most common form of skin cancer . we present a case of cutaneous leishmaniasis resistant to standard intralesional glucantime injection in an immunocompromised patient , which was proved to be bcc after surgical excision .

Case Report Discussion Authors Contributions

a 60-year - old man was admitted because of visual disturbances and ocular pain following penetrating keratoplasty in the right eye for a corneal chemical burn . on slit lamp examination , we observed a feather - like corneal opacity , corneal infiltrations , and epithelial defects . subconjuctival amphotericin b ( 1 mg ) , topical amphotericin b 0.125% , and levofloxaxin 0.5% were subsequently administered every hour for 16 weeks . the patient was restarted on and maintained on a topical amphotericin b treatment ( 0.125% twice daily ) for 1 year . in addition , levofloxaxin 0.5% ( three times daily ) and fluorometholone 0.1% ( twice daily ) were administered for 1 year . the lesion did not progress , but there was no change in corneal infiltration . on examination one year later , an elevated corneal lesion and increased corneal infiltration with a yellow color were observed despite the topical amphotericin b treatment . seven days later anterior chamber hypopyon and vitreous opacity on a b - scan were observed ( fig . we immediately performed an intravitreal injection of amphotericin b 5 g/0.1ml and vancomycin 1 mg/0.1ml . intravitreal voriconazole 80 g/0.1ml and ceftazidime 2 mg/0.1ml were injected and topical voriconazole 1% was administered every 2 hours . the patient discontinued the oral voriconazole because of abdominal pain , dry mouth , and scaling of the oral mucosa . the patient was treated topically for 6 weeks , after which the corneal lesion and endophthalmitis had improved . the patient subsequently underwent another penetrating keratoplasty and demonstrated a visual acuity of 0.1 at 10 months follow - up with no signs of recurrence . a 60-year - old man was referred to us because of a lack of improvement after a month of empiric treatment with topical amphotericin b for fungal keratitis . on our initial examination after 10 days the culture revealed no growth and the corneal epithelium appeared healed ; however , there was no change in corneal infiltration ( fig . because of a lack of improvement in the corneal lesion a month later , a new treatment regimen was initiated with topical voriconazole 1% administered every hour . six weeks later we observed a decreasing density of the infiltrate and healing of the corneal epithelium ; the topical voriconazole was decreased to twice daily . following completion of the therapy , complete healing of the corneal epithelium and resolution of the corneal infiltrate were observed ; however , the corneal opacity persisted ( fig . 2c ) voriconazole , a derivative of fluconazole , is a new triazole antifungal agent.4 like other triazoles , this voriconazole inhibits cytochrome p450 demethylase , which is essential for the synthesis of ergosterol . it is hypothesized that this adversely affects the permeability of the fungal cell membrane.7 voriconazole has excellent oral bioavailability and a broad spectrum of activity . therapeutic aqueous and vitreous levels are achieved after oral administration of voriconazole.8 voriconazole showed lower mics compared to other antifungal agents when tested against five corneal isolates of scedosporium apiospermum,2 and it had the best in vitro susceptibility profile for 34 common fungal pathogens compared to other antifungal agents.9 gao et al.10 reported that direct intravitreal voriconazole injections of 25 g / ml ( equivalent to 100 mg per injection in a human eye ) caused no electroretinographic or histopathologic abnormalities in rodent retinas . kramer et al.11 reported that intravitreal injection of voriconazole ( 100 g/0.1ml ) with pars plana vitrectomy was an effective therapy for aspergillus endophthalmitis . lee et al.7 reported a case of drug - resistant penicillium endophthalmitis that was treated with intravitreal voriconazole injection ( 50 g/ 0.1ml ) . in these cases , voriconazole has been shown to be highly effective against filamentous organisms and is more potent in invasive aspergillosis than amphotericin b.12,13 according to a report by jang et al.,5 voriconazole is also effective against candida chorioretinitis . ozbek et al.14 reported one case of alternaria keratitis that showed improvement with 1% topical voriconazole . in contrast , giaconi et al.15 reported two cases of fungal keratitis caused by fusarium oxysporum and colletotrichum dematium that did not respond to treatment with 1% topical voriconazole . our report indicates that 1% voriconazole is an effective treatment for candida spp . and unknown fungal keratitis . in conclusion , voriconazole is a new , promising therapy for fungal keratitis refractory to standard antifungal agents . nevertheless , more clinical trials will be necessary to investigate the effectiveness of systemic voriconazole and corneal transplantation .

we describe two patients with fungal keratitis refractory to standard antifungal therapy whose conditions were managed with voriconazole.the first case is a patient with endophthalmitis and corneal ulcer due to candida parapsilosis after receiving a corneal transplant . the patient was treated with amphotericin but showed no signs of improvement . topical voriconazole , oral voriconazole , and intravitreal voriconazole yielded signs of improvement . the second case is a 63-year - old male who underwent a month of empiric treatment with 0.2% topical amphotericin for fungal keratitis but showed no signs of improvement . treatment was then provided with 1% voriconazole . both cases showed effective treatment with voriconazole.voriconazole may be considered as a new method to treat fungal keratitis refractory to standard antifungal therapy .

Case 1 Case 2 Discussion

liver plays a pivotal role in detoxification of xenobiotics , environmental pollutants , and chemotherapeutic agents . for this reason , this organ acetaminophen ( apap ) is a widely used antipyretic and analgesic which is currently the most frequent cause of drug - induced hepatic failure in the united states with intentional and unintended overdose . cytochrome p450 enzymes convert a relatively minor portion of apap to the highly reactive intermediate metabolite n - acetyl - p - benzoquinone - imine ( napqi ) . under normal physiological conditions , apap overdose increased napqi formation which exceeds the rate of detoxification by gsh . in recent times , much attention was attracted to herbal medicines as alternative medicines . quite a few plant remedies have been examined for the treatment of liver disorders for centuries prosopis farcta ( pf ) [ figure 1 ] from leguminosae and sub - family mimosoideae has some medicinal properties such as antiinflammatory effects , treating gastric ulcers , fetus abortion , dysentery , arthritis , larynx inflammation , heart pains , and asthma . this plant is the indigenous to dry and semi - dry areas of america , asia , and africa some of the compounds existing in prosopis plant are : quercetin ( flavonoids ) , tryptamine , apigenin 5-hydroxytryptamine ( alkaloids ) , l - arabinose , and lectin . moreover , a number of phenolic compounds with strong antioxidant activity has been identified in extracts of this plant such as vicenin-2 , apigenin c - glycoside , iso - orientin , vitexin , luteolin 7-o - glucoside , isovitexin , quercetin 3-o - glucoside , rutin , kaempferol 3-o - rutonoside , caffeic acid derivative , and luteolin . antioxidants play an important role in inhibiting and scavenging free radicals and thus , providing protection against infections and degenerative diseases . there is no in vitro or in vivo report to show an academic research to evaluate the protective effect of this plant so far . since pfe has some antioxidant property and may alleviate hepatotoxicity , this study evaluated the possible preventive hepatoprotective effect of pfe on liver injury induced by apap in rat . thirty - six male rats of wister strain , weighing 220 30 g were randomly assigned to six groups of six . all animals were housed individually with a 12 h alternating light - dark cycle and had free access to food and water . ambient temperature in the animal facility was kept at 22c 2c with humidity between 30% and 50% . pf beans were collected from outskirts of qom in iran and were coded with the university of birjand , iran herbarium ( herbarium code 1952 ) . the fruit of pf was crushed to moderately coarse powder and for the extraction , 50 g of powder dissolved in 1000 ml ethanol 80% and placed on a shaker for 24 h. after 24 h , the solution is passed through a filter paper . to remove the solvent , the solution was filtered and placed in the oven for 1 - 2 days at 40c . after evaporation of the solvent , the samples maintained at 20c . a stock solution of 1 g / ml in 0.9% ( w / v ) normal saline after 2 weeks of adaptation , animals were orally administered pfe ( 50 or 75 mg / kg ) or saline once daily for 7 consecutive days . to induce hepatotoxicity , 1-h after the last pretreatment by pfe 5 - 6 ml of blood was obtained by cardiac puncture 24 h after induction of hepatotoxicity and plasma samples were separated by the heparinized syringe and analyzed for various biochemical parameters . total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , total protein , albumin , alanine aminotransferase ( alt ) , and aspartate aminotransferase ( ast ) concentrations were measured using standard techniques and commercial kits ( pars azmoon co. , iran ) with an auto analyzer ( gcsan chem 2000 , spain ) . very low density lipoprotein ( vldl ) was calculated by deduction of the sum of the cholesterol fractions from the tc plasma concentration as described by salau et al . statistical significance between two groups of parametric data was analyzed using one - way analysis of variance followed by tukey 's multiple comparisons test . the experiment was approved by the animal welfare committee of the agriculture faculty of birjand university ( research project number : 2131336 ) . tables 1 and 2 reveal that plasma ast and alt levels were increased in rats administered with apap when compared with controls indicating reliable induction of hepatotoxicity by apap ( p < 0.01 ) . while the groups received pfe at each dose alone showed no significant changes in any of the biochemical parameters . treatment of rats with pfe before apap administration , dose - dependently prevented the increase in ast and alt . furthermore , administration of apap significantly decreased plasma total protein and albumin levels ( p < 0.05 ) as well . as shown , pretreatments with different doses of the extract attenuated the reduction in plasma levels of total protein and albumin to some extent . moreover , plasma cholesterol , tg , ldl , and vldl levels markedly increased in rats administered with apap when compared with controls ( p < 0.05 ) . pretreatment with pfe , especially at 75 mg / kg , provided marked protective effect and comparable improvement on these biochemical indices . effect of prosopis farcta on liver enzymes in rats effect of prosopis farcta on liver enzymes and total protein , albumin and lipid profile in rats the present study for the first time brings about the potential hepatoprotective activity of pfe against apap - induced hepatotoxcity . the results of the study indicated that the pfe significantly reversed the enhancement of ast and alt levels . necrosis or membrane damage releases the intracellular enzymes into circulation and hence , it can be measured in the plasma . elevated levels of plasma enzymes are indicative of cellular leakage and loss of functional integrity of the cell membrane in liver prior to other indicators of liver function tests . the most important enzymes are alt and ast that are present in the liver parenchyma . alt predominantly found in liver unlike ast which is also abundantly present in other organs namely , cardiac muscle and kidneys . for this reason , alt is more specific indicator of liver inflammation than ast , though the parallel increases in alt and ast often observe . in the present study , this enhancement may be due to disruption of hepatic cell as a result of necrosis or a consequence of altered membrane permeability . pretreatment with oral doses of p. farca extract attenuated the enzyme activities which are indications of the protective activities of the pfe against apap hepatotoxicities . the most critical assessment if an intervention is used as a pretreatment is the evaluation of metabolic activation ( gsh depletion and protein adduct formation ) which has not been assessed in this study . metabolism of paracetamol produces a highly toxic metabolite , napqi via the cytochrome p450 pathway which induces the process of lipid peroxidation . although , accumulation of napqi occurs when the rate of formation exceeds the rate of detoxification by gsh or the depletion of gsh stores . in addition , covalent bindings of napqi to hepatocyte macromolecules cause necrosis of liver cell and consequent release of the cytosolic liver enzymes particularly the aminotransferases that are regarded as signs of hepatocellular injury . given the large number of compounds present in the extract , it is highly likely that several p450 inhibitors are present . quercetin , a flavonoid antioxidant , is one of the compounds found in prosopis with an excellent radical scavenger activity which attenuated oxidative stress induces by apap , as well as other chemical . it is possible that presence of polyphenols and flavonoids in pf might be responsible for its protective effect on apap - induced liver damage in rats . concerning plasma lipid profile , oral administration of apap increased plasma cholesterol , tg and ldl with no significant alteration in plasma hdl level . lipid deposition in the liver may be as a result of excessive supply of lipids to the liver or interference with lipid deposition . formation of phenoxyl radicals in the presence of peroxidases act as an ldl prooxidant is responsible of ldl elevation in apap animals . it is documented that lipid - lowering drugs have antioxidant properties which prevent ldl peroxidation . pf with antioxidant property is capable of preventing ldl peroxidation and inhibiting elevation of ldl by apap . three molecules of acetyl coa combined and produce 3-hydroxy-3-methyl - glutaryl - coa , which it affected by different enzymes converted to malonic acid , and during different reactions , convoluted and converted to cholesterol . hdl - c is responsible for reverse transport of cholesterol from peripheral cells to the liver cells . cholesterol is transformed to bile acids , which are excreted into the intestine via the biliary tract . the findings of omidi et al . , while preliminary , suggests that the efficacy of pf beans powder as a beneficial agent to decrease ldl - c and increase hdl - c concentration . it produces proteins for its own cellular needs , as well as secretory proteins that are released into the circulation . one of the most important of these secretory proteins is albumin . in this study , the decreased total protein and albumin levels by apap were significantly improved in pfe - treated groups indicating of its hepatoprotective effect . based on the results of the present study , it can be concluded that the aqueous extracts of pf beans exhibit hepatoprotective activity against apap . however , the mechanism by which pfe exhibited protection is not clear by the present study . further investigations on cellular and molecular mechanism of the plant may throw more light on the use of pf for hepatoprotective activity .

background : hepatotoxicity by acetaminophen is the most frequent cause of acute liver failure in many countries . prosopis farcta beans extract ( pfe ) has some antioxidant property and may alleviate hepatotoxicity . therefore , the aim of this study was to evaluate effects of pfe against acetaminophen - induced hepatotoxicity.methods:thirty-six male wistar albino rats weighing 220 30 g were distributed into six groups . two groups were pretreated with pfe ( 50 and 75 mg / kg ) for 7 days before administration of acetaminophen ( 600 mg / kg ) . two were given acetaminophen or pfe ( 50 and 75 mg / kg ) alone , and the control received normal saline . one day after acetaminophen , administration blood samples were collected by cardiac puncture to determine liver function enzymes markers ; aspartate aminotransferase and alanine aminotransferase ( ast and alt ) , cholesterol , triglyceride ( tg ) , high , low , and very low density lipoproteins ( ldl and vldl).results : in acetaminophen - treated rat plasma ast ( 314 18.54 vs. 126.37 4.13 ) , alt ( 304 49.24 vs. 187.33 3.71 ) , cholesterol , tg , ldl , and vldl were increased by 149 , 160 , 37 , 92 , 60 , and 94% , respectively . pfe at both doses significantly ( p < 0.05 ) attenuated the above biochemical indices to near normal.conclusions:prosopis farcta beans extract ( 50 and 75 mg / kg ) exhibited hepatoprotective activity against apap .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSIONS

melamine ( 2,4,6-triamino-1,3,5-triazine , cas # 108 - 78 - 1 ) was first prepared and described by liebig in 1834 ( 1 ) and has since become an increasingly important chemical commodity . most of the melamine production is used in the fabrication of melamineformaldehyde resins ( 2 ) . the first analytical methods related to food were therefore developed to detect melamine migration from resins used in food contact materials ( 3 ) . recently , melamine has become infamous as being one of the most effective adulterants used to increase the nitrogen content in foods and feeds . melamine contains about 66.6% nitrogen , and the addition of 1% melamine to protein leads to a false increase in the kjeldahl protein content by 4.16% ( 6 ) . the first cases of melamine adulteration were detected in fish meals from italy in the late 1970s ( 7,8 ) , and methods to detect melamine in potato proteins were developed in switzerland in the 1980s ( 6 ) . since then , melamine adulteration cases have not been reported in the literature until 2004 and 2007 , when melamine was found in pet foods , causing renal failure in dogs and cats ( 9,10 ) . nephrotoxicity also appears to be the major toxic effect in humans ( 11,12 ) . with the intentional adulteration of human foods , including baby foods , with melamine , the economically motivated fraud in the food chain has reached a new dimension , for which food control systems were unprepared . the problem first became evident in china as an increase in urinary tract stone formation in infants beginning in the spring of 2008 ( 13 ) . more than 294,000 children have reportedly been affected by adulterated formula , with over 50,000 hospitalized , and at least 6 deaths ( 14 ) . twenty - two dairy companies were implicated in the melamine fraud , with the sanlu company being identified as the one that had most seriously violated the law ( 13 ) . apparently , the adulterations occurred at the raw milk collection stations , for which no systematic state surveillance had been implemented ( 13 ) . the adulterators ostensibly used relatively sophisticated techniques such as special protocols for premixes and were said to have provided training regarding the use of those premixes after diluting the milk with water ( 15 ) . because of globalization and the worldwide food trade , the melamine contaminated foods were detected in a large number of countries , including the united states ( 14 ) and the european union ( 16 ) . some commentators expect similar cases in the future with even improved techniques ( 15 ) . this crisis has highlighted not only the hazards of an increasingly globalized food chain but also the weaknesses in the control systems ( 17 ) . we think that in order to improve consumer protection in the future , completely new strategies for food surveillance must be developed . the current food control system is generally target - oriented , meaning that quantitative analyses of certain health - relevant compounds such as heavy metals , pesticides , or carcinogenic contaminants are conducted ( 18 ) . while the current system has not paid great attention to intentional adulterations ( 17 ) , testing laboratories are also faced with the problem of choosing parameters for spot checks , as systematic full analyses are not possible for economical reasons ( 19 ) . therefore , analysis parameters are usually chosen by purely arbitrary choices ( 19 ) or by a risk - oriented approach ( 20 ) , both of which most certainly would not include novel high - tech adulterants ( 15 ) chosen specifically with the intent of evading the food control system . for all of these reasons , we propose that future food control must implement what we call a nontargeted approach . this is an approach that must be able to detect deviations in food composition even by compounds that are not specifically being searched out or ones that are unknown to the analyst . this would enable food control to work proactively , rather than being one step behind the adulterators , as is currently the case . the most preferred form of nontargeted analysis would be a screening technique that would allow measurement of a large number of samples in a short time frame . nuclear magnetic resonance ( nmr ) spectroscopy has been proposed as probably the best nontargeted technique for use in screening food extracts ( 21 ) . in a comparison of spectroscopic screening techniques , much richer information was provided using nmr in comparison to near - infrared or fourier transform infrared spectroscopy , and selective and sensitive qualitative and quantitative information could be gathered from the spectra ( 22 ) . even though 400 mhz nmr machines are still expensive , compared with the cost of other analytical systems , the cost per sample for nmr can be very low , depending on the turnover to be achieved . the successful application of nontargeted screening approaches for food analysis has been previously demonstrated for a number of food matrices ( 21 ) . however , the focus was always placed on authenticity control and not on the detection of health - relevant compounds . in this study , on the basis of our experience with beer ( 23 ) and fruit juice screening ( 19,24 ) , we demonstrate for the first time that nmr is useful for nontargeted screening of baby food formula for health - relevant compounds such as melamine and also for labeling controls ( e.g. , lactose - free products ) . additionally , we provide information that the 400 mhz nmr spectra of liquid sample extracts can also be used for quantification of melamine . using 700 mhz high - resolution magic angle spinning ( hrmas ) nmr , we demonstrate that melamine can be quantitatively detected in complicated food matrices without any sample preparation . authentic chinese infant formulas ( n = 9 ) , including one contaminated sanlu infant formula , were purchased in beijing ( china ) in september 2008 during the melamine crisis . german infant formulas ( n = 13 ) were sampled in the context of official food control by governmental food inspectors in the german federal state baden - wrttemberg in december , 2008 . melamine - positive chinese candy from the german market was also included in the study . all nmr measurements were performed on a bruker avance 400 spectrometer ( bruker biospin , rheinstetten , germany ) equipped with a 5-mm bbi inverse probe with z - gradient coils , using bruker automatic sample changer ( b - acs 60 ) . all spectra were acquired at 300.0 k. h spectra were acquired using one - dimensional ( 1d ) nuclear overhauser enhancement spectroscopy ( noesy ) pulse sequence implemented with a low - power continuous wave presaturation during the relaxation delay and during the mixing time . the relaxation delay and mixing time the data were acquired automatically under the control of icon - nmr ( bruker biospin , rheinstetten , germany ) , requiring about 12 min per sample . the proton hrmas experiments were carried out using a 700 mhz av iii nmr spectrometer ( bruker biospin , rheinstetten , germany ) with a 4 mm hrmas standard bore probe ( h , c , p , and h - lock ) and gradient . the temperature was controlled at 300.0 k using a bcu05 precooling unit for the n2 bearing gas flow and a temperature control unit ( bvt300 mas ) . the rotor spin rate was 7000 hz ; therefore , the spectral window was free from spinning side bands . a single pulse proton experiment with a 30 flip angle was executed with 4 s acquisition time , a relaxation delay of 4 s , and a sweep width of 20 ppm . the number of scans for the standard experiments was ns = 64 ( experimental time 9:15 min ) . the processing was done with zero - filling to 128k data points and an exponential multiplication with lb = 1 . the chemometrical analyses of the spectra were performed using matlab ( the mathworks , version r2008a ) routines developed in - house . for the quantitation of the melamine concentrations , the signal of the nh2-groups at about 5.93 ppm ( figure 1 ) was investigated . its intensity was derived by fitting a theoretical signal on the spectrum , which minimized the sum of squares of the residues and therefore optimally explained the melamine influence on the nmr spectrum . the integral value of this fitted signal was linearly dependent on the melamine concentration and did not require a calibration for each sample when fixed acquisition and preparation parameters were used . the fitting was done in a fully automated mode using a nonlinear bound optimization algorithm , which optimized parameters such as line shape , position , height , and signal - underground ( baseline ) . comparison of melamine - contaminated sanlu sample with the reference distribution of a collection of infant formulas ( measurement in dmso - d6 at 400 mhz ) . the nontargeted analysis of the data was performed using simple robust techniques to describe the univariate distributions of the nmr spectra at each ppm value . figure 1 illustrates this method which uses the quantiles of the distributions of the reference samples as an estimate of the medians and the corresponding variances at each chemical shift . since this procedure yields a robust description , a few atypical samples in the reference group the melamine signal of the sanlu infant formula was easily detected ( manually and in automation ) since its intensity translates into a z - score larger than 56 ( medianreference + z stdreference ; a z - score of about 3 would cover 99% of all samples under normal distribution assumption ) . this approach emphasizes deviating spectral regions and signals , which can then be investigated with sophisticated nmr experiments or other conventional analyses . nondeviating samples will pass this screening , and adulterations up to certain limit can be ruled out . in the first stage , for nontargeted analysis , 20 0.4 mg of infant powder was weighed and dissolved in 2 ml of waterbuffer solvent ( 90% distilled water and 10% buffer ; 1.5 m kh2po4/d2o and 0.1% tsp at ph 7.0 ) to obtain a final concentration of 10 0.2 this solution was shaken for 1 min on a vortex mixer and 3 min in an ultrasonic homogenizer . then 600 l of homogenized solution was transferred to a standard 5 mm nmr tube for analysis . when it became evident that the melamine peak itself could not be measured in water , the sample preparation was repeated in 99.8% dmso - d6 ( deutero gmbh ) , while 20 0.4 mg of infant powder was dissolved in 2 ml of dmso - d6 . for calibration , melamine standards in dmso - d6 at a concentration range between 2 and 200 mg / l were measured . to determine the limit of detection , a separate calibration curve in the range between 0.2 and 2 was filled into the 50 l hr - mas rotor , and 45 l of dmso - d6 was added and stirred . the rotor was prepared with a 50 l upper spacer placed at a 3.0 mm depth and sealed with a teflon screw . the material from the white rabbit candy was scraped off and powdered in a mortar before analysis . for hrmas calibration , a blank matrix ( nestl student sweetened milk powder , nestl china , absence of melamine was confirmed by reference analysis ) was filled in the rotor , and 45 l of melamine standard solution in dmso - d6 was added . the calibration range was 9 to 900 mg / kg , and the second curve for the determination of limit of detection was in the range between 0.9 and 9 mg / kg . for infant formula , 1 g of sample was added to a centrifuge tube , and 10 ml of acetonitrile was added . ten milliliters of deionized water , a small amount ( covering the tip of a spatula ) of ammonium acetate , and 0.2 ml of glacial acetic acid were then added to the tube . the solution was again vortexed for 1 min and centrifuged for 10 min at 4000 g . a 2.5 ml aliquot of the supernatant was placed into a conditioned solid - phase extraction ( spe ) tube ( strata - x - c 200 mg/6 ml tubes , phenomenex , aschaffenburg , germany ) . for candy , a different sample preparation had to be conducted because they were not soluble in cold solvents . therefore , 1 g of candy sample was dissolved in 50 ml of hot water after the addition of 1 ml of acetic acid . after cooling , 30 ml of acetonitrile was added , and the solution was ultrasonicated for 15 min . after adjustment with acetonitrile to 100 ml , an aliquot of the extract was centrifuged and transferred to an spe tube as described above . the spe tubes were conditioned using 15 ml of methanol followed by 15 ml of water . cleanup was conducted using 6 ml of hcl ( 0.1 m ) and 3 ml of methanol . after each elution step , the extract was dried at 5060 c using a nitrogen stream . then , 500 l of acetonitrile / water ( 1:1 ) was added , and the solution was membrane filtered ( 0.2 m ) and used for chromatography ( injection volume 20 l ) . the lc / ms / ms system consisted of an agilent ( waldbronn , germany ) 1100 hplc system ( binary pump , degasser , and autosampler ) coupled with a thermo fisher scientific ( formerly thermo finnigan ) ( dreieich , germany ) tsq 7000 mass spectrometer . , 3 m , 125 , c18 column ( phenomenex , aschaffenburg , germany ) at 30 c , using mobile phase a ( acetic acid , 0.1% ) and mobile phase b ( acetic acid in methanol , 0.1% ) in the following gradient program . flow rate : 0.2 ml / min , 05 min , 90% a ; 55.5 min , 90% a to 20% a ; 5.57.5 min , 20% a ; 7.58 min , 20% a to 90% ; 813 min , 90% a. atmospheric pressure chemical ionization ( apci ) used a capillary temperature of 270 c , a vaporizer temperature of 400 c , and a corona discharge current of 2.0 a . the sheath gas was nitrogen at 50 psi , and argon was used as the collision gas . the collision cell of the triple quadrupole was operated with a collision energy of 35 ev for all transitions . for quantitative analysis , the following fragmentations were monitored in the selected reaction monitoring ( srm ) mode : m / z 127 85 , m / z 127 68 , and m / z 127 43 for melamine . in the calibration range between 5 to 1000 ng / ml , melamine exhibited good linearity , with regression coefficients greater than 0.99 . the limit of detection was 10 ng / ml and the limit of quantitation was 30 ng / ml . the recovery was 105 5% in spiked baby formula ( n = 8) , 81 13% in spiked ready - to - eat baby purees ( n = 6 ) , 98 3% in different spiked egg powders ( n = 4 ) , or 92 6% in candy ( n = 11 ) . the precision ( expressed as coefficient of variation ) was 7.5% ( n = 6 , bakery products ) , 5.1% ( n = 8 , baby formula ) , or 6.4% ( n = 11 , candy ) . at this point in time , only one study in the literature has presented h nmr data on melamine in the context of a kinetic study ( 25 ) . the signal assignment ( dmso - d6 , 6.02 , s , nh2 ) reported previously corresponds excellently with the findings in our study . the identification of the singlet peak of nh2 at 5.93 ppm in dmso - d6 for melamine was confirmed using measurements of pure standards as well as standard addition to positive samples . other studies have used hrmas nmr to study the structure of a cyanuric acidmelamine system ( 26 ) or the formation of melamine condensation products ( 27 ) ; however , no h data were presented in either study that could be used as a comparison with our results . table 1 shows a comparison between the sensitivities of the different methodologies under investigation . from the methods we evaluated , the reference lc / ms / ms procedure was the most sensitive , followed by 700 mhz hrmas , and then 400 mhz liquid nmr . this was not unexpected , on the basis of the physical characteristics of the methods . the results clearly show that nmr can be used for screening purposes at levels down to the lower mg / kg range . for example , the who melamine recommendations of 1 mg / kg in powdered infant formula and 2.5 mg / kg in other foods ( 28 ) can be reached by hrmas . the sensitivity of the 400 mhz procedure could be increased if an optimized sample extraction or sample preconcentration step was included ( e.g. , a spe procedure similar to that conducted prior to lc / ms / ms ) ; however , it was our intention to leave the sample preparation as simple as possible . in contrast to the previous belief that nmr is only suitable for structure verification , elucidation , and purity analysis ( 24 ) , our results clearly demonstrate that nmr can be used for quantitative analysis of foods for health - relevant compounds . of course , the power of nmr resides in the nontargeted approach ( see below ) , but as we also had previously demonstrated in beer and fruit juice analysis ( 19,23,24 ) , the same spectra can be used to acquire quantitative data along with the results from multivariate analysis . determined according to din 32645 using a separate calibration curve in the range of lod . measuring solution is the final sample extract filled into the nmr tubes or injected into the lc / ms / ms system . calculated for a sample weight of 10 mg in 1 ml of dmso - d6 . determined using a hrmas rotor filled with 10 mg of authentic matrix . calculated for a sample weight of 1 g reconstituted in 500 l after spe cleanup . regarding the precision of the methods , the 400 mhz method showed a coefficient of variation of 3.2% ( n = 9 ; replicated measurement of authentic contaminated sanlu sample ) , while the coefficient of variation of the hrmas 700 mhz method was 2.6% ( measurement of spiked infant formula , n = 11 ) . the precision of nmr was judged to be sufficient for the purposes of food analysis . the lc / ms / ms procedure , in comparison , also had precisions of this order of magnitude or above ( due to the complicated sample preparation procedure ) . regarding accuracy , the nmr results were compared to the lc / ms / ms reference procedure . for the contaminated sanlu formula , the 400 mhz nmr , the 700 mhz nmr , and the lc / ms / ms melamine results were 412 mg / kg , 460 mg / kg , and 470 mg / kg , respectively , and for the contaminated candy , the melamine results were 47 mg / kg , 74 mg / kg , and 68 mg / kg , respectively . the 400 mhz procedure showed lower results than either of the other procedures , which was judged to be due to an incomplete extraction during sample preparation . this exemplifies the advantages of the hrmas procedure , which gave results very similar to those of the reference procedure . nevertheless , we also judge the 400 mhz procedure in the current form as suitable for food screening because any amount of melamine is of course not admissible so that even a qualitative result would be sufficient ( e.g. , in the context of raw product screening in industry ) . our study is the first to apply hrmas nmr to quantitatively determine melamine in authentic food matrices . hrmas appears to be eminently suitable for analyzing complicated matrices , such as solid or semisolid foods , that are difficult to extract by conventional means . many of these foods form emulsions or are not completely soluble in organic solvents , as in the case of the candy . hrmas is a form of gel - phase nmr , where the sample is mixed with a solvent that swells the matrix . using rotation of the sample , we can obtain high resolution h spectra in a routine manner ( 29 ) . a major application of hrmas appears to be the detection of tumor tissues ( 3033 ) . so far , relatively few applications in the field of food control have been presented , and these have involved the authentication of cereal foods ( 34,35 ) , cheese ( 3638 ) , or beef ( 39 ) . lc / ms / ms with spe extraction is clearly the preferred method for melamine quantification in all kinds of foods ( 4043 ) . according to our own experience the advantage of nmr and especially hrmas is the minimal time for sample preparation compared to that in the tedious lc / ms / ms procedure . unfortunately , melamine contamination is not the first instance of unsafe milk formula in china . in 2003 , 12 children died of malnutrition in anhui province as a result of being fed infant formula of poor quality . in this incident , 55 different infant formulas , 40 corporations , and 10 provinces were involved , which exposed key public health gaps in food safety and public protection ( 44 ) . this incident might even have indirectly led to the melamine crisis because government directives against diluted preparations were implemented . the fact that melamine could increase the apparent protein content and , furthermore , could make the product look milky may have been irresistible to those who would adulterate milk formulas ( 14 ) . as we have detailed in , consumer safety can not be ensured only by targeted analysis of compounds such as melamine because the adulterators are always one step ahead . in our research to establish a quantitative method for nmr analysis of melamine , it became quickly evident that even by a simple visual inspection of the nmr spectra , the sanlu sample showed differences . these were observed in several spectral ranges , even in the analysis of the aqueous solutions , in which the melamine peak at 5.93 ppm is not visible because of rapid proton exchange on the nh2 groups with the solvent protons . for example , the sanlu sample showed deviations in the range between 3.6 and 4.2 ppm . a search in our library of nmr spectra of pure compounds showed that this spectral pattern can be assigned to sucrose ( figure 2 ) . upper panel : comparison of melamine - contaminated sanlu sample with the reference distribution of a collection of infant formulas ( measurement in water at 400 mhz ) . lower panel : reference measurement of sucrose from the spectral library . in the same fashion , we found that one of the german infant formula samples diverged from all other samples . in this case this finding shows that the nmr method can also allow one to check recipes and labeling claims ( such as lactose - free , hypoallergenic , etc . ) . confirming these factors is comparably important to ensure the health of those subgroups of consumers with special nutritional needs . we therefore conclude that the routine application of nmr in the screening of baby food products would provide a considerable improvement in consumer protection . similar to what we have shown in the screening of fruit juices or beer , our chemometric approach allows the establishment of a standard model of a typical infant formula . this model can then be used without manual intervention to provide a judgment about a sample , regarding whether it does or does not correspond to the typical composition . in the case of noncompliance , the large depth of spectral information of nmr then allows one to make the assignment of compounds using databases ( as in the case of lactose or sucrose ) . in the case of compounds not in databases ( e.g. , such as melamine when we started our study ) , the option for nontargeted screening , along with the structural information inside the spectra , is certainly the major advantage of nmr over other techniques that have been recently proposed . for example , surface desorption mass spectrometry ( 4547 ) or enzyme - linked immunosorbent assays ( 48 ) again have the disadvantage that they are focused on sensitive target analysis . these will certainly detect melamine but will miss novel adulterants that might be used in its place in the future . the only other proposals in the literature that might be usable for a nontargeted approach are several infrared spectroscopy methods ( 4951 ) . multivariate regression analysis was able to derive models to quantify melamine from raman , mid , or near - infrared spectra . so far , only nmr can be seen as a holistic approach for detecting adulteration in infant formula .

the recent melamine crisis in china has pointed out a serious deficiency in current food control systems , namely , they specifically focus on selected known compounds . this targeted approach allowed the presence of melamine in milk products to be overlooked for a considerable time . to avoid such crises in the future , we propose that nontargeted screening methods need to be developed and applied . to this end , nmr has an extraordinary potential that just started to be recognized and exploited . our research shows that , from the very same set of spectra , 1h nmr at 400 mhz can distinguish between melamine - contaminated and melamine - free infant formulas and can provide quantitative information by integration of individual lines after identification . for contaminated chinese infant formulas or candy , identical results were obtained when comparing nmr with spe - lc / ms / ms . nmr was found to be suitable for routine nontargeted and targeted analyses of foods , and its use will significantly increase food safety .

Introduction Materials and Methods Results and Discussion

fatigue is a transitional state between awake and sleep which manifests itself as lack of alertness and deteriorated mental or physical performance and often associated with drowsiness . road accidents due to fatigue are often much more severe than other crashes , since the driver reaction time increases ( 1 ) . driver fatigue is deemed to account for up to 40% of road accidents ( 2 ) . it is conjectured that 1030% of road deaths are related to driver fatigue ( 3 ) . mental fatigue causes reactions become prolonged , more fluctuable , and more error tending ( 4 , 5 ) . grandjean defined fatigue as a state with decreased efficiency and lack of general willingness to work ( 6 , 7 ) . these can be classified as subjective , psychological , performance and physiological methods . in subjective methods , standard questionnaires such as f - vas and moreover , the use of behavioral and psychological techniques in mental fatigue investigation has been adopted in several preceding studies ( 1417 ) . among these studies , a set of video recordings of facial expressions , mannerisms and personality traits questionnaires were common methodologies with high reliability and validity ( 17 ) . in addition , some fatigue studies on driving simulator exploited performance features such as steering wheel angle and lane departure ( 1820 ) . some other researchers have focused on driver s physiological changes , such as the measurement of eye activity , heart rate , skin electrical potential and specially eeg activity as a means to detect cognitive states ( 21 , 22 ) . although several physiological indices available for assessing the alertness level , eeg signal may be one of the safest and most predictive , ( 2325 ) , since it immediately reflects brain activity . driving involves several tasks such as motion , reasoning , visual and auditory processing , decision - making , perception and cognition . driving is also under the influence of emotion , anxiety and many other psychological factors ( 24 ) . the brain electrical activity rhythms are classified according to frequency bands including delta , theta , alpha and beta waves ( 26 ) . alpha rhythm has the frequency range from 813 hz , which occurs during wakefulness , especially in the occipital cortex area of the brain . it typically appears during eye closure and reduced when eyes open and attenuates severely during attention tasks ( 27 ) . in previous researches , it is known that increase in delta power during internal processing is associated with mental task performance ( 28 ) . other researchers have reported that an increase in eeg theta power depends on decreased performance in monotonous tasks ( 29 ) . although some definite trends were observed in the delta , theta and alpha power frequency bands during fatigue , the results of different studies may be influenced by inter - individual and intra - individual variations in eeg data ( 30 ) . changes in eeg power spectra are associated with fluctuations in the alertness state ( 31 ) . monitoring physiological signals while driving can provide the possibility to detect and warn fatigue ( 32 ) . most investigations revealed that changes in delta and theta activity are related to the transition to fatigue . therefore , eeg monitoring during driving may be a promising variable for using in fatigue countermeasure devices ( 24 ) . it has been suggested that during driving at night , delta band varies significantly with the degree of fatigue ( 33 ) . some researchers have presented an eeg - based cerebral workload index based on the increase of eeg power spectra in the theta band over prefrontal areas and the immediate decrease of eeg power spectra over parietal areas in alpha band during driving ( 34 ) . while physiological mental fatigue level increases , the relative power of theta , alpha and beta rhythms decrease , but the relative power in delta rhythm increases ( 35 ) . the relative power of alpha increased while the attention level of the driver decreased ( 36 ) . however , research on the physiological links specially eeg frequency bands to driver fatigue is still exploratory and is an important area that needs further investigation . therefore , this study attempted to detect the driver mental fatigue through changes in eeg alpha power activity . twelve healthy male volunteer car drivers ( ranging 20 to 30 years old ) participated in an overnight study from 2 to 6 a.m. in virtual reality laboratory of khaje nasir toosi university of technology in 2013 . all the subjects held valid driving license with at least 2 years driving experience and had no history of prior brain injuries . the epworth sleepiness scale was used to measure the participants for any trait sleepiness . after taking informed written consent , the subjects were requested to stay awake 18 h before the experiments and refrain from caffeinated drinks or any other stimulant as well as cigarette smoking for 12 h prior to the experiments . the drivers should have regular sleep pattern ( i.e. sleeping not later than 1 a.m. and awaking not later than 9 a.m. ) and not get used to daily napping which was studied through sleep diary for one week before the experiment . this research employed a fixed - based car driving virtual reality simulator ( ci004 semi ) in a calm controlled room with fixed temperature and illumination conditions . 1 shows a snapshot of the car - driving simulator developed in mechatronics department of k.n . snapshot of the car - driving simulator used for the implementation of the proposed protocol a set of 110 km road sceneries had been designed and simulated . the first 90 km of the road was monotonous and straight with minimal side components to induce more fatigue and the last 20 km was winding mountainous road ( see fig . 2 ) . a scenery of the road captured from the car simulator lcd the designed road was from the most dangerous parts of real road pattern of iran , so called haram to haram road , photographed through google maps using autodesk autocad civil 3d ( version 2013 ) and autodesk 3ds max ( version 2012 ) softwares . a portable g.usb amp bio - signal amplifier with 16 channels was used with 256 hz sampling rate and 24 bit quantification with active electrodes for signal acquisition . during driving on the simulator , eeg and the electrode positions on different areas of the scalp were based on the 1020 international electrodes placement guideline . the main eeg signal channels were o1 , o2 , p3 , p4 , p7 , p8 , oz , fp1 , fp2 , cz , fz , t7 and t8 . the eeg data analysis involved pre - processing , artifact removal , and features extraction . the eeg signal was first processed executing a band pass filter between 0.5 and 60 hz . eeglab ( version 10.2.5.6a ) was utilized to remove muscular and ocular artifacts by visual inspection . all the signals were sequenced to epochs each lasting two seconds ( 1 epoch consisting of 512 samples = 2 seconds ) . this research exploits power spectrum density and fast fourier transform ( fft ) analyzing technique to determine the absolute and relative powers of alpha frequency band . relative alpha power was computed as the ratio between absolute alpha power and the total spectral power of the signal . we compared the absolute and relative alpha power variations in the first and last 10 minutes of driving with the f - vas scores . for further certainty , this study employed the double check validity method by comparing the extracted features from eeg signals with the video rating scores . before resuming the experiments , then , eeg and eog signals were recorded from the subjects in relaxed sitting posture for 3 minutes with closed eyes ( imagining driving on a highway ) and 3 minutes with open eyes ( looking at a road picture on the screen ) as a baseline for alertness . the subjects were asked to drive the monotonous road at a constant speed of 90 km / h . at the same time , a continuous eeg and eog records were taken during the driving on the car simulator . meanwhile , the drivers faces and behaviors were monitored via video recordings in lateral and front views . these videos were used to rate the fatigue level of drivers on a four - point scale from 1(alert ) to 4 ( very tired ) by two trained observers . moreover , the self - rating of fatigue was performed during the driving using fvas scored from 0 ( no fatigue or energetic ) to 10 ( worst possible fatigue ) in every 10 minute interval . the descriptive statistics such as central and scattered indices were computed in order to describe the variables . pearson and spearman correlation coefficients were employed to explore the associations between f - vas scores and video ratings , fvas and absolute and relative alpha powers . paired - sampled t - test was utilized to compare the means of f - vas scores , video ratings , absolute and relative alpha powers in the initial and final 10 minutes of driving . twelve healthy male volunteer car drivers ( ranging 20 to 30 years old ) participated in an overnight study from 2 to 6 a.m. in virtual reality laboratory of khaje nasir toosi university of technology in 2013 . all the subjects held valid driving license with at least 2 years driving experience and had no history of prior brain injuries . the epworth sleepiness scale was used to measure the participants for any trait sleepiness . after taking informed written consent , the subjects were requested to stay awake 18 h before the experiments and refrain from caffeinated drinks or any other stimulant as well as cigarette smoking for 12 h prior to the experiments . the drivers should have regular sleep pattern ( i.e. sleeping not later than 1 a.m. and awaking not later than 9 a.m. ) and not get used to daily napping which was studied through sleep diary for one week before the experiment . this research employed a fixed - based car driving virtual reality simulator ( ci004 semi ) in a calm controlled room with fixed temperature and illumination conditions . 1 shows a snapshot of the car - driving simulator developed in mechatronics department of k.n . snapshot of the car - driving simulator used for the implementation of the proposed protocol a set of 110 km road sceneries had been designed and simulated . the first 90 km of the road was monotonous and straight with minimal side components to induce more fatigue and the last 20 km was winding mountainous road ( see fig . 2 ) . a scenery of the road captured from the car simulator lcd the designed road was from the most dangerous parts of real road pattern of iran , so called haram to haram road , photographed through google maps using autodesk autocad civil 3d ( version 2013 ) and autodesk 3ds max ( version 2012 ) softwares . a portable g.usb amp bio - signal amplifier with 16 channels was used with 256 hz sampling rate and 24 bit quantification with active electrodes for signal acquisition . during driving on the simulator , eeg and the electrode positions on different areas of the scalp were based on the 1020 international electrodes placement guideline . the main eeg signal channels were o1 , o2 , p3 , p4 , p7 , p8 , oz , fp1 , fp2 , cz , fz , t7 and t8 . the eeg data analysis involved pre - processing , artifact removal , and features extraction . the eeg signal was first processed executing a band pass filter between 0.5 and 60 hz . eeglab ( version 10.2.5.6a ) was utilized to remove muscular and ocular artifacts by visual inspection . all the signals were sequenced to epochs each lasting two seconds ( 1 epoch consisting of 512 samples = 2 seconds ) . this research exploits power spectrum density and fast fourier transform ( fft ) analyzing technique to determine the absolute and relative powers of alpha frequency band . relative alpha power was computed as the ratio between absolute alpha power and the total spectral power of the signal . we compared the absolute and relative alpha power variations in the first and last 10 minutes of driving with the f - vas scores . for further certainty , this study employed the double check validity method by comparing the extracted features from eeg signals with the video rating scores . before resuming the experiments , the car driver s sleepiness propensity was measured by the epworth sleepiness scale . then , eeg and eog signals were recorded from the subjects in relaxed sitting posture for 3 minutes with closed eyes ( imagining driving on a highway ) and 3 minutes with open eyes ( looking at a road picture on the screen ) as a baseline for alertness . the subjects were asked to drive the monotonous road at a constant speed of 90 km / h . at the same time , a continuous eeg and eog records were taken during the driving on the car simulator . meanwhile , the drivers faces and behaviors were monitored via video recordings in lateral and front views . these videos were used to rate the fatigue level of drivers on a four - point scale from 1(alert ) to 4 ( very tired ) by two trained observers . moreover , the self - rating of fatigue was performed during the driving using fvas scored from 0 ( no fatigue or energetic ) to 10 ( worst possible fatigue ) in every 10 minute interval . the descriptive statistics such as central and scattered indices were computed in order to describe the variables . pearson and spearman correlation coefficients were employed to explore the associations between f - vas scores and video ratings , fvas and absolute and relative alpha powers . paired - sampled t - test was utilized to compare the means of f - vas scores , video ratings , absolute and relative alpha powers in the initial and final 10 minutes of driving . the drivers mean age was 23.8 years ( sd = 1.44 years ) with mean body mass index ( bmi ) of 21.57 ( sd = 2.01 ) . other demographic characteristics and work - related information of the drivers drivers demographic and background characteristics data as shown in table 2 the self - report scale measured by means of f - vas suggested that the participants had not been fatigued during the first 10 minutes of driving and moderately to extremely fatigue during the final section of driving . compared with the initial section , the f - vas scores increase significantly ( p = 0.001 ) during the final section of driving task , which demonstrates that continuous monotonous driving may predispose the subjects to cognitive fatigue . different fatigue measures in i10d and f10d i10d : the initial 10 min of driving ; f10d : the final 10 min of driving furthermore , table 2 presents the video scores rated by trained observers in the initial and final 10 minutes of driving . paired - sampled t test suggests significant increase during the final 10 minutes of driving ( p = 0.001 ) . the results showed a mild , but significant correlation between f - vas and video rating scores in the initial 10 minutes of driving ( r = 0.404 , p = 0.001 ) . meanwhile , spearman s correlation test showed a relatively strong and significant association between f - vas and video rating scores in the final 10 minutes of driving ( r = 0.62 , p = 0.001 ) . table 2 also indicates the absolute and relative alpha powers in the initial and final 10 minutes of driving . there was a significant increase in the absolute alpha power during the final 10 minutes of driving ( p=0.006 ) . moreover , our findings showed no differences in absolute alpha power in different areas of the scalp in the initial and final 10 minutes of driving , except in the parietal area of the brain ( p=0.005 ) . paired - sampled t test suggests statistically significant difference in p4 site between absolute alpha powers in the initial and final 10 minutes of driving ( p = 0.026 ) . this study attempted to present a simple and reliable method for early detection of driver mental fatigue based on eeg alpha power in healthy sleep deprived drivers . in our experiment , different fatigue outcome measures including subjective self - report of fatigue ( f - vas ) , video ratings as well as eeg alpha powers were employed to evaluate the change in fatigue for drivers during the initial and final 10 minutes of driving . the experimental results suggested a significant increase in the subjective self - report of fatigue ( fvas scores ) during the final 10 minutes of driving . the same results were obtained from zhang ( 2008 ) study which found that the level of both subjective sleepiness and fatigue increase significantly from pre - task to post - task ( 37 ) . they studied the impacts of visual display terminal ( vdt ) task on autonomic nervous system and central nervous system through subjective self - report of sleepiness and some physiological measures such as power spectral indices of hrv and wavelet packet parameters of eeg . it should be regarded that our findings from fvas scores showed lowered variability ( all participants reached the state of fatigue and extremely fatigue ) in the final section of driving . this may be because a subject who is fatigue will be less likely to evaluate his / her state sufficiently . in other words , mental fatigue may impact the drivers judgment of their existing state . the findings of this preliminary research indicated a significant increase in absolute alpha power in the final section of driving . this is consistent with the known aspects of eeg alpha rhythm ( 38 , 39 ) . the increase in alpha rhythm in the final 10 minutes of driving depicted the decrease in the level of alertness and attention and the commencement of fatigue and drowsiness . the findings of the present study are well be in line with the findings of researchers in which they found significant difference between the alpha frequency band in the first and fifth section of driving ( 40 ) . in another study , delta and theta activity increased in the final 3 hours of 7 truck drivers during night driving ( 41 ) . nevertheless , our results revealed no significant difference between the relative alpha powers in the initial and final 10 minutes of driving . the reason for this may be due to the increase in delta and theta rhythms , which causes less relative alpha variability in the final section of driving . in our study , most changes occurred in the right parietal region ( p4 ) which was in contrast with the schier ( 2000 ) study that showed the greatest changes in the right frontal region ( f4 ) of the scalp ( 36 ) . however , this is consistent with the reported role of the right hemisphere via blood flow measurement in attention demanding tasks ( 42 ) . since the subjective self - report of fatigue was measured in the initial and final 10 minutes of driving , sudden variations can not be detected using f - vas technique . another limitation to employing this scale is that drivers verbal expression of their level of fatigue may alert them , thereby decreasing their fatigue level . meanwhile , it is almost unfeasible to obtain fatigue feedback from a driver in real driving conditions unless significantly distracting the driver attention from the driving task . our study benefited from using video rating of facial expressions and driver behaviors by trained observers that might not be able to see what goes on behind the facial behavior and expression . placing the electrodes on the scalp to obtain signals is an intrusive technique that should be addressed as another limitation of researches using wired biological signals . therefore , future projects should make the use of less - intrusive systems such as wireless electrodes and benefit the strengths of the various fatigue monitoring methodologies into a hybrid system to develop an efficient fatigue detection device in real driving situations . the present study specifically deals with eeg alpha power in the initial and final section of driving in partially sleep - deprived drivers - a very prominent aspect of driver fatigue analysis . the results of this study may serve as a baseline for the development of anin - vehicle device preventing fatigue related crashes on monotonous roads and greatly improving the transport industry in terms of socio - economic benefits and safety . this study suggests that alpha brain wave rhythm can be a good indicator for early prediction of driver fatigue . meanwhile , image processing of facial expressions may be a complementary method for road accident prevention . these indicators can be incorporated to develop a fatigue countermeasure device to prevent road accidents and reduce fatigue related costs . 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 : driver fatigue is one of the major implications in transportation safety and accounted for up to 40% of road accidents . this study aimed to analyze the eeg alpha power changes in partially sleep - deprived drivers while performing a simulated driving task.methods:twelve healthy male car drivers participated in an overnight study . continuous eeg and eog records were taken during driving on a virtual reality simulator on a monotonous road . simultaneously , video recordings from the driver face and behavior were performed in lateral and front views and rated by two trained observers . moreover , the subjective self - assessment of fatigue was implemented in every 10-min interval during the driving using fatigue visual analog scale ( f - vas ) . power spectrum density and fast fourier transform ( fft ) were used to determine the absolute and relative alpha powers in the initial and final 10 minutes of driving.results:the findings showed a significant increase in the absolute alpha power ( p = 0.006 ) as well as f - vas scores during the final section of driving ( p = 0.001 ) . meanwhile , video ratings were consistent with subjective self - assessment of fatigue.conclusion:the increase in alpha power in the final section of driving indicates the decrease in the level of alertness and attention and the onset of fatigue , which was consistent with f - vas and video ratings . the study suggested that variations in alpha power could be a good indicator for driver mental fatigue , but for using as a countermeasure device needed further investigations .

Introduction Materials and Methods Participant selection Driving simulator preparation EEG and EOG data acquisition: Research protocol Statistical data analysis Results Discussion Conclusion Ethical considerations

there are many published reports of malfunction of the inner tube of the bain co - axial circuit , with potentially lethal complications for the patient . the following case report describes a case where profound hypercapnia occurred consequent to avulsion of the inner tube of the co - axial circuit at the machine end of the circuit , which was neither apparent nor visible to the anaesthesiologist . this report therefore emphasizes the need for testing the co - axial circuit for any circuit malfunction before each use . a 30 year - old male presented as an emergency for repair of a crush injury of the left hand . induction was done with thiopentone sodium 5 mg / kg and fentanyl citrate 1.5 mg / kg . after achieving muscle relaxation with succinylcholine 1 mg / kg , anaesthesia was maintained with oxygen , nitrous oxide , isoflurane , and intermittent doses of vecuronium bromide . the vital signs monitored were blood pressure , oxygen saturation , electrocardiogram , temperature , and end - tidal carbon dioxide ( etco2 ) . the gas flows , airway pressure , temperature , chest expansion and bilateral air entry in to lungs were all checked and found to be normal . we changed the d - fend of our side - stream capnograph for a new one but the etco2 continued to rise . the heart rate rose from the baseline of 80 per minute to 110 per minute and blood pressure rose from a baseline of 110/70 mmhg to 140/94 mmhg ; however , the oxygen saturation was 100% . not finding any cause for the rise of etco2 , we decided to change the circuit . following this , the etco2 curve began to fall and reached the normal value of 40 mmhg over the next couple of minutes and the inspiratory carbon dioxide baseline returned to zero . close examination of the original circuit revealed that the inner tubing of the co - axial tube had become disconnected from its seat at the machine end of the circuit [ figure 1 ] . this was a previously unused bain circuit and we had not checked the integrity of the inner tubing before inducing the patient . the inner coloured tube carries the inspiratory gases . if this tube should become disconnected or develop any breach in its integrity there will be a huge increase in dead space , with consequent hypercapnia and its complications . four main causes for development of hypercapnia during anaesthesia have been described : ( 1)reduced alveolar ventilation , with rise in partial pressure of co2(2)inhalation of exhaled co2 ( as has been noted in association with a defective fresh gas flow tube of a bain circuit)(3)inhalation of exogenous co2(4)increased metabolic rate ( as in malignant hyperthermia ) reduced alveolar ventilation , with rise in partial pressure of co2 inhalation of exhaled co2 ( as has been noted in association with a defective fresh gas flow tube of a bain circuit ) inhalation of exogenous co2 increased metabolic rate ( as in malignant hyperthermia ) hypercapnia leads to sympathetic system stimulation , with tachycardia , hypertension , arrhythmias , excessive sweating , and peripheral vasodilatation , which may lead to excessive intraoperative blood loss . our patient had significant increase in heart rate and blood pressure , which was initially erroneously attributed to the light plane of anaesthesia . a number of tests have been described to assess any co - axial circuit malfunction ; these include : ( 1)visual inspection of tubing for any obvious disruption or obstruction.(2)pethick test : this tests the low - pressure system and the integrity of the inner tube . collapse of the reservoir bag due to the creation of venturi effect in the outer tube is an indication that the inner tube is intact . this test will not detect a system in which inner tube is omitted or does not extend to the patient port or one that has holes at the patient end of the inner tube.(3)foex - crampton - smith manoeuvre : this manoeuvre assesses the gas flow line from the flowmeters of the machine to the patient end of the circuit . with an oxygen flow at 2 litres / minute , the patient end of the inner tube is occluded briefly for 23 seconds using the forefinger . a positive test is indicated by descent of the rotameter bobbin due to back pressure ; with removal of finger , the bobbin ascends to its original position . ghani suggested the use of the plunger of a 3-ml syringe to occlude the inner tube more precisely . pethick test : this tests the low - pressure system and the integrity of the inner tube . collapse of the reservoir bag due to the creation of venturi effect in the outer tube is an indication that the inner tube is intact . this test will not detect a system in which inner tube is omitted or does not extend to the patient port or one that has holes at the patient end of the inner tube . foex - crampton - smith manoeuvre : this manoeuvre assesses the gas flow line from the flowmeters of the machine to the patient end of the circuit . with an oxygen flow at 2 litres / minute , the patient end of the inner tube is occluded briefly for 23 seconds using the forefinger . a positive test is indicated by descent of the rotameter bobbin due to back pressure ; with removal of finger , the bobbin ascends to its original position . ghani suggested the use of the plunger of a 3-ml syringe to occlude the inner tube more precisely . this case report highlights the possibility of severe hypercapnia due to dead - space rebreathing as a result of disconnection of the inner tube of the co - axial circuit . we present this as a warning and reminder that the integrity of the co - axial circuit must be always checked visually as well as mechanically . in this regard , the foex - crampton - smith manoeuvre with the ghani modification appears to be most satisfactory method of assessing the integrity of the gas line from the flowmeter up to the patient end of the bain circuit .

the bain co - axial circuit is fully established in general anaesthesia practice . a major concern is the potential malfunctioning of the circuit due to avulsion of the inner fresh gas delivery tube at the machine end of the circuit . the following case report presents a case in which a patient connected to the bain circuit developed severe hypercapnia in the early intraoperative period due to the above mentioned defect .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

we attempted to estimate the perceived degree of urgency of the visit and to identify reasons for seeking non - urgent care in the ped by patients and parents . a prospective survey was completed by parents ( for children 17 and younger ) and patients ( 18 - 21 ) presenting to a suburban academic ped that sees approximately 15,000 patients per year . three hundred and five of 334 surveys were completed ( 91% response rate ) over a 3-month period . twenty - four percent of the chief complaints were perceived by those surveyed as emergent or possibly life - threatening , 23% were felt to be very urgent , and 52% were deemed somewhat urgent or minor . twenty - five percent of those with minor or somewhat urgent complaints arrived by ambulance . overall , 79% of those surveyed identified a primary care provider ( pcp ) for themselves or their child . of those , 54% had attempted to contact the pcp prior to coming to the ped . six percent of those who attempted to reach their primary care providers were able to contact them and 52% were told to come to the ped . more than half of patients and parents presenting to the ped believed they had minor or somewhat urgent complaints . while the majority of patients have a regular provider , limited access to timely primary care and convenience may make the ped a more attractive care option than primary care for many parents and patients .

objectives : pediatric emergency department ( ped ) patients often present with non - urgent complaints . we attempted to estimate the perceived degree of urgency of the visit and to identify reasons for seeking non - urgent care in the ped by patients and parents.methods:a prospective survey was completed by parents ( for children 17 and younger ) and patients ( 18 - 21 ) presenting to a suburban academic ped that sees approximately 15,000 patients per year . a convenience sample of participants was enrolled.results:three hundred and five of 334 surveys were completed ( 91% response rate ) over a 3-month period . twenty - four percent of the chief complaints were perceived by those surveyed as emergent or possibly life - threatening , 23% were felt to be very urgent , and 52% were deemed somewhat urgent or minor . twenty - five percent of those with minor or somewhat urgent complaints arrived by ambulance . weekend visits and minority race correlated with a lower degree of perceived urgency . overall , 79% of those surveyed identified a primary care provider ( pcp ) for themselves or their child . of those , 54% had attempted to contact the pcp prior to coming to the ped . six percent of those who attempted to reach their primary care providers were able to contact them and 52% were told to come to the ped.conclusions:more than half of patients and parents presenting to the ped believed they had minor or somewhat urgent complaints . while the majority of patients have a regular provider , limited access to timely primary care and convenience may make the ped a more attractive care option than primary care for many parents and patients .

Objectives: Methods: Results: Conclusions:

suicide of which the literal meaning is to kill oneself is an act of intentionally causing one 's own death . the reasons are mostly related with some mental disorders such as depression , bipolar disorder , schizophrenia , alcoholism , or sometime drug abuse . the triggering factor is stress which is many times related with either financial difficulties or troubles within their personal relationships . it is 10 leading cause of death worldwide in which around 800,000 to a million people die every year and an estimated 10 - 20 million nonfatal attempted suicides occur each year . in india , most suicides are under - reported and very little data is available about attempted suicides . indian union health ministry estimated that around 1.2 lakh people commit suicide , and over 4 lakhs people attempt suicide in india . unfortunately , 11% of these cases are reported from andhra pradesh state of india . national crime record bureau of india reported that in last three and half decades an increase in 175% of suicidal rate is observed and one suicide occurs in every 5 min in india although the suicidal act is made illegal in the country under the section 309 and 306 indian penal code ( ipc ) and even attempt to commit suicide is part of criminal activity under section 309 of ipc . number of suicidal incidences is proportional to attempted suicide cases hence if attempt cases reduced number of suicidal death can also be decreased and for that purpose risk factors should be identified and reduced . there are number of risk factors for suicide which varies according to country , culture , religion , gender , age , and social values . hence , the present study is planned to identify risk factors among lower socioeconomic rural population of both genders of surrounding areas of hyderabad region of india as little data is available . moreover , hyderabad is a very important densely populated metropolitan city of south india . surrounding rural population including rural population of adjoining districts therefore , it is important to properly understand the risk factors for attempted suicide cases of little studied lower socioeconomic class rural population so that suitable policies and programs can be initiated for prevention . this was a prospective study in which all the suicide attempt cases reported at bhaskar medical college and general hospital were included . it was conducted during the period of january 2013 to july 2013 . studied population belongs to surrounding rural areas of hyderabad region of india . all the cases were diagnosed and managed by emergency and general medicine department of the institute and as per the routine standard protocol , were referred to psychiatric outpatient department for further evaluation and intervention , once the patients were medically became fit for the psychiatric interview . all the patients clearly making an attempt on their life by themselves or assisted by someone were included , and accidental cases of poisoning were excluded from the study . these patients have undergone a detailed psychiatric interview including their demographic details and complete suicide risk assessment was done using beck 's suicide intent scale . the end point of the psychiatric management was counseling and treatment in the form of medication and follow - up for further management . in this study , a total of 36 patients were studied in which 20 were females , while 16 were males . mean age was 25 years for female , and 34 years for males , the age difference was statistically significant ( p < 0.05 ) in them . minimum age of the female was 16 years and the maximum was 40 years , while in the male it was 18 years and 70 years respectively . in both the groups , most of the cases were in between the age group of 21 and 30 years [ table 1 ] . age group distribution female ( n=20 ) , male ( n=16 ) more than 50% subjects were uneducated in both groups . in females majority of the subjects ( 75% ) were in the category of uneducated to secondary education while in males same trend is seen , but the numbers were much higher ( 93.75% ) . moreover , in the female group , substantial number of subjects ( 20% ) was intermediate passed , while in males , no intermediate qualified subject was seen . as far as a profession is concerned the subjects were housewife , laborer , students , and low income self - employed or were farmers . about 25% male subjects were farmers and 50% were laborer , while in the female group 35% were housewife and 45% were laborer [ table 2 ] . their married life ranged in between < 1 and > 10 years of marriage . in the male group almost 40% subjects majority of females ( 75% ) and males ( 56.25% ) subjects had dependent children between 1 and 3 kids [ table 3 ] . marital status and children majority of subjects attempted suicide 1 time although 10% female and 6.25% male attempted 2 time . most of the females ( 75% ) were free from any kind of substance use , and only 25% of them occasionally took toddy . in case of males , most of them ( 62.5% ) were consuming alcohol regularly , although 25% of males are nonalcoholics and the rest ( 12.5% ) were occasional users . apart from alcohol and toddy , subjects in both groups were not involved in any kind of drug or substance use . at the time of attempt , attempts , substance use , substance influence most of the males took organophosphorus ( 87.5% ) for suicide attempt followed by calotropis ( 12.5% ) while the majority of females also took organophosphorus ( 45% ) followed by rat poison ( 15% ) , known / unknown tablets ( 15% ) , phenyl ( 10% ) , animal / plant energy mix ( 10% ) and calotropis ( 5% ) [ table 5 ] . methods / substances for suicide attempt among the study population , 10 individuals met the criteria for alcohol dependence syndrome and 14 had depression . most of them had depressive disorder ( 38.9% ) , followed by alcohol dependence syndrome ( 27.8% ) . both of them together were present in 13.8% of the individuals [ table 6 ] . prevalence of psychiatric disorders on the suicide assessment scale in isolation parameter , score 2 was predominantly observed in females ( 70% ) , while in males scores 2 and 3 was equal ( 43.8% each ) . on timing parameter , score 2 was mainly seen in both the genders ( 65% females and 56.3% males ) , while in precautions against discovery / intervention parameter , score 1 was predominant in both the genders ( 70% females and 68.8% males ) . score 2 was also common in acting to get help during / after attempt parameters in both the groups [ tables 7 and 11 ] . scores on suicide assessment scale score 1 was mainly observed in final acts in anticipation of death ( female 95% , male 93.8% ) , active preparation for attempt ( female 90% , male 56.3% ) and a suicide note ( 100% in both groups ) parameters although 31.3% males scored 2 on active preparation for attempt parameter . most of the female and male subjects score 2 on overt communication of intent before attempt parameter ( female 80% , male 75% ) . on alleged purpose or intent parameter , score 3 was common in females ( 70% ) while in males score 3 was observed in 50% subjects and score 2 in 43.8% individuals [ tables 8 and 11 ] . scores on suicide assessment scale on expectations of fatality parameter , score 3 was more common in females ( 60% ) while in males score 2 was common ( 68.8% ) . on the conception of method 's lethality parameter , score 2 was common in both the genders ( females 65% , males 68.8% ) and also in seriousness of attempt parameter score 3 was more common in both the genders ( females 60% , males 68.8% ) . we observed score 3 predominantly in females ( 60% ) and males ( 60% ) on the attitude toward living / dying parameter although score 2 was also seen in the high number of subjects ( females 40% , males 43.8% ) . score 2 was the predominant in both the genders ( females 80% , males 68.6% ) although the number of female subjects were higher in comparison to their male counterparts on the parameter of conception of medical rescuability . most of the subjects of both the genders ( females 75% , males 68.6% ) scored 1 on the degree of premeditation parameter [ tables 9 and 11 ] . scores on suicide assessment scale total score on suicide assessment scale scores on suicide assessment scale when we assessed total score of all the parameters of suicide assessment scale it was found that in the female group equal number of ( 50% each ) cases were in medium ( total score between 20 and 28 ) and high risk ( > 28 ) categories . in the male group , number of males with medium risk were slightly more ( 56.3% ) in comparison to high risk group ( 43.8% ) . in both groups , not a single case was found to be in low risk category [ tables 10 and 12 ] . this prospective study was conducted to explore the sociodemographic variables in individuals with suicidal attempt in which females ( n = 20 ) have outnumbered males ( n = 16 ) . kessler et al . , also found female in dominance in their study , but das et al . , have reported a higher incidence of suicide attempts in males when compared to females in indian population , which is contrast to our finding in south india . das et al . , suggested that the trend might be because of higher responsibility of males in the family financial issues that contributes to increased exposure to stressful events . bashir et al . , and kiran et al . , also observed male dominance in their respective studies in tribal and urban regions of india . but in our study , this difference could be because of the study population considered . most of our study population is from the rural background and both husband and wife work as daily laborers , sharing equal amount of stress at work . besides this females have additional family responsibilities at home which in fact make them more prone for stress than males . joseph et al . , also found female dominance in rural regions of india . with increasing age , there was a decline in the number of the individuals who attempted suicide . people in 21 - 30 years age group had more suicidal rate than those belonging to other age groups . with the increasing age , individuals develop more adaptability to the stressful conditions , helping them to encounter challenging situations in a better way , thus contributing to lesser incidence of suicides . , on incidence rates of suicide in west bengal , which concluded that the vulnerable age group being those between the ages of 18 and 30 years . high proportion ( 87.5% ) of the individuals who committed suicide was married and out of total married females 15% were pregnant at the time of the attempt . marital conflicts , family stress and financial issues would have contributed to the increased number of suicides in married group . ramdurg et al . , also found predominance of married individuals in their study of which was conducted to find out sociodemographic profile clinical factors and mode of attempt of suicide attempt . , observed that most of people of developing countries live in rural regions and they are involved in agriculture and farm small areas of land . these farmers keep all their agriculture materials including pesticides , commonly within , or close to , the household . the easy availability of these pesticides when the individual is impulsive would have contributed to the high number of them attempting by this method . most of the individuals ( 62.5% ) had regular intake of alcohol and 31.25% of the study population attempted suicide under the influence of alcohol . this could be probably because of the impulsivity , lack of ability to think logically during the intoxicated state . hufford , in his study found that alcohol intoxication increases suicide risk up to 90 times , in comparison with abstinence . , also reported that , constructs related to aggression and impulsivity confers additional risk for suicidal behavior among persons with alcohol dependence . this is in accordance with the study conducted by potukuchi and rao in which there was a high prevalence of alcohol intake in the females of rural population of this region . in this study , diverse precipitating factors for suicide attempt were observed . including quarrel with close relatives , husbands or fathers were alcoholic and abusive , over all family disputes stand as a major reason for the suicidal attempt . this finding is in harmony with the findings of siwach and gupta , who reported that marital disharmony , economic hard ships and disagreement with other family members as the major precipitating factors for suicide . depressive disorders were diagnosed in 38.9% of individuals and stands as the most common associated disorder . parkar et al . , in mumbai region of indian also observed similar trend , as in their studied population 40% of individuals with intentional self - harm were suffering from depression and significant number of them also had alcohol use related disorders . increased risk of suicide with alcohol intake the individuals included in the study were referred from other departments of the institute . therefore , this may not represent all the individuals who had attempted suicide as there is a chance of people not consulting psychiatry department . a few individuals were referred to other health care institutes or expired during the medical management were not included in the study . we conclude that in the studied population risk of suicide attempt is almost equal in terms of medium and high category of suicide assessment scale in both the genders . females in between the age group of 20 and 30 years , uneducated , married and daily laborers by occupation are involved more than males and thus such category of females have greater risk for suicide attempt . depressive disorders are most common associated psychiatric disorders in both the genders , followed by alcohol use related problems . we suggest that all the individuals with alcohol related disorders must be screened for suicidal ideation , so that appropriate methods can be adopted to reduce the risk .

background : suicide is an act of intentionally causing one 's own death . number of suicidal incidences is proportional to attempted suicide cases hence if attempt cases are reduced , number of suicidal death can also be decreased and for that purpose risk factors should be identified and reduced . therefore , this study is planned to identify risk factors among lower socioeconomic rural population of surrounding areas of hyderabad in india.materials and methods : this was a prospective study in which all the suicide attempt cases reported at bhaskar medical college and general hospital were included . the study period was from january 2013 to july 2013 . they were undergone a detailed psychiatric interview , including their demographic details , and complete suicide risk assessment was done using beck 's suicide intent scale.results:it was found that females in the age group of 20 - 30 years , uneducated , married and daily laborers by occupation had higher incidence of suicidal attempts . depressive disorder is the most common associated psychiatric disorder in both the genders , followed by alcohol use related problems . family disputes are the other major risk factors . most common mode for attempt was organophosphorous poisoning followed by ingestion of calotropis.conclusion:risk of suicide attempt is almost equal in terms of medium and high category of suicide assessment scale in both genders . we suggest that all individuals with alcohol related disorders must be screened for suicidal ideation so that appropriate methods can be adopted to reduce the risk .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LIMITATIONS CONCLUSIONS

single - cell microorganisms ( scm ) constitute an emerging alternative to source high - value lipids for a series of growing markets demanding low - cost , high - quality alternatives . scm as a broad class display a series of advantages when compared to plants and animals as lipid sources . in addition to being more genetically accessible , scm are capable of producing greater diversity and storing higher percentages of lipids . therefore , their productivity per volume and energy input can be up to 5 or 6 times that of plants and even more when compared to animal sources . additionally , single - cell microorganism architecture and circuitry tend to be more straightforward than those in plant or animal cells . for example , there are fewer organelle compartments in fungal cells than in plant cells , and some microalgal metabolic pathways ( e.g. , fatty acid synthesis ) are encoded by single - copy genes , whereas plants have multiple genes encoding proteins with redundant enzymatic activities . scm can be readily grown under controlled conditions , and the key metabolic and biochemical questions can be answered more simply and quickly than in complex multicellular systems . in principle , scm can achieve greater sustainability to alleviate the increasing problem of sourcing oils for both the fuel and human consumption markets , thus mitigating the continuous increase in commodity oil prices . microbial lipids can also become sources of safe and clean biomaterials ( e.g. , biosurfactants ) at reduced costs and continuous availability . a complete mechanistic understanding of cellular machinery is the key to moving from principle to practice in lipid production . the purpose of this paper is to provide an overview of the current knowledge of the different cellular and biochemical mechanisms involved in neutral and polymeric lipid accumulation within four main single - cell microbial groups : yeasts , microalgae , bacteria , and archaea . special emphasis is given to the production of triacylglycerols ( tag ) and polyhydroxyalkanoates ( pha ) as the hallmark lipids accumulated in eukaryotes and prokaryotes , respectively . insights are provided on the mechanistic differences existing in each group during the transformation of substrates ( e.g. , acetyl coenzyme a ) , formation of intermediate pools ( e.g. , fatty acyl chains ) , allocation to intracellular lipid pools ( e.g. , polar lipid pools or neutral lipid pools ) , and the architecture of lipid accumulation storage ( e.g. , lipid droplets ) . in microorganisms , different types of neutral lipids are accumulated by different types of scm . for example , in oleaginous yeast , tag and/or sterol esters ( se ) are the primary neutral lipids accumulated inside the cell , whereas in specific types of bacteria and archaea polyhydroxyalkanoates ( pha ) , and to a lesser extent tag and wax esters , are preferentially stored . these lipids are currently viewed as valuable potential biofuel sources , high - value compounds in the food and pharmaceutic industries , building blocks for biomaterials , potential tools to treat chronic diseases , and natural alternatives for the production of oleochemicals , among many other uses . neutral lipids follow distinct cellular accumulation strategies compared to polar lipids , such as phospholipids ( pl ) , galactolipids , and sulfolipids among others . however , in most cases , both classes share key biosynthetic steps . depending on cell needs , the cell is capable of transforming polar lipids into neutral lipids and vice versa due to the different pools present in strategic cellular locations . for instance , polar lipids are pooled and may be mobilized from the different cell membranes . key membranes involved in the accumulation of lipids include the plasma membrane in both eukaryotes and prokaryotes , and in the case of eukaryotes the endoplasmic reticulum ( er ) , peroxisome , mitochondrial , plastidial ( and their variants , such as apicoplastidial ) , or even thylakoid membranes , depending on the organism . key intermediates , such as acetyl coenzyme a ( ac - coa ) , malonyl coenzyme a ( mal - coa ) , acyl coenzyme a ( acyl - coa ) , and glycerol-3-phosphate ( g3p ) are also pooled in strategic cellular locations such as the cytosol , plastidial stroma , and mitochondrial intermembrane space . the distribution of the different biosynthetic enzymes plays a key role in achieving the correct lipid traffic toward accumulation . the accumulation of intracellular neutral lipid is a highly reductive process , requiring more nadph and atp than the storage of glucose and glucose derivatives . accumulation of lipids is a biological process that fulfills several roles in microorganisms . storage of lipids contributes to cell growth , cell division , stress response , and as energy storage for survival . in some cases , some scm have the capacity to accumulate lipids > 20% of their weight when exposed to an environmental stress , such as the lack of a key nutrient . when growing on limiting concentrations of a key nutrient ( typically nitrogen ) and with sufficient or excess carbon sources , oscm will stop their replication processes and utilize the available carbon to synthesize and store it in the way of reduced lipids . in the case of tag and se , oscm shift their metabolic machinery to generate a pool of ac - coa , as well as nadph , which is the reducing power driving fatty acid ( fa ) synthesis forward the strategies vary among different classes of oscm , and details will be discussed in the following sections . in the second stage , ac - coa is carboxylated to produce mal - coa , transferred to acyl carrier protein ( acp ) , and further transformed into an acyl acp by sequential turns within the fatty acid synthase ( fas ) . there are two types of fas : type i is an integrated enzyme complex ( common in eukaryotic cytoplasm ) , and type ii is a dissociated version in which all subunits are independent ( common in prokaryotes and in organelles of prokaryotic origin in primary and secondary endosymbionts , such as plastids and mitochondria ) . type i can be subdivided in type ia , present in fungi ( 66 complex in the cytoplasm ) , and type ib , present in animals ( as cytoplasmic 2 dimers ) . for reviews describing type i fas and type ii fas , the reader is referred to the works of schweizer and lu et al . , depending on the cell s needs and the type of scm , the acyl chain might end in a specific type of lipid pool , such as membrane lipid ( coupling the acyl chain to g3p , ac - coa , or other glycerol - based backbone to form pl or other types of polar lipid ) or can eventually be incorporated into a neutral lipid droplet core ( via the construction of tag or se molecules ) . the nature , location , and dynamics of this stage vary among species and are explained more in detail in the following sections . in this stage , further elongation and desaturation of the acyl chains may also occur , and these phenomena vary throughout the different domains of life . finally , the fourth stage involves the formation of intracellular lipid droplets ( ld ) . variations occur depending on the organism and the environmental conditions . in the case of prokaryotes , accumulation of tag or se is less common . in some members of the gram - positive actinomycete group , such as mycobacterium , rhodococcus , nocardia , dietzia , and streptomyces , tag interestingly , bacterial tag biosynthesis occurs frequently in the environment because actinomycetes are the most abundant microorganisms in soil . accumulation of tag in gram - negative bacteria has been reported only in species from the genus acinetobacter , but only as a minor component of the neutral accumulated lipid . wax esters are the main compound stored in these species . for the rest of the gram - negative bacteria , however , several studies have successfully transformed model bacteria such as escherichia coli by inserting the required genes to become oleaginous , with remarkable success . in addition , tag have been reported as major components of the neutral lipid of the cyanobacterium nostoc commune , although no indications were obtained for the storage of lipid inclusions in the cells . in the case of archaea , it is more common in prokaryotes to accumulate pha , comprising specialized lipids such as poly(3-hydroxybutyrate ) ( p3hb ) , polyhydroxyvalerate ( phv ) , or copolymers such as poly(3-hydroxybutyrate - co-3-hydroxyvalerate ) ( phbv ) . pha possess thermoplastic and elastomeric properties and are recyclable materials that can be easily degraded into carbon dioxide and water . the most promising are as film formation , paper coating , foils and diaphragms , and multiple - use packages , because the melted polymers have low viscosity , permitting the injection molding of objects with thin walls . the end product is very hard and can be used at temperatures from 30 to 120 c . chemical structures of three different types of pha , synthesized and accumulated in certain prokaryotes . the first stage involves the generation of a suitable cytoplasmic ac - coa pool . the second one involves the synthesis of the hydroxyalkanoate ( ha ) monomer or monomers , which can change depending on the species and substrates . the third stage consists of polymerizing or copolymerizing the monomers into pha chains , and the fourth one involves the formation of the intracellular pha ld . in both prokaryotes and eukaryotes , accumulation of neutral lipids results in the formation of intracellular fat bodies typically called lipid droplets ( ld ) . ld are also called lipid bodies , fat bodies , or adiposomes , and in the case of plants they have also been termed oleosomes , spherosomes , or plastoglobules , the latter if their location is inside the plastid . for the present discussion , the term lipid droplet will be used . in most of the eukaryotes ld are intracellular sphere - like particles composed of a core of neutral lipids , such as tag or se or both , coated by a monolayer of polar lipids , mainly pl , and decorated with a series of proteins . the polar heads face the cytoplasm , and the nonpolar tails face the inside , where the neutral lipids reside . the inserted proteins within the polar lipid monolayer play functional , structural , and regulatory roles in the life cycle of ld . in the case of polyhydroxyalkanoate - accumulating prokaryotes , they form ld , which are also called lipid granules or carbonosomes . in some higher organisms ( e.g. , humans ) , lipid droplet interaction with other cellular organelles , and their dynamics is closely related to progression of metabolic diseases , such as obesity , fatty liver , type 2 diabetes mellitus , and atherosclerosis . therefore , it has become apparent that ld are not just a static storage compartment in the cell , but rather a dynamic organelle that interacts with other organelles within the cell and actively participates in the intricate cellular processing symphony , to the point that some authors consider ld as specialized organelles in cells . as described in later sections , strategies for lipid accumulation in scm vary among the different types of scm , and it is important to understand these differences to improve and/or create productive strategies that can become economically as well as technologically feasible for the generation of biofuels as well as nutritional lipid metabolites such as long - chain polyunsaturated fatty acids ( lc - pufa ) . thus , they are capable of metabolizing carbon from simple sugars or from other simple compounds such as glycerol . when oleaginous yeasts encounter an environmental stress , such as a limiting nutrient ( e.g. , nitrogen ) , they shift their metabolic machinery to stop synthesizing proteins and nucleic acids and , thus , begin to allocate the available carbon in the form of reduced lipids . other examples of limiting nutrients can be phosphorus or magnesium . in practice to date , nitrogen has been extensively used because it seems to yield higher lipid accumulation when compared to other nutrients . the carbon to nitrogen ratio is a useful tool to construct nitrogen - rich or nitrogen - depleted media . for example , it has been shown that for yeasts , poor lipid accumulation occurs in media in which the carbon to nitrogen ( c : n ) ratio is < 20 , whereas ideal lipid production occurs in a c : n ratio range of 3080 . the optimal c : n ratio for lipid accumulation varies greatly with the microbial species , strain , and carbon sources present in the growth medium . in yeasts , oleaginicity depends on the ability to produce ac - coa , the necessary precursor of fatty acids , in an effective manner . the conversion of 1 mol of ac - coa to fatty acids requires the formation of 2 mol of nadph , which constitutes the reducing power necessary to drive the reaction forward . the metabolic steps for lipid accumulation in yeast can be divided into four main stages , as mentioned before ( see figure 2 ) . the diagram also includes the connection between fatty acid synthesis , triacylglycerol synthesis , and lipid droplet formation . abbreviations : me , malic enzyme ( cytosolic ) ; me 2 , malic enzyme ( er membrane , responsible for fatty acid desaturation ) ; acl , atp : citrate lyase ; i d , isocitrate dehydrogenase ; nld , nascent lipid droplet ; ld , lipid droplet ; er , endoplasmic reticulum ; lpa , lysophosphatidic acid ; pa , phosphatidic acid , fas i , fatty acid synthase i ; dag , diacylglycerol ; tag , triacylglycerol ; ampd , amp deaminase . the cell responds by activating amp deaminase and inducing an acute decrease of cellular amp content . in mitochondria of oleaginous species , the decrease in cellular amp levels causes isocitrate dehydrogenase ( idh ) activity to decrease or even stop . as a consequence , the production of -ketoglurarate drops and the tricarboxylic cycle ( tca ) is dramatically reduced or even stopped . to reverse this situation , aconitase transforms isocitrate back to citrate ( cit ) , leading to an accumulation of cit in mitochondria . cit is transported by an antiport protein , known as citrate / malate translocase ( cmt ) , from the mitochondria to the cytoplasm . in the cytoplasm , cit is cleaved to form oxaloacetate and ac - coa , by atp : citrate lyase ( acl ) . cytosolic malate is converted to pyruvate by malic enzyme ( me ) , encoded by me1 . this reaction generates nadph and is coupled to a series of parallel reactions as well . the first is the carboxylation of pyruvate to form oxaloacetate by pyruvate carboxylase inside mitochondria . this nadph is the reducing power required to convert ac - coa into fatty acids . thus , pyruvate carboxylase , md , and me are known as the transhydrogenation machinery . the resulting pyruvate completes the cycle and goes inside the mitochondria ( figure 2 ) . me is present in all fungi , but its role in supplying nadph for de novo lipogenesis is preponderant in oleaginous species . it has been found that , upon nitrogen limitation , me changes from isoform d to isoform e , which supplies nadph for de novo lipogenesis . ac - coa is converted by acetyl - coa carboxylase ( acc ) to mal - coa , which is then used to synthesize fatty acids . in yeast , cytosolic acc the mitochondrial version closely resembles in both its molecular mass and amino acid sequence the cytoplasmic one . acc uses biotin as a cofactor to transfer co2 to ac - coa in a two - step process . it is a trifunctional enzyme , harboring a biotin carboxyl carrier protein domain , a biotin carboxylase domain ( where co2 binds to biotin ) , and a carboxyl - transferase domain ( where co2 is transferred to ac - coa to yield mal - coa ) . the fatty acyl chain is built in a type i cytosolic fatty acid synthase ( fas i ) . it is an 66 complex encoded by two genes , fas1 ( subunit ) and fas2 ( subunit ) . it is suggested to contain 6 equivalent sites of fa synthesis with a total of 42 catalytic domains , organized in a ring - like structure . however , variations to this architecture exist and probably may affect the performance in de novo lipogenesis . for example , ac - coa is always the basis of fa biosynthesis and is the substrate of -ketoacyl acp synthase ( ksa ) in bacteria . the sequence of reactions in yeast fas ii is condensation of ac - coa with mal - coa ( via ks ) , reduction ( via ketoacyl reductase , kr ) , dehydration ( via a dehydratase , dh ) and further reduction ( via enoyl reductase ear ) in a repetitive manner until palmitoyl - acp is formed . release of the acyl moiety is a key step that differs from species and is currently a subject of intense research . yeasts employ malonyl - palmitoyl transacylase ( mpt ) to transfer the acyl chain from acyl acp to acyl - coa . in algae , the acyl chain can be released in three ways , depending on the cell s needs . first , it can be hydrolyzed from acp as a free fatty acid by means of a thioesterase ( te ) . second , it can be transferred either to g3p or monoacylglycerol-3-phosphate ( mag3p ) through an acyltransferase ( at ) in the chloroplast . lastly , if the acyl chain is destined to leave the plastid , it can also be released in the acyl - coa form by acyl - coenzyme a synthetases ( acs ) . it has been found that acs play a key role in regulating in each compartment the internal acyl - coa pools by esterification of fa to coa . the localization of the pools is maintained due to acyl - coa s not being able to cross the intracellular membranes . the final fatty acid composition of different types of microalgae is in great part dependent on the activity of these enzymes . for example , microalgal chain length specific te have been reported , which can release acyl moieties of specific length ( e.g. , c 12:0 or even c 8:0 ) . this type of fatty acid is more suitable for the production of gasoline and jet fuel . in contrast , gram - positive bacteria first form acyl - phosphate from acyl - coa ( via plsx ) , which is then transferred onto g3p by plsy . gram - negative species use plsb acyltransferase only to load an acyl group directly onto g3p from acyl acp . in yeast , acyl - coas released from cytosolic fas i system there , a series of esterifications to a g3p backbone occur , also known as the kennedy pathway . the first step consists of esterifying the acyl moiety from acyl - coa to g3p or dihydroxyacetone 3-phosphate via glycerol-3-phosphate acyl transferase ( g3pat ) , on the sn-1 position . g3pat is encoded in yeast by gat1 and ayr1 , when the acceptor is g3p or dihydroxyacetone 3-phosphate , respectively . gat1 does not exhibit a particular preference for acyl - coa , whereas ayr1 prefers palmitoyl - coa , thus defining fa composition . a second acyl moiety can be attached on the sn-2 position to generate phosphatidate ( pa ) , catalyzed by acyl - coa : lysophosphatidic acyltransferase ( lpaat ) . pa plays a key role in the regulation of acc1 , fas1 , and fas2 genes , as it is involved in an autoregulatory loop directed by the concentrations of inositol and choline in the cytosol and pa in the er membrane . expression of upstream activating sequence ( uasino)-operated genes changes in parallel to pa , inositol , and choline concentrations . in the case when pa is going to be transformed into tag first , the phosphate group can be directly hydrolyzed by a phosphatidate phosphatase ( pap ) to create dag . the second one involves synthesizing cytidine diphosphate dag ( cdp - dag ) from pa , catalyzed by cdp - dag kinase , encoded by cds1 . cdp - dag is the precursor of the different pl in the er membrane . it thus stays as either phosphatidylcholine ( pc ) , phosphatidylethanolamine ( pe ) , or another type of pl . eventually , dag is generated by cleavage of the pl via phospholipase c. both alternatives are in metabolic interlock with each other . this creates a positive feedback system that channels pa into the tag pathway if cdp - dag concentration is higher than dag concentration or directs pa to the membrane pool in the opposite situation . lastly , dag is transformed into tag by esterifying an acyl moiety from acyl coa catalyzed by an acyl coa : dag acyltransferase ( adat ) , encoded by either dga1 or lro1 , depending on whether dag is generated directly from pa via pap or it comes from the er membrane pl pool , respectively . another me , different from the cytosolic malic enzyme , catalyzes the conversion of malate to pyruvate and the consequent reduction of nadp to nadph . nadph then couples to a series of electron transfer reactions involving cytochrome b5 reductase and further activating the desaturase , which adds a double bond on the fatty acid chain at the expense of converting 1 mol of oxygen into a mole of water . , ergosterol is the major sterol present in yeast . for a review in the biosynthesis of ergosterol backbone yeast se are synthesized via transesterification of ergosterol with acyl - coa . in yeast , two acyl - coa cholesterol acyltransferase ( acat ) related enzymes , are1p and are2p ( encoded by are1 and are2 ) , catalyze the reaction . microscopic localization of green fluorescent protein hybrids and enzyme measurements showed that both are1p and are2p are localized to the er . are1p esterifies ergosterol and its precursors with nearly equal efficiency with a slight preference for lanosterol , whereas are2p uses ergosterol as a preferred substrate . an acyl - coa - independent pathway for the formation of ses has not been identified in yeast . lipid droplet biogenesis is an area of intensive current research , and there is emerging scientific evidence showing that in yeast it takes place between the two membrane leaflets of the er . by mechanisms still not clearly understood there are spots in the er membrane where there is a concentration of adats dga1 and lro1 . the first one is responsible for tag synthesis in the outer leaflet of the endoplasmic reticulum , whereas the second is responsible for tag synthesis in the inner leaflet of the er . the synthesized tag begin to accumulate , generating a lens - like protrusion and promoting the recruitment of structural proteins . in yeast , pat proteins ( from the initials of perilipin , adipocyte differentiation - related protein , and tip47 ) accumulate in the outer leaflet ( see figure 3 ) , whereas in plants this role is played by oleosins . when there is enough accumulation of tag between the leaflets , the outer buds off and the lipid droplet is formed ( figure 3 ) . model of lipid droplet formation in the er membrane . adapted from ref ( 11 ) . there seems to be a functional relationship between the lipid droplet membrane and the er membrane , because several studies have shown that certain functional proteins can migrate between the lipid droplet and er membranes by mechanisms that do not require energy expenditure . in yeast , for example , experiments using yeast mutants unable to synthesize tag revealed that ld were not formed . however , the proteins that were present in the wild forms in ld were also present in the er of the yeast mutants , suggesting a relationship between er proteins and lipid droplet proteins . in some cases , lipid droplet - localized proteins can relocate back to the er , indicating that some continuity between the two organelles is maintained , even if only transiently , in a way that allows the two - way partitioning of proteins between the two compartments . in describing the functions of proteins embedded in the droplet monolayers , two mechanisms have been proposed . examples of this strategy are the multifunctional caveolin protein , which has been localized to both the plasma membrane and ld , and dga1 , the major enzyme catalyzing triacylglycerol synthesis . their long internal hydrophobic stretch may enable them to be embedded in either bilayers or monolayers . the second mechanism is best represented by the previously mentioned pat protein family . they display a four - helix bundle with great similarity to the n - terminal domain of apolipoprotein e. upon binding to lipids , the apoe four - helix bundle opens to expose amphipathic helices that can bind the monolayer surfaces of lipoproteins . in an analogous manner , pat proteins may bind to lipid droplets by embedding hydrophobic helices into the droplet surface . pat proteins also share a common structural element : n - terminal 11-mer repeats that have an amphipathic helical structure . it is still unclear why or how they do it . in the specific case of perilipins ( members of the pat family ) , there are five groups , and their abundance has been correlated with the abundance of tag in the lipid droplet as well . it is important to mention that perilipin - like proteins are not found in plants . as was already mentioned , functionally similar oleosins and caleosins are present in plants instead . the accumulation of lipids that promote concavity decreases the energy burden required for budding . it has been shown that lipid droplet pl contain more lysophospholipids and less sphingomyelin and pa compared to the total membrane . in silico studies proposed a packing parameter s to quantify the degree of convexity / concavity in pl . lysophosphatidylcholine and phosphatidylinositol ( pi ) promote a convex shape where their lipid footprint areas are much smaller than their headgroup areas . lipids with s values < 1 adopt a convex surface , favoring lipid droplet formation . configurations adopted by different phospholipids , which can affect the curvature during lipid droplet formation . eukaryotic microalgae are classified into nine divisions : glaucophyta , rhodophyta ( red algae ) , heterokontophyta , haptophyta , cryptophyta , dinophyta ( dinoflagellates ) , euglenophyta , chlorarachniophyta , and chlorophyta ( green algae ) all of these divisions include single - cell strains , which can be either motile , nonmotile , or both . in terms of their nutritional strategies , microalgae can be divided into obligate heterotrophs , obligate photoautotrophs , facultative mixotrophs , and obligate mixotrophs . most algal divisions contain colorless heterotrophic species that can obtain organic carbon from the external environment either by taking up dissolved substances ( osmotrophy ) or by engulfing bacteria and other cells as particulate prey ( phagotrophy ) . in most cases , lipid accumulation strategies in microalgae and some single - cell protists ( both primary and secondary endosymbionts ) follow the four - stage lipid accumulation strategy described for yeast . , further studies are demonstrating that lipid biosynthesis pathways in microalgae are not a simple mirror image of what happens in higher plants , which have been more thoroughly studied . it seems likely that regulation of triacylglycerol synthesis and breakdown in microalgae tends to obey a stress response phenomenon , whereas in plants it follows a developmental phenomenon . for a review comparing lipid metabolism between microalgae and plants , see liu . microalgae differ from yeast in the location of the acetyl - coa pools within the cell . microalgae display plastidial and cytosolic acetyl - coa pools , which are key for lipid accumulation . in contrast , yeast s main acetyl - coa pool used for lipid accumulation is located in the cytosol . there is an additional acetyl - coa pool present in mitochondria as well as a mitochondrial lipid biosynthesis pathway employing a type ii fas ( different from plastidial fas ii ) both in algae and in yeasts . they play important roles in different cell processes , such as rna processing , mitochondrial lipoic acid synthesis , and protein lipoylation . however , mitochondrial lipid synthesis is not the main avenue for lipid accumulation and will not be covered in the present work . for a review of mitochondrial lipid biosynthesis and its relevance in cell function , plastids present in algae play a key role in de novo lipid biosynthesis ( for a review of plastid evolution and diversification , see the work of keeling ) . in photoautotrophic microalgae , photosynthesis provides an endogenous source of plastidial acetyl - coa , although more than one pathway may contribute to maintaining the acetyl - coa pool . for instance , in photoautotrophic microalgae plastidial pyruvate can be sourced via transformation of photosynthesis - derived glyceraldehyde-3-phosphate to phosphoenolpyruvate ( pep ) . pep is irreversibly converted to pyruvate ( see figure 5 ) by pyruvate kinase ( pk ) . finally , the plastidial pyruvate dehydrogenase complex ( pdh ) catalyzes the oxidative decarboxylation of pyruvate to produce plastidial ac - coa , co2 , and nadh . pdh contains three components : e1 ( pyruvate dehydrogenase , composed of e1 and e1 subunits ) , e2 ( dihydrolipoyl acyltransferase ) , and e3 ( dihydrolipoamide dehydrogenase ) . it is possible that photosynthesis - derived pyruvate is the major contributor to plastidial ac - coa for de novo fatty acid synthesis . however , in mixotrophic grown cultures of heterokonts such as nannochloropsis sp . , incorporation of acetate directly into lipids occurs . the acetyl - coa synthetase ( acsin ) converts acetate to plastidial ac - coa . additionally , a study showed that under nitrogen deprivation chlamydomonas is capable of changing its metabolism from converting acetate to glucose to a more direct incorporation of acetate into fatty acids by down - regulating glyoxylate cycle activity and gluconeogenesis . abbreviations : er , endoplasmic reticulum ; accase , acetyl - coa carboxylase ; acp , acyl carrier protein ; dagat , diacylglycerol acyltransferase ; dhap , dihydroxyacetone phosphate ; enr , enoyl - acp reductase ; fat , fatty acyl - acp thioesterase ; g3pdh , glycerol-3-phosphate dehydrogenase ; gpat , glycerol-3-phosphate acyltransferase ; hd , 3-hydroxyacyl - acp dehydratase ; kar , 3-ketoacyl - acp reductase ; kas , 3-ketoacyl - acp synthase ; lpaat , lysophosphatidic acid acyltransferase ; lpat , lysophosphatidylcholine acyltransferase ; mat , malonyl - coa : acp transacylase ; pdh , pyruvate dehydrogenase complex . in microalgae , the cytosolic ac - coa pool is mainly fueled by the release of mitochondrial cit to the cytosol and further cleaved into oxaloacetate and ac - coa by acl . cytosolic ac - coa is the building block used for lc - pufa elongation in the er . concomitant production of nadph by me generates the reductive power necessary to drive plastidial de novo fatty acid synthesis forward . availability of nadph can increase the reaction velocity of acc 2 ( see stage 2 ) and acl . fatty acid synthesis is an energy - demanding process due to the activity of elongases and desaturases . for instance , the formation of a c18 fa requires 54 nadph from oxygenic photosynthesis . other functions of malic enzyme in algae may include delivery of co2 from the tca for the plastidial ribulose-1,5-bisphosphate carboxylase ( rubisco ) . studies also suggest the existence of a plastidial me ( absent in yeast ) , which can provide electrons for plastidial fa synthesis . for a review about some microalgae are capable of accumulating intracellular starch , such as the thoroughly studied microalgal model chlamydomonas . starch synthesis is an example of how carbon partitioning might play a key role in lipid accumulation . one study showed that in wild - type chlamydomonas , tag accumulated only after the maximum amount of starch was reached , whereas starchless chlamydomonas mutants initiated tag accumulation earlier and reached a higher level than wild - type strain . therefore , it is possible to engineer high tag algal strains by eliminating competing carbon utilization pathways such as starch synthesis to maximize lipid biosynthesis . in algae , the committed step in plastidial fatty acid synthesis is the conversion of plastidial ac - coa to mal - coa by acc1 . the three domains of the homomeric acc1 are located on a multifunctional polypeptide encoded by a nuclear gene . others , such as t. pseudonana and p. tricornutum , contain two homomeric accases , acc1 ( described above ) and a cytosolic acc ( acc2 ) , which uses cytosolic ac - coa to generate mal - coa . the latter plays a role in lc - pufa elongation in the er membrane . fas are synthesized in microalgae s plastid via a dissociated type ii fas , containing discrete , monofunctional enzymes encoded by distinct genes . in plastidial fas ii mal - coa is loaded to acp via malonyl - coa : acp transacylase encoded by fabd . malonyl - acp is used in the cyclic condensation reactions to extend the acyl group to palmitoyl acp or stearoyl acp . the acyl acp can be released from fas ii in several ways : it can be hydrolyzed by a fatty acyl - acp thioesterase located in the chloroplast envelope , forming a free fatty acid , or it can be transesterified from acp to coa via ( acs ) , or it can even be coupled to either g3p or mag3p through an at in the chloroplast . there are some heterotrophic microalgal species that contain a cytosolic type i fas , synthesized from one or two polypeptides , different from plastidial type ii fas . for example , aurantiochytrium(100 ) contains a type i fas , which synthesizes saturated c14:0 and c16:0 . the synthesized free fas and the absence of genes homologous to a type ii te may indicate integration of te activity into the synthase . several attempts have been made to overexpress specific enzymes in the lipid biosynthetic pathways . in the cases of acc and ks ( ks iii ) , overexpression failed to increase lipid accumulation . the released acyl moieties as free fatty acids destined to stay within the plastid may be further desaturated ( typically to hexadecatrienoic acid hdt , c16:3n-4 ) and coupled to a monogalactosyldiacylglycerol ( mgdg ) backbone . mgdg , along with other types of galactosylglycerides ( gg ) , is a major component of photosynthetic membranes in microalgae and in some cases may be even more abundant than pl . in plants , the fa combination of gg can be traced back to their biosynthetic pathways ; the so - called eukaryotic molecular species ( c18/c18 ) of gg are synthesized outside the chloroplast in the eukaryotic pathway , and the prokaryotic molecular species ( c18/c16 ) are synthesized in the plastid via the prokaryotic pathway . microalgae differ from plants in that most c20 fas are synthesized outside the chloroplast and are present in both the eukaryotic - like ( c20/c20 , c18/c18 ) and prokaryotic - like ( c18/c16 , c20/c16 ) molecular species . c20/mlc ( where mlc means medium to long chain ) rather than eukaryotic- and prokaryotic - like gg . when acyl acp s are esterified to either g3p or mag3p , they can join the plastidial pl pool . in plants two ats the second one resides on the inner chloroplast envelope membrane and preferentially selects palmitoyl - acp . in some algal species , such as c. reinhardtii and p. lutheri , pc is absent in both plastidial and extraplastidial phospholipid pools . instead , they contain the non - phosphorus betaine lipid diacylglyceryl - n , n , n - trimethylhomoserine ( dgts ) , which has similar physicochemical properties , as a major membrane component . it has been suggested that dgts may have a role in lipid droplet formation similar to that of pc in higher plants . however , they contain other types of phospholipids , such as pa , pe , and pi . acyl acps synthesized in plastidial type ii fas can be transesterified with coa via acs . thus , stearoyl acp can leave the fas ii complex via transesterification by a series of acs responsible for maintaining both intraplastidial and cytosolic acyl - coa pools . in both cases , acyl - coa is destined to leave the plastid and enter the er membrane for further oxygen - dependent elongation and desaturation , to become long - chain polyunsaturated fatty acids ( lc - pufa ) , such as eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) . the first are front - end desaturases containing an n - terminal cytochrome b5 domain and insert the new double bond between the fa carboxyl group and a possible existing double bond . one example is the high substrate specific plastidial - located 12 desaturase , identified in p. tricornutum , which desaturates palmitoleic acid c16:1n-7 to hexadecadienoic acid 16:2n-4 . the second group comprises the less common 6/3 desaturases capable of inserting a new double bond between the fa methyl end and a pre - existing double bond . stearoyl coa can be desaturated by an er membrane - bound 9 desaturase to oleyl - coa and then linked to a glycerol backbone for further processing . some microalgae contain a plastidial 9 desaturase , capable of direct desaturation of stearoyl acp to be then transesterified to coa and sent to the er for elongation and desaturation . in most microalgae , n-3 lc - pufa are more abundant than n-6 lc - pufa , whereas in filamentous fungi , such as mortierella and mucor , n-6 lc - pufa are more common . in microalgae the most common pathways for epa and dha synthesis are the n-3 and n-6 pathways , but variations of the theme occur . in the n-3 pathway , oleic acid already linked to a glycerol backbone in the er membrane is desaturated to linoleic acid ( la ) , via a 12 desaturase ( encoded by fad2 ) . a third double bond inserted by an n-3 15 desaturase gives -linolenic acid ( lna ) , which is further desaturated to produce stearidonic acid ( sa ) by a 6 desaturase . sa is then elongated to c20:4 n-3 ( eicosatetraenoic acid , ea ) and finally desaturated by a 5 desaturase to produce epa . in most dha - producing microalgae this is a different , less complicated strategy for dha synthesis from epa , compared to the sprecher pathway , present in mammals , which involves an additional elongation and a -oxidation . in contrast to the n-3 pathway , some microalgae synthesize epa via the n-6 pathway , following 6 desaturation of la to -linolenic acid ( gla ) , elongation to dihomo--linolenic acid ( dgla ) , creation of a fourth double bond by 5 desaturase to produce arachidonic acid ( ara ) , and a final desaturation to create epa via a n-3 17 desaturase . some microalgae express 9 elongase and 8 desaturase , allowing them to synthesize epa in a different way compared to the n-3 and n-6 pathways . in a variation of the n-3 pathway , lna is elongated by a 9 elongase to eicosatrienoic acid ( eta ) and desaturated by the 8 desaturase , creating ea . on the other hand , a variation of the n-6 pathway using this pair of enzymes involves 9 elongation of la to eicosadienoic acid ( eda ) and 8 desaturation to dgla . thus , microalgae can switch from n-6 fatty acids to n-3 fatty acids via the n-3 15 desaturase and the n-3 17 desaturase . in some species of the thraustochytrids , it is also possible to switch from n-6 docosapentaenoic acid ( dpa ) to dha , via an n-3 4 desaturase . in addition to these pathways , there is a different n-3 lc pufa biosynthetic pathway present in one of the three genera of heterotrophic thraustochytrids , namely auranthiochytrium , based on an anaerobic polyketide synthase pathway ( pks ) . a large multifunctional enzyme complex carries out the multitude of individual reactions , utilizing mal - coa and producing free n-3 lc - pufas . the major free fas dpa ( 22:5n-6 ) and dha ( 22:6n-3 ) are then activated to acyl - coa and incorporated into tags . because pks does not require aerobic desaturation , the pathway is energetically favorable compared to the membrane - bound desaturases and elongases . tag synthesis in microalgae follows the kennedy pathway in the er in a similar fashion as yeast ( see figure 5 ) . however , an acyl - coa - independent mechanism for triacylglycerol synthesis in some plants and yeast has been reported . this pathway uses pl as acyl donors and dag as the acceptor , and the reaction is catalyzed by the enzyme phospholipid : dag acyltransferase . there are two diacylglycerol acyltransferase families identified in chlamydomonas , involved in the final step of triacylglycerol synthesis : type one ( dgat ) , encoded by dgat1 , and type two ( dgtt ) , encoded by five dgtt genes , which do not share sequence similarity . two independent studies showed the expression levels of dgat1 and dgtt1 increased considerably following nitrogen deprivation . however , it remains unclear which of these dgats are primarily responsible for the accumulation of tags under this condition or whether individual isoforms have specific roles . triacylglycerol synthesis can also occur within the plastid , through a series of acyl - acp esterifications to plastidial g3p , catalyzed by plastidial ats similar to the kennedy pathway in the er . the involvement of gg ( the major polar lipid family in plastidial membranes in several microalgae ) in this process remains to be elucidated . during light dark cycles , many microalgae initiate triacylglycerol storage during the day and deplete those stores at night to support cellular atp demands and/or cell division . this cycling has to be taken into account when scaling up processes for production of lipids from algae . this variable may be key to the overall success of an open pond process or a closed photoreactor process . they can grow facing the cytosol or toward the inside of the plastid , facing the stroma , which is the major aqueous fluid surrounding the thylakoids inside the chloroplast . the mechanisms underlying the orientation of lipid droplet growth in plastid membranes are not well understood . when ld grow toward the inside of the plastid ( facing the stroma ) , they can also be called plastoglobules ( ptg ) . ptg can be considered to be functionally equivalent to cytosolic ld , but differ in three major respects . second , they can assume several different forms including rods , fibers , and globules ; and third , they are bound by a specific family of proteins , variously termed plastoglobulins , plastid lipid - associated proteins , and fibrillins . oleosins ( the major lipid droplet proteins present in plants ) are not present in green algae , but a major lipid droplet protein was identified by applying proteomics in chlamydomonas and shown to modulate lipid droplet size . a wide variety of parameters affect the abundance of ld in chlamydomonas , and there is ample evidence that turnover of ld plays crucial roles in cellular lipid or carbon homeostasis . in bacteria , the most frequent types of neutral and polymeric lipids synthesized and accumulated are pha , tag , wax esters ( we ) , and , to a lesser extent , se . however , a special situation occurs in bacteria such as rhodococcus ruber , and other related bacteria , capable of accumulating both types of lipids from unrelated carbon sources such as glucose . the mechanisms of wax ester accumulation have been reviewed elsewhere . in a nutshell , an acyl - coa is transformed to an aldehyde via an nadph - dependent acyl - coa reductase ( encoded by acr1 ) , which is then reduced to an alcohol via an also nadph - dependent fatty aldehyde reductase . the alcohol is then transesterified to an acyl - coa via an enzyme displaying both wax ester synthase ( ws ) and acyl - coa : diacylglycerol acyltransferase ( dgat ) activities ( abbreviated ws / dgat ) . the only report of the presence of an sterol ester - synthesizing enzyme in prokaryotes was provided by thornton et al . to date , triacylglycerol biosynthesis has been detected only in aerobic heterotrophic bacteria and in cyanobacteria . in most bacteria , accumulation of tag and other neutral lipids , such as we , is stimulated by a carbon source present in excess , together with limited nitrogen in the medium . structurally , fluorescence staining experiments showed that lipid biosynthesis starts at peripheral lipid domains close to the cytoplasm membrane . biochemically , fatty acid biosynthesis begins in bacteria with acc , a heterotetrameric enzyme encoded by four genes , acca , accb , accc , and accd . ac - coa is converted to mal - coa and transferred to acp by malonyl - coa : acp transacylase ( fabd in bacteria ) to form malonyl - acp . a first condensation of malonyl - acp with acetyl - coa by -ketoacyl - acp synthase iii ( fabh ) to form -ketobutyryl - acp and co2 initiates a cycle that can elongate the fatty acyl - acp by two carbon units for each cycle until a saturated fatty acid of 16 or 18 carbons is made . ksi ( fabb ) and ksii ( fabf ) are responsible for the subsequent elongation cycles of the growing acyl - acp chain . to balance chain initiation with growth and utilization in bacterial fasii , ksiii , enoyl - acp reductase ( fabi in bacteria ) , and -ketoacyl - acp reductase ( fab g in bacteria ) are under negative feedback control by long - chain acyl - acps . long - chain acyl - acps directly control reductase activities ; consequently , fasii is biased to catalyze forward if these products are withdrawn from the system by any conversion , including phospholipid or triacylglycerol synthesis . in some bacteria , fas ii yields unsaturated fatty acids , which play a key role in bacterial membrane fluidity and function . unlike er desaturases present in microalgae or yeast , which introduce double bonds into the completed fatty acid chains at the expense of oxygen , the bacterial fas ii system can also desaturate fatty acids anaerobically , because it does not require molecular oxygen . faba introduces the double bond at the 10-carbon intermediate , forming cis-2-decenoyl - acp . it additionally isomerizes it into trans-3-decenoyl - acp , which is further elongated by fabb . two genes , faba and fabb , are the key players in this pathway and occur together in bacteria that produce unsaturated fatty acids . some rhodococcus and nocardia bacteria are capable of incorporating branched or phenylic groups into their intracellular tag if the corresponding substrate is fed to the medium . for example , nocardia globerula strain 432 accumulated tag containing the branched fatty acid 4,8,12-trimethyltridecanoic acid after cells were fed pristane ( a branched alkane ) , and tag with a subfraction containing phenyldecanoic acid residues were detected in cells of r. opacus pd630 after phenyldecane was fed as sole carbon source . the enzymes involved in the esterification of the glycerol moiety probably act via sequential acylation of the sn-1 , -2 , and -3 positions of g3p , with the removal of the phosphate group occurring before the final acylation step . the first esterification is catalyzed by a g3pat using either acyl - coa or acyl acp to form lysophosphatidic acid . a second acylation to lysophosphatidic acid gives pa , which is the first branchpoint for the synthesis of tag and pl , because it can be converted to cdp - dag , the precursor of the different pl species in bacterial membranes . dag itself is also at a metabolic branchpoint that divides phospholipid and triacylglycerol formations , because it acts as a precursor for tag , pc , and pe biosynthesis . in addition , dag can also be derived from pl by the action of phospholipase c the distribution of acyl groups on the hydroxyl groups of the glycerol backbone is nonrandom , as has been demonstrated for r. opacus pd630 . the shorter and saturated fatty acids were esterified to the hydroxyl group at position 2 , whereas unsaturated fatty acids were preferentially found at position 3 . this distribution in bacterial tag is different from the tag of mammals and plants , where the longer unsaturated fatty acids are found at position sn-2 . the final step in wax ester and triacylglycerol biosyntheses in bacteria is catalyzed by ws / dgat . ws / dgat is encoded by atfa and is not related to any known at involved in the formation of tag and we in eukaryotes . it has also been shown that ws / dgat is localized at the bacterial cytoplasmic membrane , presumably attached to the inner leaflet of the membrane , probably via ionic interactions . for a detailed review about ws / dgat , the reader is referred to the work of waltermann et al . alternative pathways for triacylglycerol synthesis that do not involve dag in bacteria have also been reported and may involve an enzyme similar to that reported by dalquist in yeast , which catalyzes the formation of tag from the transesterification of an acyl donor ( e.g. , acyl - coa ) to pl . this follows observations that following a double knockout of ws / dgat genes in a. borkumensis , cells were still capable of substantial triacylglycerol accumulation in ld . triacylglycerol / wax ester lipid droplet biogenesis presumably begins by allocation of newly formed tag in a hydrophobic zone within ws / dgat . as time goes by , more ws / dgat attaches to the membrane , and cumulative synthesis advances , presumably leading to the formation of very small triacylglycerol agglomerates , depicted by wltermann et al . as small lipid droplets ( sld ) . sld apparently recruit pl from the membrane by a mechanism that is still not well understood , forming a layer toward the cytoplasmic side ( see figure 6 ) . the agglomeration of phospholipid - coated sld appears in microscopy as an oleaginous layer just parallel to the membrane . then , accumulation of sld in a given point gives birth to ld , which are coated by pl and detach from the oleaginous layer . it has been suggested that acquisition of additional tag is by merging with other ld freshly synthesized from the oleaginous layer via coalescence and/or through a protein identified as tada . an interesting study by ding and co - workers examined the proteome of rhodococcus sp . rha 1 ( a gram - positive bacteria capable of accumulating tag ) grown both in nitrogen - abundant ( not favoring lipid accumulation ) and nitrogen - depleted conditions ( favoring lipid accumulation ) . using a combination of techniques , including lc - ms , sds - page , and immunoblot assays , they reported 228 lipid droplet - associated proteins , which clustered primarily into metabolism - related enzymes , transcriptional regulators , ribosome proteins , and cell division - related proteins . interestingly , they identified two major proteins , ro02104 and pspa , which constituted about 15% of the total lipid droplet protein . according to their findings , the structure predicted for ro02104 resembles that of apolipoproteins , the structural proteins of plasma lipoproteins in mammals . suggested mechanisms of neutral ld formation in bacteria . adapted from ref pha comprise a complex class of storage polyesters , with almost 150 different hydroxyalkanoic acids known as constituents . a wide variety of gram - positive as well as gram - negative bacteria synthesize pha . examples include pseudomonas , bacillus , ralstonia , aeromonas , and rhodobacter , among others . pha are divided into two groups on the basis of the number of constituent carbon atoms in their monomer units : short chain length phas ( scl pha ) and medium chain length phas ( mcl pha ) . monomers of scl pha are 35 carbon atoms long , compared to 614 carbon atoms in mcl pha . in addition , scl pha are stiff and brittle with a high degree of crystallinity , whereas mcl pha are flexible and have low crystallinity , tensile strength , and melting point . accumulation of pha starts with the creation of hydroxyalkanoate monomers from three different biosynthetic pathways . the first one involves incorporation of two acetyl - coas to form acetoacetyl - coa , by the enzyme -ketothiolase . this pathway creates hydroxybutyrate ( hb ) monomers exclusively and is used by bacteria such as cupriavidus necator and azotobacter beijerinckii ( see figure 7 ) . on a second pathway involving de novo fatty acid biosynthesis , monomers of different lengths can be formed by transesterifying 3-hydroxyacyl - acp , an intermediate of fas ii to 3-hydroxyacyl - coa , presumably by the enzyme acyl - acp - coa transacylase , encoded by phag . this enzyme is the key link between de novo fatty acid synthesis and polyhydroxyalkanoate biosynthesis . this pathway is of biotechnological interest because it helps generate monomers for polyhydroxyalkanoate synthesis from structurally unrelated and simple , inexpensive carbon sources such as glucose or related simple sugars . on a third embodiment , monomers of different lengths can also be sourced from fatty acid -oxidation pathway either by conversion of 2-enoyl - coa by an r - specific enoyl - coa hydratase , encoded by phaj , or by reduction of 3-ketoacyl - coa , presumably by fabg or phab . in this case the ability of microorganisms to synthesize a particular form of polyhydroxyalkanoate is mainly due to the substrate specificity of polyhydroxyalkanoate synthases . these enzymes are divided into four classes , depending on their structure and specificity . class i enyzmes utilize coa thioesters of 3-hydroxyalkanoates ( 3-has ) , 4-has , and 5-has comprising three to five carbon atoms . members of class ii display major specificity for monomers ranging from 6 to 14 carbon atoms . enzymes from both classes consist of a single subunit of an average size of 6070 kda and are encoded by phac . in contrast , class iii and class iv synthases are encoded by two genes , phac / phae , and phac / phr respectively , and consist of two subunits . class iii members are capable of polymerizing preferably monomers ranging from three to five carbons , yet can utilize monomers from six to eight carbons as well . species such as allochromatium vinosum contain class iii synthases , whereas class iv has been reported only in bacillus sp . all polyhydroxyalkanoate synthases share a conserved cysteine as a catalytic site to which the growing polyhydroxyalkanoate chain is covalently attached . the active - site cysteine , histidine , and aspartate constitute a catalytic triad similar to esterases . two models currently exist that may explain the formation of in vivo polyhydroxyalkanoate ld ( which are also called granules or carbonosomes ) : the micellar and the budding models . the first one is based on the assumption that the polyhydroxyalkanoate synthase is present in the cell as a soluble enzyme , distributed throughout the cytoplasm . once polymerization of substrate molecules ( coa - thioesters of suitable hydroxyalkanoic acids ) starts , the nascent polyester chain converts the initially soluble enzyme into an amphipathic molecule and the increasingly hydrophobic polyhydroxyalkanoate chains aggregate into a micelle - like structure . polyhydroxyalkanoate synthase remains attached to the surface of the granule and therefore becomes insoluble ( see figure 8) . in this model , pl and proteins of the surrounding layer would gradually become incorporated as the self - assembled polyhydroxyalkanoate inclusion increases in size . this model requires the polyhydroxyalkanoate granule to be localized in the cytoplasm at all stages of formation . in contrast , the budding model assumes that polyhydroxyalkanoate synthase is associated with the inner face of the cytoplasmic membrane , either inherently or as soon as a polyhydroxyalkanoate chain emerges from the enzyme . in this case , biosynthesis of the polyester would be directed into the intermembrane space where the extending chains would accumulate until eventually the granules detach from the membrane and polyhydroxyalkanoate - specific surface proteins can be attached to the growing granules . although the micelle model is supported by the fact that polyhydroxyalkanoate granules can be produced in vivo in the absence of membranes , most of the recently emerging evidence is in favor of the budding model . phas may constitute approximately 5% ( w / w ) of total cellular proteins , and they play a main structural role in preventing polyhydroxyalkanoate granules from aggregating and in preventing the nonspecific attachment of other proteins to polyhydroxyalkanoate granules . in addition , phasins are presumably involved in the regulation of polyhydroxyalkanoate synthesis , polyhydroxyalkanoate degradation , polyhydroxyalkanoate granule size control , the formation of networks on the polyhydroxyalkanoate granule surface , and the distribution of polyhydroxyalkanoate granules during cell division . another important group of proteins present in the external granule layer is the depolymerases , which are responsible of catalyzing polyhydroxyalkanoate breakdown . this is a relevant step in the role of polyhydroxyalkanoate accumulation as a survival mechanism in the absence of suitable energy / carbon sources , as was demonstrated many years ago in r. eutropha . phazs have been investigated much less than extracellular depolymerases , and the mechanism by which intracellular native polyhydroxyalkanoate granules can be reutilized is still not well understood . interestingly , the gene coding for phazs is located between two copies of phac1 and phac2 ( both polyhydroxyalkanoate synthase genes ) in all investigated bacteria that accumulate mcl pha . in contrast , secreted depolymerases are used by most bacteria to assimilate pha present in the environment from , for example , other nonliving cells . proposed mechanism of pha containing lipid droplet formation in bacteria ( micelle model ) . adapted from ref ( 22 ) . polyhydroxyalkanoate granule synthesis and phasin production are tightly regulated by the effectiveness of the transcriptional regulator phar . genes encoding proteins homologous to phar are widely distributed among scl pha producing bacteria , indicating an important role in the regulation of scl pha biosynthesis . the discovery of the archaea domain in 1977 revealed a novel class of microorganisms encountered in exceptional ecological niches such as high ( thermophiles and hyperthermophiles ) or low ( psychrophiles ) temperatures , acidic media ( acidophiles an thermoacidophiles ) , anaerobic atmosphere ( methanogens ) , and high salinity ( halophiles ) . the unique chemical structure of their core membrane lipids is in part responsible for their adaptation to such hostile environments . archaeal membrane lipids , in contrast to those of bacteria and eukaryotes , are made up of saturated chains containing methyl branches , attached to glycerol by ether linkages with a stereochemistry in the 2-position of the glycerol opposite that of conventional mesophilic lipids . ( for a review on archaeal ether lipid structures , the reader is referred to the work of jacquemet et al . ) moreover , archaea do not synthesize fatty acyl esters , which are the most common constituents of ld ; instead , their lipids are based on isoprenoid chains . therefore , no accumulation of tag has been reported yet in archaea . despite these differences , however , evidence of polyhydroxyalkanoate accumulation was first reported in haloarchaea back in 1972 . the strains were called at that time halobacterium sp . from the dead sea , but later identified as haloarcula marismortui . since then , strains of several other haloarchaeal genera , including haloferax , halobiforma , and haloquadratum , have been found to accumulate pha . as in bacteria , archaea produce pha under conditions of nutrient limitation but where carbon is available in excess . the mechanisms of polyhydroxyalkanoate accumulation within archaea are beginning to be understood , and work is underway to elucidate the type of proteins involved in archaeal polyhydroxyalkanoate accumulation . genes involved in polyhydroxyalkanoate biosynthesis in haloarchaea were not recognized until recently , when the polyhydroxyalkanoate synthase genes were identified and characterized for haloarcula marismortui and haloferax mediterranei . these archaeal polyhydroxyalkanoate synthases are all composed of two subunits , phae and phac , and they are homologous to class iii bacterial polyhydroxyalkanoate synthases but have a longer c - terminal extension in the phac subunit . the close similarity of archaeal and bacterial type iii polyhydroxyalkanoate synthase genes and the lack of other polyhydroxyalkanoate gene types in archaea suggest that archaeal pha originated from the horizontal transfer of an ancestral type iii gene from a bacterium . some authors suggest this transfer to have occurred already before permian times . genome - wide analysis of h. marismortui atcc 43049 revealed eight paralogues of a short - chain dehydrogenase / reductase , responsible for reduction of acetoacetyl - coa to ( r)-3-hydroxybutyryl - coa ( 3-hb coa ) , a monomer used by polyhydroxyalkanoate synthase to produce polyhydroxybutyrate ( phb ) . another study demonstrated that a similar paralogue in h. hispanica , namely fabg1 , encodes a pha - specific acetoacetyl - coa reductase responsible for providing 3-hb - coa for polyhydroxyalkanoate biosynthesis in haloarcula species . the authors concluded that the polyhydroxyalkanoate biosynthesis pathway from ac - coa , catalyzed by -ketoacyl thiolase , acetoacetyl - coa reductase , and polyhydroxyalkanoate synthase , as distributed in bacteria , likely also exists in the domain of archaea . nonetheless , they pointed out that for phb - accumulating haloarchaeal natrialba strain 56 , no enzyme activity of acetoacetyl - coa reductase or -ketoacyl thiolase was detected in the crude extract , indicating that a different metabolic route toward production of phb might be employed . polyhydroxyalkanoate synthase , putative enoyl - coa hydratase , and two structural phasin - like proteins have been identified in haloarchaeal polyhydroxyalkanoate granule surfaces . the phasin - like proteins in haloarchaea share some structural features with bacterial phas , such as the presence of hydrophobic domains and a high -helix content . after a genome - wide investigation into the 12 haloarchaea that harbored the phap gene , most of these archaea were found to possess a similar pha cluster , with five genes , namely , maoc - gap12-phap - phae - phac , having the same organization as that in h. mediterranei . the extensive existence of this pha gene cluster was suggested as an indication of an evolutionarily conserved pha gene cluster unique to haloarchaea . a promising approach is to develop archaeal species as industrial scale polyhydroxyalkanoate producers . in particular , several halophilic archaea have the advantages of utilizing much cheaper carbon sources ( including waste materials ) , as well as having less strict sterilization requirements , plus easier and more efficient methods for polyhydroxyalkanoate extraction . microorganisms provide an exciting platform for the development of lipid technologies . in the course of evolution , they have developed elegant pathways to synthesize a wide array of lipids , providing a versatile and cost - effective approach for sourcing lipids to virtually all sectors of industry . the understanding of the biochemical and cellular mechanisms of lipid production , accumulation , and secretion will provide valuable insights on innovations to overcome the hurdles in microbial lipid utilization . ongoing studies using different omics approaches will provide a holistic view of flows and interactions between the different metabolic pathways involved in lipid accumulation . tools such as next - generation sequencing , transcriptome analysis , proteomics , and mass spectrometry are already clearing out the missing links in single - cell lipid biosynthesis . this information will permeate in the creation and scaleup of more efficient processes . it is thus important to project the potential applications and envision the frontiers to which this valuable toolset can lead the diverse fields of lipid technology . for example , in the field of biotherapeutics , microbial - based approaches have the flexibility to construct platforms to manufacture personalized lipid therapies starting as simply as lc pufa combinations , appropriate to the metabolic phenotype of each individual and eventually becoming as complex and selective as personalized cancer interventions . this approach of personal medicine will carry improved benefits in the treatment of a range of conditions : in immunological diseases from infections to autoimmunities ; in metabolic conditions from diabetes to cardiometabolic diseases ; and in microbiota dysbiosis from ibs to ibd . microbial technologies can also provide an alternative pathway for sourcing promising lipids and fatty acids that are unavailable in the market at present . for example , oil - producing microbes can be considered as appropriate vehicles in which foreign ( plant ) genes could be cloned for the production of commercially attractive fatty acids such as nervonic acid ( c24:1 15 ) obtained from honesty ( lunaria ) , which is used in small amounts in the treatment of particular neuropathies . another example is sterculic acid { -(2n - octylcycloprop-1-enyl)-octanoic acid } , which has been considered as a treatment for certain cancers of the bowel . one more promising example is represented by the non - methylene - interrupted fatty acids ( e.g. , c20:3 5 , 11 , 14 ) , which can be obtained from juniperus chinensis seed oil and is known to reduce the amount of arachidonic acid in certain phospholipid pools and , thus , act to alter eicosanoid signaling . in the field of energy and biofuels , understanding the mechanisms by which each group of microorganisms generates highly combustible lipids will foster the production of cost - effective fuels through sustainable processing and with improved performance . it is yet to be tested whether the biochemical mechanisms for amphipathic lipid secretion present in some microorganisms would work for the secretion of neutral lipids . this concept , if brought to practice , will significantly reduce the processing costs and render high - quality combustible lipids for the production of biofuels . in the case of the food industry , a microbial - based approach will allow food companies to expand their core businesses by creating new product portfolios out of their current byproducts . two promising examples are the production of cocoa butter analogues and the production of high - value oils out of spent agricultural materials . in the field of biomaterials , microbial lipids are already being used for producing biosurfactants and bioplastics , replacing synthetic analogues due to their improved biodegradability and reduced cost . products such as metabolix pha and biomer are examples on how microbial - derived plastics are gaining momentum in the market . their specific modes of action , low toxicity , relative ease of preparation , and widespread applicability are increasing their use in applications such as emulsifiers , wetting and foaming agents , functional food ingredients , detergents in petroleum , petrochemicals , environmental management , agrochemicals , cosmetics and pharmaceuticals , commercial laundry detergents , mining and metallurgical industries ( for an overview of microbial surfactant applications , see mukherjee ) . the scope of applications goes to such an extent that the petroleum extractive industry is already researching the use of microbial lipid - based biosurfactants for increasing oil recovery yields from subterranean depots . the great era of chemistry culminating in the restructuring of the human condition in the 20th century has been defined by the principles of reductionism and simplicity . in contrast , the 21st century heralds the era of complexity for which the inherent biological diversity and information content of microorganisms will be key to adding value to all industrial chains . a search performed on july 6 , 2013 , with the terms microbial lipids in the u.s . patent publication since 2008 , showing the increasing interest in the generation of intellectual property around this area of research . the estimated patents by the end of 2013 according to the current trend will be around 1420 , almost doubling the number registered in 2009 . the next step in microbial lipid technology is the integration of processes and applications to generate comprehensive , sustainable solutions . the immediate challenge is to combine desired metabolic pathways present in different species or strains to generate superior microbial species . benefits will include improved yields , targeted lipidomic profiles , and structural features to synthesize in a sustainable and cost - effective way the lipid structures necessary to satisfy the breadth of needs of 21st century industry . for example , an appropriate integration of microbial - based lipid technologies will allow delivery of smart biofuels and even personalized edible oils derived from microorganisms and reporter - equipped biodegradable containers also derived from microorganisms . currently the most used packaging material for commercial edible oils is polyethylene terephthalate , which is virtually a nondegradable plastic . with the appropriate use of microbial pha , it could be possible to see in the same facility the production of high - value microbial oils ( rich in a combination of lc pufa ) , along with the production of polyhydroxyalkanoate - based bottles for its commercialization . furthermore , additional microbial biotechnology platforms will be integrated to transform the byproducts either into biomass nutrients to make the process sustainable or even into another portfolio of high - value products , as a way to increase the business profitability ( figure 10 ) . conceptual flowchart for an integral , sustainable microbial - based edible oil process . microbial lipid technologies are a valuable toolset available for the future generation of scientists , which will help them push the boundaries of production solutions ultimately toward a more sustainable society .

in recent years attention has been focused on the utilization of microorganisms as alternatives for industrial and nutritional applications . considerable research has been devoted to techniques for growth , extraction , and purification of high - value lipids for their use as biofuels and biosurfactants as well as high - value metabolites for nutrition and health . these successes argue that the elucidation of the mechanisms underlying the microbial biosynthesis of such molecules , which are far from being completely understood , now will yield spectacular opportunities for industrial scale biomolecular production . there are important additional questions to be solved to optimize the processing strategies to take advantage of the assets of microbial lipids . the present review describes the current state of knowledge regarding lipid biosynthesis , accumulation , and transport mechanisms present in single - cell organisms , specifically yeasts , microalgae , bacteria , and archaea . similarities and differences in biochemical pathways and strategies of different microorganisms provide a diverse toolset to the expansion of biotechnologies for lipid production . this paper is intended to inspire a generation of lipid scientists to insights that will drive the biotechnologies of microbial production as uniquely enabling players of lipid biotherapeutics , biofuels , biomaterials , and other opportunity areas into the 21st century .

Introduction Accumulation of Lipids in Single-Cell Microorganisms: An Overview The Case of Yeast The Case of Microalgae The Case of Bacteria The Case of Archaea Future Perspectives

large elastic arteries in the central region and medium - sized muscular arteries have two functions , i.e. , they act as low resistance conduits and as flow pulsation buffers ( 1 ) . moreover , a reduction in buffering capacity may increase systolic blood pressure ( bp ) , left ventricular afterload , and pulsatile flow in capillary beds and reduce the diastolic contribution to blood flow in the coronary artery ( 2 ) . arterial stiffness is determined by the properties of the arterial wall matrix and by vascular smooth muscle tone , and may be changed immediately by an alteration in vascular smooth muscle tone caused by exercise ( 3 ) . exercise training - induced alterations in arterial stiffness would be of great benefit to those with coronary artery disease ( cad ) , and would potentially reduce myocardial oxygen demand and ischemic symptoms ( 4 ) . in the present study , we investigated the effect of short - duration exercise on arterial stiffness in patients with coronary artery disease , by repeatedly measuring brachial - ankle ( ba ) pulse wave velocity ( pwv ) ; an established non - invasive means of assessing arterial stiffness . fifty patients that underwent percutaneous coronary intervention ( cad group ) and 50 patients without a history of cardiovascular disease ( control group ) who were referred for treadmill testing by physicians mostly due to atypical chest pain , were prospectively enrolled . patients who were positive for myocardial ischemia on treadmill tests or those with comorbid conditions that limited exercise were excluded to ensure adequate exercise duration . thus , patients with residual ischemia after pci was excluded from cad group and patients with overt clinical coronary artery disease was excluded from control group . brachial - ankle pwv was measured using an automatic pwv measurement system ( form - pwv / abi , colin , komaki , japan ) in both brachia and ankles before treadmill exercise testing . this instrument simultaneously records bapwv , and brachial and ankle blood pressures on left and right sides , and provides an electrocardiogram and heart sounds . after baseline measurements , each subject performed symptom limited treadmill exercise testing according to the bruce protocol . at 10 min after the completion of exercise , bapwv was remeasured . heart rate and blood pressure ( bp ) were continuously monitored during exercise . for the analysis , statistical analysis was performed using sas ( sas system for windows 9.00 , cary , nc , u.s.a . ) . the chi - square test and the unpaired t - test the paired t - test was used to compare pre- and post - exercise results , and the independent t - test was used to compare the effect of exercise in both groups . multiple linear regression analysis was used to evaluate associations between bapwv changes and independent variables , and stepwise regression was used to select independent variables . fifty patients that underwent percutaneous coronary intervention ( cad group ) and 50 patients without a history of cardiovascular disease ( control group ) who were referred for treadmill testing by physicians mostly due to atypical chest pain , were prospectively enrolled . patients who were positive for myocardial ischemia on treadmill tests or those with comorbid conditions that limited exercise were excluded to ensure adequate exercise duration . thus , patients with residual ischemia after pci was excluded from cad group and patients with overt clinical coronary artery disease was excluded from control group . brachial - ankle pwv was measured using an automatic pwv measurement system ( form - pwv / abi , colin , komaki , japan ) in both brachia and ankles before treadmill exercise testing . this instrument simultaneously records bapwv , and brachial and ankle blood pressures on left and right sides , and provides an electrocardiogram and heart sounds . after baseline measurements , each subject performed symptom limited treadmill exercise testing according to the bruce protocol . at 10 min after the completion of exercise , bapwv was remeasured . heart rate and blood pressure ( bp ) were continuously monitored during exercise . for the analysis , statistical analysis was performed using sas ( sas system for windows 9.00 , cary , nc , u.s.a . ) . the chi - square test and the unpaired t - test the paired t - test was used to compare pre- and post - exercise results , and the independent t - test was used to compare the effect of exercise in both groups . multiple linear regression analysis was used to evaluate associations between bapwv changes and independent variables , and stepwise regression was used to select independent variables . the clinical characteristics and laboratory findings of the study subjects are shown in table 1 . mean age was higher in the cad group , and the cad group contained more male patients and hypertensive patients than the control group . the cad group had a lower mean left ventricular ejection fraction , a lower mean ldl cholesterol , a higher bapwv , and a shorter treadmill exercise duration than the control group . the patients with cad took more medicines , such as aspirin , -blockers , renin - angiotensin system inhibitors , and hmg - coa reductase inhibitors . baseline bapwv values were found to correlate significantly with age , systolic bp ( sbp ) , mean arterial pressure ( map ) , and diastolic bp ( dbp ) ( table 2 ) . brachial - ankle pwv values were significantly lower at 10 min after exercise than at baseline in both groups . however , this decrease was significantly larger in the cad group , thus bapwv in the cad group was initially higher than in the control group but became similar after exercise . in the control group , sbp and map were significantly lower at 10 min after exercise than at baseline , but dbp was not . in the cad group , map was significantly lower at 10 min after exercise than at baseline ( table 3 ) , whereas sbp was marginally lower , and dbp was not significantly different . heart rates were higher at 10 min after exercise than at baseline in both groups ( table 3 ) . by multivariate analysis , the cad group showed a larger decrease in bapwv after exercise than the control group after adjusting for age , bmi , sbp , map , map reduction , and baseline bapwv ( table 4 ) . arterial stiffness increases left ventricular afterload and alters coronary perfusion ( 5 ) , and has been independently associated with target organ damage and increased cardiovascular morbidity and mortality ( 6 ) . brachial - ankle pwv is a simple marker of arterial stiffness ( 7 , 8) and mainly reflects large artery stiffness , although it has also been reported to reflect endothelium - dependent peripheral vasodilation . changes in vascular wall distensibility may be induced by changes in the quality and quantity of vascular fibrous matrix ( e.g. , elastic fibers and collagen fibers in media : an organic factor ) and by changes in smooth muscle tone ( a functional factor ) . elastic fiber is the primary determinant of vascular distensibility under physiologic conditions ( 9 , 10 ) . moreover , the elastin - collagen compositions of arterial walls represent a more chronic component of arterial stiffness and changes only over years , thus it is unlikely that short - duration aerobic exercise changes these structural components ( 11 ) . instead , arterial compliance is probably altered in the short terms , or even acutely , via the modulation of the sympathetic - adrenergic tone of smooth muscle cells in arterial walls ( 12 ) , which is affected by autonomic nervous activity and vasoactive agents derived from vascular endothelial cells , e.g. , nitric oxide ( no ) , prostacyclin , and endothelium - derived hyperpolarizing factor ( 13 ) . in particular , the production of no is important , because it is a potent endothelium - dependent vasodilator and reduces vasoconstrictor response to -adrenergic receptor stimulation ( 14 ) . moreover , pulsatile flow in the aorta associated with exercise training might evoke the acute release of no , upregulate no production , and increase the productions of other vasodilatory factors ( 15 - 17 ) . in cad patients , endothelial dysfunction develops secondary to reduced no production and early reactivation by reactive oxygen species ( 18 ) . in the present study , bapwv , which was higher in the cad group at baseline , was found to be reduced significantly at 10 min after exercise in both groups , and because this decrease was larger in the cad group , no difference in bapwv was observed between the two groups after exercise . these observations suggest that short - duration exercise affects arterial stiffness even in patients with cad . we speculate that the mechanism involved may be related to the restoration of an equilibrium between no production and inactivation by reactive oxygen species , which also appears to be the primary mechanism underlying exercise training - mediated perfusion improvements in cad patients ( 18 ) . bp and age have been reported to be important determinants of bapwv in healthy individuals ( 7 , 19 ) , which is consistent with the findings of the present study . exercise duration is significantly different between the two groups which might be a confounding factor . however , exercise duration is longer and the proportional change of bapwv is smaller in control group , and moreover exercise duration is not correlated to both absolute and relative change of bapwv ( data not shown ) . thus it is not probable that different exercise duration is a significant confounding factor . because , as mentioned above , cad group members were taking more medications , it is unclear whether our findings suggest that patients with stable cad have the potential to reverse arterial stiffness with exercise despite the presence of disease or whether they reflect an effect of the medications taken , such as aspirin , -blocker , renin - angiotensin system inhibitor , and hmg - coa reductase inhibitor . the lower level of ldl - cholesterol in the cad group was probably due to hmg - coa reductase inhibitors . however , considering the higher baseline bapwv value in the cad group , it appears that medications do not completely normalize arterial stiffness in cad patients and that short - duration exercise seems to independently improve arterial stiffness immediately . because majority of patients in cad group and only a small number of persons in control group was taking one or more of the drugs which influence endothelial functions , statistical adjustment is not feasible with our data . complete exclusion of the effects of medication probably can only be performed by experimental design in which normal control group is given the same medications . still , it is rather puzzling that cad patients showed more prominent reduction in bapwv than control group and resulted in similar level of bapwv after exercise . this apparent ' reversibility ' of arterial stiffness might be related to the duration and extent of atherosclerosis . as discussed above we speculate that those in relatively early course of cad may have more reversible component related to endothelial dysfunction and less irreversible component such as structural change of vascular wall . this group of patients has higher baseline bapwv due to the endothelial dysfunction but much of this functional abnormality might be reversible by some intervention , such as short - duration exercise in this study . patients in the cad group had undergone percutaneous coronary intervention and those with findings of residual ischemia were excluded by exercise test . this exclusion may have resulted in selection of patients with lower risk and less extensive coronary artery disease . however , as a limitation of this study , we do not have enough information on the duration and extent of cad in the patient group . also , it is not possible to investigate this speculation further by measuring biomarkers of no production and oxidative stress . if markers of central blood pressure ( 20 , 21 ) had been measured , different findings might have been found . in previous studies , aerobic exercise improved peripheral arterial stiffness but not central arterial stiffness ( 22 ) , and central arterial stiffness was a better prognostic factor than peripheral arterial stiffness ( 23 ) . however , this does not mean that peripheral arterial stiffness is meaningless , which has been shown to have prognostic value in another study ( 24 ) , and also shown to be correlated to central arterial stiffness ( 8) . it is likely that central arterial stiffness is a better index but peripheral arterial stiffness measured by bapwv is a very convenient alternative . another weakness of the study is that we do not have data on the heart rate and blood pressure at the time of pwv measurement . because these variables acutely influence bapwv , this can be a potential source of confounding . however , we assume that heart rate and blood pressure was probably not different from the baseline at 10 min post - exercise . though our study showed immediate short - term response to exercise , evidence is scarce on whether repeated short - duration exercise may result in a persistent and long - term improvement of arterial stiffness . further study is needed on this question , considering that frequent short - bout exercise is being discussed as a practical alternative to conventional long - duration exercise ( 25 , 26 ) . in conclusion , in the present study , we prospectively investigated the effects of short - duration exercise on arterial stiffness by measuring bapwv in patients with or without cad . a significant reduction in bapwv was observed at 10 min after short - term aerobic exercise in both groups , and in the cad group this decrease was more prominent . these observations suggest that short - duration exercise training may be effective at improving arterial stiffness even in patients with coronary artery disease , at least in short - term . clinical study is needed to see whether repeated short - duration exercise will improve arterial stiffness in cad patients in long - term .

arterial stiffness is an important contributor to the development of cardiovascular disease . we investigated the effect of short duration exercise using the treadmill test on arterial stiffness in the presence of coronary artery disease . we enrolled patients with and without coronary artery diseases ( cad and control group , 50 patients each ) referred for treadmill testing . brachial - ankle pulse wave velocity ( bapwv ) were measured before and after treadmill testing . values of bapwv were significantly reduced at 10 min after exercise in both groups , more in the cad group than in the control group ( baseline bapwv and post - exercise change [ cm / sec ] : 1,527245 and -132155 in the cad group , 1,439202 and -7793 in the control group , respectively , p for change in each group < 0.001 , p for difference in changes between the two groups < 0.001 ) . these findings persisted after adjusting for age , body mass index , systolic blood pressure , mean arterial pressure ( map ) , map decreases , and baseline bapwv . significant post - exercise bapwv reductions were observed in both groups , and more prominently in the cad group . this finding suggests that short - duration exercise may effectively improve arterial stiffness even in patients with stable coronary artery disease .

INTRODUCTION MATERIALS AND METHODS Subjects Measurements Statistics RESULTS DISCUSSION

with cataract surgery regarded as the most widely performed surgical procedure , a demand exists for continued innovation and technology . the latest advances evolved through application of well - defined principles to current surgical goals and patient expectations . for example , femtosecond laser technology emerged after fifty years of employing laser technology in ophthalmology . theodor scheimpflug described the principle of scheimpflug images in 1904 , but he was actually an austrian army captain who spent his life 's work dedicated to designing methods and tools to create maps depicting aerial photography . application of these principles to ophthalmology in the last few years has advanced our understanding of corneal biomechanics . the latest highlights in technology include advances in preoperative and intraoperative diagnostics , femtosecond laser - assisted cataract surgery ( flacs ) , and a new generation of intraocular lenses ( iols ) . more than ever , patients have the desire to reduce their dependence on spectacles after cataract surgery . physicians now have access to advanced diagnostics that can better quantify conditions such as dry eye , light scatter , and posterior corneal astigmatism . mcdonald recently reported that the postoperative prevalence of dry eye related symptoms is approximately 88% . analysis and optimization of dry eye preoperatively and postoperatively has a beneficial impact on visual outcomes after cataract surgery . therefore , increased interest among ophthalmologists to utilize objective measurements to assess the ocular surface exists . the acutarget hd ( visiometrics sl , spain ) assesses the objective scatter index , which can objectively evaluate dry eye disease severity using the degradation of image quality over time ( figure 1 ) . the keratograph ( oculus , germany ) noninvasively measures tear break up time , tear meniscus height , and meibography , providing a functional and qualitative analysis of the corneal surface and tear film [ 8 , 9 ] . the tearlab osmolarity system ( tearlab corporation , san diego , california ) uses a small tear sample to measure tear osmolarity using a microelectrode . compared to other commonly used diagnostic tests for dry eye disease , test results were better at predicting dry eye severity . the lipiflow ( tearscience , morrisville , north carolina ) combines heat and eyelid pressure to treat dry eye disease due to meibomian gland dysfunction . recent studies showed consistent improvement in meibomian gland function up to 12 months after the treatment . evaluation of optical quality also aids in decision making between corneal or lens - based procedures . the c - quant ( oculus , germany , optikgenrate gmbh ) assesses straylight subjectively by utilizing a compensation comparison method . the acutarget hd ( visiometrics sl , spain ) uses a double pass system to measure point spread function ( psf ) , modulation transfer function ( mtf ) , strehl ratio , and intraocular scattering of the light . these data allow clinicians to evaluate the quality of a patient 's optical system objectively . another objective functional diagnostic is the salzburg reading desk ( srd vision , vienna , austria ) , which allows measurement of the variable read print sizes and distances with differences in contrast sensitivity and luminance . both anterior and posterior corneal astigmatism should be taken into account in iol planning , particularly in patients desiring astigmatic correction . inaccuracies arise when posterior corneal astigmatism is measured based on the assumption of a fixed - ratio relationship with the anterior curvature . the cassini corneal shape analyzer ( i - optics bv , the hague , the netherlands ) is a new topographer that uses led ray tracing technology with 700 diode lights to measure anterior and posterior corneal astigmatism . these advances in cylinder and axis measurement precision can be useful for preoperative planning of toric iol implants and in postrefractive surgery patients [ 1517 ] . patients with a history of corneal refractive surgery expect reduced dependence on spectacles after cataract surgery . the optiwave refractive analysis ( ora , alcon , fort worth , tx ) uses wavefront interferometry to produce a fringe pattern , and distortions in this pattern are translated into refractive values and aphakic and pseudophakic readings ( figure 2 ) . studied mean postoperative residual refractive astigmatism in patients receiving toric iols with power selection aided by intraoperative aberrometry . surgeons altered cylindrical power 24% of the time and spherical power 35% of the time . patients were 2.4 times more likely to have less than 0.50 d of residual refractive astigmatism when intraoperative aberrometry was used . in contrast , huelle et al . published a study where aphakic spherical equivalent- ( se- ) based iol formulas were generated from repeated intraoperative wavefront aphakic measurements of se . the agreement of repeated aphakic se readings ranged from 0.69 diopters to + 0.66 diopters . the authors concluded that measurement precision is limiting reliability of intraoperative aberrometry and application to routine cataract surgery . however , it may be useful in guiding limbal relaxing incision enhancements and has resulted in the need for fewer subsequent laser enhancements . this technology is particularly useful in postrefractive patients and those with astigmatism uncertainty or other corneal pathology . other intraoperative inconsistencies include cyclotorsion , variable anterior chamber depth and intraocular pressure , variability in wound hydration , and use of viscoelastic device versus balanced salt solution . although limitations may exist in quality and measurement precision , the future of this technology is promising . the use of the electroretinogram ( erg ) has been well described and may have a novel application for refractive cataract surgery . richard mackool described the use of flash erg testing with office - based electroretinography ( diopsys , pine brook , new jersey ) in preoperative cataract evaluation . it can provide an objective evaluation of macular function and could be useful in influencing lens selection for patients with conditions such as epiretinal membrane , diabetic retinopathy , and age - related macular degeneration . more studies evaluating ergs in preoperative cataract assessment need to be done to further assess its value and implications . noninferiority has been established relative to manual cataract surgery , and some reports have suggested superiority relative to manual methods . potential advantages include customized corneal incisions and capsulotomy position , precision in shape and size of capsulotomy , custom lens fragmentation patterns , endothelial cell loss reduction , and better refractive stability and predictability . after the food and drug administration ( fda ) approval of laser - assisted capsulotomy and lens fragmentation in 2010 , five platforms have been released : lensx by alcon ( fort worth , tx ) ; the lensar by lensar ( orlando , fl ) ; the catalys by abbott / optimedica ( north chicago , il ) ; the victus by bausch and lomb ( rochester , ny ) ; and the ldv z8 by ziemer ( port , switzerland ) ( figure 3 ) . the docking process using the femtosecond laser - eye interface uses a suction ring to stabilize the eye , thereby allowing imaging and laser delivery through a clear optical pathway . considerations for docking include complete coupling , patient comfort , intraocular pressure elevation , and minimal distortion of anatomy to avoid disruption of the beam path . in a study using alcon 's lensx platform to compare curved direct contact and modified soft interfaces ( softfit by alcon ) , mayer et al . showed that redocking was unnecessary when a modified soft interface was used , even though some cases resulted in incomplete incisions requiring manual opening . schultz et al . found significantly fewer intraocular pressure elevations after docking using a liquid interface ( liquid optics interface , catalys precision laser system ) in comparison to flat and curved interfaces . while docking is a necessary step with femtosecond laser technology , laser incisions are optional in flacs . comparatively analyzed femtosecond laser incisions and manual incisions and cited better tunnel morphology with flacs incisions . flacs theoretically decreases endothelial cell loss relative to manual techniques by reducing the use of ultrasound energy . however , krarup et al . compared endothelial cell loss rates between phacoemulsification and flacs and showed there were no differences between both modalities . published similar findings but did cite a difference in favor of flacs that was limited to the early postoperative period . they also showed that laser corneal incisions themselves may influence endothelial cells , as there may be a disturbance in the postoperative inflammatory response after laser application [ 3032 ] . new surgical techniques , in combination with more advanced lens fragmentation patterns , will allow the lens to be extracted through an aspiration mechanism that may reduce endothelial cell loss . the size , shape , and position of a capsulotomy should theoretically lead to a more predictable lens position by enhancing uniform capsule - optic overlap , thereby reducing the incidence of lens tilt and leading to an overall better effective lens position and visual outcome ( figure 4 ) . recently , toto and colleagues found no difference in prediction error when comparing traditional phacoemulsification with flacs but did find higher refractive stability and iol centration with flacs . this similarity in prediction error may be a consequence of unexplored potential with iol calculations and algorithms . ma approached the prediction of true lens position using an algorithm based on oct anterior segment 3-d reconstruction . this prediction model could have great potential once there is consistent alliance of oct measurements with flacs to provide more precise outcomes . this is particularly relevant with premium iols , as there is a lower tolerance threshold for minor unanticipated miscalculation and decentration . okulix ( tedics peric & joher gbr , dortmund , germany ) is an innovative software program that calculates iol power using ray tracing combined with corneal topography . evaluated its accuracy in post - lasik eyes in comparison with camellin - calossi , shamas - pl , haigis - l formulas and double - k srk - t method . they reported that this technology provides sufficient predictability outcomes in postrefractive myopic lasik , even though a small hyperopic shift tendency was noted in the study . g6 lens professional ( ziemer , port , switzerland ) is an optical biometer that integrates placido rings with a dual rotating scheimpflug camera as well as an optical coherence tomography based a - scan in a single device . shin et al . compared its accuracy with the lenstar ls 900 ( haag - streit , koeniz , switzerland ) , for intraocular lens ( iol ) power calculation . they noted that axial length , lens thickness ( lt ) , and white - to - white ( wtw ) values were statistically different . thus , even though high repeatability was present , and the iol powers were not statistically different between the two devices , the values provided by the galilei g6 were not interchangeable with the lenstar in the clinical setting . the goal of appropriate iol selection is to provide the best visual outcome that meets a patient 's individualized goals and expectations . variability in materials , optical properties , and designs are important factors to consider in the patient - specific selection of an iol . advancements in iol technology aim to improve visual functionality by creating customized iols or modifying optical power postoperatively . the concept of adjustable iols involves the correction of residual refractive error postoperatively or customization after lens implantation . this new paradigm in iol manufacturing may be subdivided into two major categories : a modular multicomponent category requiring a separate intraocular procedure and another category where the optic is adjusted postoperatively with a secondary device . the first category includes multicomponent iols ( clarvista harmoni modular iol system , clarvista medical , aliso viejo , ca ; and omega lens , omega ophthalmics , lexington , ky ) ; infinite vision iol ( infinite vision optics , france ) ; and mechanically adjustable iols ( acritec ar-1 pc / iol , acri.tec , hennigsdorf , germany ) . the second category includes magnetically adjustable iols ( university of missouri - rolla , rolla , and eggleston adjustable lens , st . louis , mo ) , light adjustable iols ( calhoun vision , pasadena , ca ) , and the perfect lens ( perfect lens , llc , irvine , ca ) . the latter is a novel platform , which can be adjusted with the femtosecond laser based on the concept of refractive index shaping . its mechanism involves the use of infrared light to polymerize photosensitive silicone macromers , which results in changes in lens morphology and optical properties . a new generation of optics with extended depth of focus , multifocal rotational symmetry and asymmetry , and accommodating capabilities offers promising strategies to advance functional vision in refractive cataract surgery . the tecnis symfony ( amo ) is an extended depth of focus iol that works by correcting chromatic aberration using diffractive optics and reduces glare and halo symptoms classically associated with conventional multifocal iols . multifocal lenses with bifocal and trifocal designs include the rotationally asymmetric lentis mplus ( topcon europe medical bv , the netherlands ) and the rotationally symmetric finevision ( physiol , liege , belgium ) and at lisa ( zeiss , oberkochen , germany ) . found that trifocal lenses provide satisfactory intermediate vision without compromising near and far distance visual acuity . the fluidvision iol ( powervision , belmont , ca ) has silicone oil inside the lens that moves in response to ciliary contraction forces . an electroactive iol with a liquid crystal that is sensitive to electric current is also in development ( sapphire autofocal iol , elenza , roanoke , va ) . dual accommodating iols designed for sulcus placement include the dynacurve iol ( nulens , israel ) and the lumina iol ( akkolens , the netherlands ) . the injectable polymer smartiol ( medennium , irvine , ca ) , which is a thermodynamic , pliable capsule - filling iol , is the only bag filling technology in development . the future of refractive cataract surgery is exciting ; in time , these new technologies may be the standard of care . with refinements of the latest technology , flacs and other parallel advances will provide surgeons with the potential to perform an even safer , predictable , and effective surgery .

technology in cataract surgery is constantly evolving to meet the goals of both surgeons and patients . recent major advances in refractive cataract surgery include innovations in preoperative and intraoperative diagnostics , femtosecond laser - assisted cataract surgery ( flacs ) , and a new generation of intraocular lenses ( iols ) . this paper presents the latest technologies in each of these major categories and discusses how these contributions serve to improve cataract surgery outcomes in a safe , effective , and predictable manner .

1. Introduction 2. Preoperative and Intraoperative Diagnostics 3. Femtosecond Laser-Assisted Cataract Surgery 4. Intraocular Lenses

epilepsy is the most common serious neurological disorder of young people affecting nearly 3.4 million individuals in europe . societal costs are considerable as individuals with medically intractable seizures make up a third of the epilepsy population . more than eu15 billion is spent annually on the treatment of epilepsy in europe , a financial burden comparable to that of lung and breast cancer combined [ 2 , 3 ] . a recent irish prevalence study estimated that up to 40,000 children and adults in ireland have the disorder , which gives a point prevalence of about 0.9% ; in line with other industrialized nations . studies in the last decade have shown that the majority of patients with epilepsy will be urgently admitted to secondary and tertiary care institutions ( mostly through the ed ) at some point in the history of their illness and a significant proportion will require multiple visits . furthermore , symptomatic seizures , secondary to acute medical or surgical illness , alcohol and drug intoxication , brain trauma and stroke , add to the burden of seizure pathology in the ed . finally , a range of mimic disorders from psychogenic nonepileptic seizures ( pnes ) to blackouts caused by impaired vascular responsiveness contribute to the diagnostic and therapeutic challenges . despite the heavy burden of seizures in the ed , international studies suggest that the majority of patients are referred unnecessarily for admission and that the acute treatment of seizures is often ineffective indicating that seizure admissions are a cause of unnecessary medical intervention , delayed diagnosis , and prolonged length of stay [ 5 , 6 ] . beginning in 2006 we first sought to determine the impact of emergency seizure admissions on the resources of a large irish teaching hospital . on the basis of two baseline audits we identified a number of areas where we could improve the quality of service to patients with seizures by the employment of an evidence - based seizure care pathway in the emergency department ( ed ) and acute medical admissions unit ( amau ) . this is a report of preliminary quality and safety metrics accrued by this intervention over a 12-month period ( november 2008 - 2009 ) . the study consisted of three parts : retrospective audit of admissions with seizures through the emergency department in 2004 . measurement of quality and safety metrics after the implementation of an evidence - based seizure care pathway from november 2008 to november 2009 . the first study group was restricted to in - patients discharged in 2004 with a diagnosis of a seizure or convulsion . these patients were identified through the hospital in - patient enquiry ( hipe ) system ( a national coding system for hospital discharges in ireland ) , which identified 341 patients with epilepsy or seizure as the primary reason for admission . particular attention was paid to investigations , diagnosis , specialist referral , and follow - up . the charts were also examined for what we designated as necessity of admission . an admission was deemed medically necessary if there was a history of prolonged ( > 5 minutes ) or clustered ( > 2 ) events , a history of status epilepticus ( > 30 minutes of seizure activity ) , or an abnormal neurological exam 90 minutes after arrival to the ed . hipe system was used to determine if any of these patients was readmitted or died over a one - year period following admission . the second study group represented all patients who attended the ed with an event that was deemed likely to have been a seizure or its aftermath over one calendar month in 2006 . patients who presented with a complaint of seizure , weakness , confusion , head injury , dizziness , and collapse of unknown cause were examined for possible inclusion in the study . in a similar vein to part 1 of the study , attention was paid to investigations , diagnosis , referral , and follow - up in all patients . after the baseline audits , a seizure care pathway was designed and implemented by the neurology service with the cooperation of the ed staff . the pathway required early rapid access ambulatory follow - up for patients fit for discharge from the ed . a rapid access clinic ( rac ) was established and run by an existing epilepsy nurse specialist ( ens ) . in addition to patients discharged from wards and the ed , this clinic also reviewed new referrals from gps and patients with an established diagnosis of epilepsy who had exacerbations of their illness , all of which were designed to avoid ed referrals . education sessions were provided by the ens who also provided a phone help - line and e - mail service to facilitate follow - up care . patients presenting to the ed or admitted to the amau overnight with seizures were seen by the seizure service fellow . patients were managed according to a locally designed , evidence - based , seizure care pathway ( figure 1 ) . the pathway clearly laid out the criteria for : admission , discharge and follow - up . the pathway was designed with reference to published international guidelines of care ( scottish intercollegiate guideline network ( sign ) 2003 ( 8) and national institute of clinical excellence ( nice ) 2005 ( 9 ) ) . all patients seen by the seizure service were provided with printed cards with full details for phone and e - mail contact . length of stay and time to ct , mri and eeg were analysed using kruskal wallis test for nonparametric data . the rate of representation was analysed using pearson chi - square test . times to follow - up the reduction in admission rates was analysed using chi - square test with yates correction . during 2004 , hipe data identified 341 admissions with a specific diagnosis of epilepsy or seizure out of a total of 11,721 admissions from all causes through the ed . in the 50 charts randomly selected out of this group , 34% of patients had a previously documented diagnosis of epilepsy at presentation . investigations performed included ct brain ( 84% ) , mri brain ( 28% ) , and eeg ( 56% ) . median delay to ct , mri , and eeg were 2 days , 5 days , and 5 days , respectively . ambulatory follow - up was evenly divided between neurology ( 28% ) , general medicine ( 28% ) , and general practitioner ( 20% ) , with a further 24% having no follow - up whatsoever . 23/50 patients ( 46% ) represented to the emergency department with further seizures over the next 12 months . using our criteria for necessity of admission , we concluded that 36% could have been discharged earlier or from the ed , had appropriate investigations and neurological opinion been available in a timely manner . of the 341 patients with a diagnosis of epilepsy , 10 died over the subsequent year but only one of those who presented with status epilepticus died as a direct result of their epilepsy . 20 ( 19% ) of the 102 patients included in the study had a previously established diagnosis of epilepsy . of the special investigations required for epilepsy , ct brain was the only one conducted on the day of admission and in only 5.8% of cases . neither mri nor eeg 's were performed on any patient on the day of presentation . ultimately of the 34 admitted , 14 ( 41% ) had an eeg , 21 ( 61% ) patients had a ct brain , and 4 ( 12% ) had an mri brain . median delay for eeg was 2 days ; ct brain was 1 day , and mri was 2.5 days . no data was collected on mortality or follow - up as it was designed primarily to gather data on patients presenting to the ed . during 2009 , there were 276 admissions with a primary diagnosis of epilepsy out of 12 , 607 admissions from all causes through the ed . 350 patients who presented to ed between november 2008 and november 2009 with seizures and other forms of collapse were referred to the seizure team for assessment and had the seizure care pathway applied . 97 patients had an established history of either generalised or focal epilepsy and 72 patients had epilepsy associated with significant medical and surgical comorbidities . 34 patients were referred with undefined collapse , 12 patients were referred with confusion , and 4 patients with myoclonic jerks . of the 181 eegs requested during the intervention study period , 99 ( 55% ) were done on the same day . 66 ( 36% ) were done within 1 to 3 days , and 16 ( 9% ) were done as outpatients within 4 weeks . 150 patients had ct brain requested and 140 ( 93% ) were performed on the same day and median delay for ct brain was again zero days . in 2008 - 2009 , 68 ( 19% ) of the total cohort of 350 , had mr imaging of the brain requested . same day mr brain acquisition however went up from 0% in 2004 to 7.2% in 2008 - 2009 and another 8.8% cases were done within 1 to 3 days . of the 57 patients with nonepileptic collapse , 12 had an eeg , 18 patients had ct brain and only one patient had an mri brain performed . 110 patients ( 31.4% ) were seen in the rapid access clinic ( rac ) . 64 patients ( 18% ) were seen in the subspecialty epilepsy clinic and median follow - up time for this more stable group was 8 weeks . 6 patients ( 1.71% ) were followed up in other hospitals 31 ( 8.9% ) of the total study group were readmitted in the 12-month follow - up period . of the patients seen during the study period , 19 ( 5.4% ) the neurological causes of death were herpes encephalitis , obstructive hydrocephalus , nonconvulsive status epilepticus and subdural haematoma . three patients died of direct seizure - related causes , two with nonconvulsive status epilepticus , and one with convulsive status epilepticus . during the intervention study period no patient who was discharged from the ed or within 2 days of admission died . the number of admissions with epilepsy or seizure dropped significantly from 341 out of 11,721 ( 2.9% ) in 2004 to 276 out of 12 , 607 ( 2.2% ) in 2009 ( p = 0.0006 ) . there was a significant reduction in median length of stay between the first 2 audits and the intervention study ( p < 0.001 ) . figure 2 summarises the median length of stay over the 3 studies there was a significant improvement in time to diagnostic investigations such as ct brain , mri brain , and electroencephalography between the first two audits and the intervention study ( p 0.001 , p 0.048 , p 0.001 ) . figure 3 summarizes the median delay to investigations on admitted patients across the three studies . there was a significant reduction in follow - up times from a median of 16 weeks to 5 weeks ( p < 0.001 ) . figure 4 shows median times to follow - up in the baseline audit in 2004 and in the intervention study in 2008 - 2009 . there was a significant reduction in readmission rates from 45.1% to 8.9% ( p 0.001 ) . figure 5 shows the change in readmission rates between 2004 and the intervention study in 2008 - 2009 the use of care pathways in modern healthcare delivery has been somewhat controversial since the expected gains are not always forthcoming . for instance a cochrane review of the implementation of a care pathway in stroke rehabilitation did not endorse any benefit to patient care . nevertheless , given the highly variable care delivered in the ed in relation to seizure care , we felt that a care pathway could provide much needed improvement . the aim of this study was to demonstrate improvements in for patients presenting to the ed with seizures and related disorders , without compromising safety by the use of an evidence - based seizure care pathway . the main quality indicators measured were requirement for admission , median length of stay , time to diagnostic tests , specialist follow - up , readmission rates , and mortality . the main findings of the study are that through the utilization of the seizure care pathway the ed and amau can reduce unnecessary admissions and safely discharge patients for early follow - up , which has a very significant impact on reducing representation rates . timely decision support has the effect of significantly reducing time to diagnostic tests , particularly eeg , and thus reducing median length of stay by up to 3 days . all of these outcomes were significant statistically and support the use of care pathways for patients presenting to the ed with seizures without any increase in mortality . it has been suggested that admission of seizure patients is only warranted in patients who are at high risk of further events , remain drowsy or comatose following a period in the ed , or in whom the neurological exam reveals signs indicative of an underlying lesion or treatable infective cause . however , international studies suggest that the majority of patients are referred unnecessarily to the in - house medical or neurological services for admission [ 5 , 6 , 9 , 10 ] . in our original retrospective audit ( part 1 ) , using a set of criteria based on the above indications for admission , we determined that 36% could have avoided admission . the intervention study showed the stability of this figure with 31% of patients actually being discharged from the ed and a further 8.5% within 24 hours of admission . comparison of hipe data between 2004 with that of 2009 following implementation of the seizure care pathway shows a reduction in the number of admissions with a specific diagnosis of epilepsy from 2.9% ( 341 ) of total hospital admissions to 2.2% ( 276 ) . this is despite an increase in overall admission rates from ed of 7.56% from 11,721 in 2004 to 12,607 in 2009 . had admissions continued at the rate of 2.9% with no seizure care pathway in place , it would have resulted in 365 epilepsy - related admissions in 2009 , suggesting that 89 epilepsy specific admissions were avoided due to implementation of the seizure care pathway in 2008 to 2009 . if we consider the median los to be 4 days ( without the seizure care pathway being applied ) this would have resulted in 356 bed days saved . combining this figure with the 478 bed days saved by an overall median reduction in los of 2 days , a total of 834 is the projected bed days saved in one 12-month period as a direct result of the implementation of the seizure care pathway . the reduction in median length of stay from 4 days in 2004 and 5 days in 2006 to 2 days during the intervention study in 2008 - 2009 was made possible by an emphasis upon early safe discharge in the pathway with an eye to reduce bed occupancy days . establishment of a separate rapid access follow - up clinic made routine and even unscheduled early follow - up possible , which increased the safety and ease of early discharge . reductions in length of stay secondary to implementation of care pathways have been reported in other areas of heath care , but not in the case of patients with seizures [ 1113 ] . of the discharged patients , the readmission rates show a significant drop from 47% to 8.9% between 2004 and 2008 - 2009 . it is possible that some unobserved bias meant that patients more likely to return were seen in the 2004 audit . it may be due to a combination of more timely and effective inpatient management including the delivery of inpatient ens education , the provision of phone and e - mail advice services , the use of rapid access to ambulatory clinics for exacerbations of existing epilepsy , and improved , timely communication with primary care teams . the reduction of median follow - up time from 16 weeks during 2004 to 5 weeks in 2008 - 09 may also have helped in reducing re - admissions to the ed . in relation to the overall safety of the seizure service with its emphasis on reducing admissions and length of stay , we found that only three of the 19 deaths in the study group were directly attributable to epilepsy . in 2004 , only one death was attributable to epilepsy . while there is a slight increase in epilepsy - related deaths , there was no excess mortality in any patients discharged from the ed or within the 2-day median length of stay window . accurate diagnosis and classification of seizure type are essential to the provision of quality patient care and good control . eeg is described as an important aid in the evaluation of seizure patients . in relation to the baseline audit the median waiting time to eeg was 5 days , demonstrating that most patients were not being tested soon after the seizure period . such waiting times observed in the baseline audits suggested that eeg contributed significantly to the length of stay of individual patients . during the intervention study the involvement of early specialist opinion allowed for the streamlining of those who required eeg which occurred on the day of admission in approximately 54% of the study group and in total over 90% had the test done in 3 days or less . neuroimaging is essential to identify structural lesions , which may result in the development of a seizure disorder . despite strong consensus within the literature for performing mri over ct especially in focal seizures [ 5 , 15 , 16 ] ct was the preferred modality for neuroimaging within all our study groups . this is largely to do with the ease of access to ct over mri at our institution , which has not changed significantly since 2004 . 84% of the retrospective sample had a ct performed , whilst only 28% had an mri . in the intervention study only 42% of patients had a ct requested reflecting the fact that in a number of cases of either established epilepsy or indeed eeg proven primary generalized epilepsy , the pathway was able to obviate the need for ct . an improvement in time to brain imaging was demonstrated in the intervention study with 93% of patients having their ct on the same day of request . unusually , the mean wait time for mri brain increased in 2009 compared to 2004 . this , we believe , was due to the steadily increasing demand for mri brain in acute presentations of seizure over those years coupled with limited availability of our mri resource ( one magnet open only during office hours ) . this has been ameliorated lately by the addition of a second scanner . as outlined by the sign and nice guidelines , adults with epilepsy should have a specialist expert opinion in an ambulatory setting including regular structured annual review . in the retrospective 2004 audit , mean time to neurology clinic follow - up was the lack of decision support and or expert neurological opinion to two - thirds of the 2004 study group may have contributed to the longer length of stay in this group as there may have been a delay in diagnosis and pursuing appropriate investigations . only 28% of that group were followed up by the neurology service in outpatient clinics , which we speculate may have contributed to return ed presentations and readmission rates . the intervention study suggests that the decision support embodied in the seizure care pathway and early follow - up contributed significantly to a reduction in readmission rates . retrospective chart reviews , which formed the basis of initial baseline data , are hazardous for deciding on service provision due to the unreliability and potential bias in the data . furthermore , the initial analysis was on a relatively small number of charts . in this study we complimented the retrospective audit with a short prospective audit , which validated some of the retrospective audit and independently verified characteristics of patients admitted with epilepsy and their course in hospital . the intervention study was large enough to draw conclusions but its comparison to the two prior audits must be done with caution as the patient characteristics may have been biased in the smaller studies . this was exemplified by the significant differences in readmission rates between the two audits and the intervention study . while some of the difference were undoubtedly due to service improvements finally , for system - wide change the decisions that contributed to the improved quality metrics would have ideally been made by ed and acute medical staff and specialist nurses applying the principles in the seizure care pathway . in this study the pathway was implemented by a specialist service and thus the generalizability of the results in unclear . however , the lack of widespread use of pathways for seizure presentations requited that a proof of principle study was required . future study should now focus on the use of an integrated care pathway ( icp ) without resource necessarily to a specialist at the ed / amau interface . it appears that a large proportion of seizure - related presentations are referred to the in - house medical or neurological teams for admission , due in large part to the lack of access to appropriate algorithms for admission and decision support for early treatment and diagnostic investigation and the difficulty in obtaining outpatient investigations and specialist epilepsy follow - up in a reasonable length of time . this study conducted over a 12 month period using baseline data collected between 2004 and 2006 shows that using an evidence - based care pathway with early specialist advice and follow - up , along with directed patients education and a range of communication tools to aid in self - management such as telephone and e - mail advice , can contribute significantly to quality and value improvements in epilepsy care without compromising safety . we recommend further study of this programme and we have embedded a continuous improvement cycle into prospective audit .

aim . to evaluate the utility of a seizure care pathway for seizure presentations to the emergency department ( ed ) in order to safely avoid unnecessary admission and to provide early diagnostic and therapeutic guidance and minimize length of stay in those admitted . methods . 3 studies were conducted , 2 baseline audits and a 12-month intervention study and prospective data was collected over a 12-month period ( nov 2008 - 09 ) . results . use of the pathway resulted in a reduction in the number of epilepsy related admissions from 341 in 2004 to 276 in 2009 ( p = 0.0006 ) ; a reduction in the median length of stay of those admittedfrom 4 - 5 days in the baseline audits to 2 days in the intervention study ( p 0.001 ) ; an improvement in time to diagnostic investigations such as ct brain , mri brain and electroencephalography ( p 0.001 , p 0.048 , p 0.001 ) ; a reduction in readmission rates from 45.1% to 8.9% ( p 0.001 ) ; and an improvement in follow - up times from a median of 16 weeks to 5 weeks ( p < 0.001 ) . from a safety perspective there were no deaths in the early discharged group after 12 months follow - up . conclusion . the burden of seizure related admissions through the ed can be improved in a safe and effective manner by the provision of a seizure care pathway .

1. Introduction 2. Methods 3. Results 4. Comparisons of Outcomes Across All 3 Studies 5. Discussion 6. Limitations 7. Summary and Recommendations

after getting the university ethics committee 's approval , a prospective case - control study was conducted . all patients who admitted to the boo - ali hospital , zahedan , southeastern iran , with a clinical suspicion of pulmonary tuberculosis or non tuberculous pulmonary infections from september 2006 to august 2007 were included for comparison . one hundred and seventy eight patients were included in the study in which 67 were documented cases of ptb and 111 were patients with pulmonary infections other than tuberculosis as control group ( mostly acute pneumonia and acute exacerbation of chronic bronchitis ) ( figure 1 ) . multiple sputum samples are usually collected from a single patient in the process of tb diagnosis . patho - tb test , ziehl - neelsen staining and culture for mycobacterium tuberculosis were done on all specimens . polymerase chain reaction based on detecting a 123-bp dna segment belonging to insertion sequence is6110 specific for mtb was just done in 42 sputum smear negative samples . analysis of data was performed in confirmed ptb patients ( 63 culture - positive and 4 pcr - positive cases ) . patients with pulmonary infections with negative acid fast bacilli ( afb ) smear and culture results were included in the control group . forty three patients in the tb group were sputum smear positive and the rest were sputum smear negative . we used the patho - tb kits ( anda - rt mycobacteria patho - tb , anda biologicals , strasbourg , france ) which were developed in collaboration with the pasteur institute of iran . the patho - tb test was performed as the manufacturer brochure 's following stages : 1- sample decontamination and neutralization : the sputum sample was treated for decontamination by the kubica method in which 2 ml of sample was put in a 50 ml conical centrifuge tube . an equal volume of solublization / decontamination solution was added and mixed well by vortexing and incubating for 20 minutes on an orbital or turning agitator . at least 0.2 ml aliquot of the re - suspended pellet was kept to perform the rapid patho - tb test and the rest was used to seed culture and perform other required diagnostic test . 2- re - suspended pellet treatment : the re - suspended 0.2 ml aliquot was diluted in 1 to 5 volumes of dns . subsequently , an equal volume of negative control and rehydrated positive control were transferred to two other boiling resistant microtubes . from dissolving solution , the rapid patho - tb test was performed with those treated samples following the procedure presented in the following section . 3- rapid test on the cartridge : only one pre - filter was placed in the center of each cartridge ( filtering unit ) . this pre - filter was essential to retain large particulate matter that would clog the filter . a funnel was made on the pre - filter by snapping it securely in place on the cartridge without damaging the pre - filter . thereafter , the funnel and the pre - filter were removed and a second wash was done with 2 drops of washing solution . then 3 drops of antibody solution was added . a red - pink color at the filter center , as mentioned earlier , indicated the presence of tuberculous bacilli in the processed sample . 4- result interpretation : the color intensity is proportional to the number of antigens in the sample . it is classified into three categories : ( + ) a visible central pinkish color , lighter than the control ; ( + + ) a pronounced central pinkish red color , similar to the control ; and ( + + + ) a central purple - red color , darker than the control . when the result of a test was compatible with the first category , the test should be repeated . the appearance of a red or pink ring with or without a red - pink central area is an artifact that does not influence the results and should be considered negative . data were analyzed using the spss software for windows , version 11.5 ( spss inc , chicago , il , usa ) . the diagnostic performances of the test characteristics including sensitivity , specificity and positive and negative predictive values were measured . a two - sided significance level of 0.05 was used . the patho - tb test was performed as the manufacturer brochure 's following stages : 1- sample decontamination and neutralization : the sputum sample was treated for decontamination by the kubica method in which 2 ml of sample was put in a 50 ml conical centrifuge tube . an equal volume of solublization / decontamination solution was added and mixed well by vortexing and incubating for 20 minutes on an orbital or turning agitator . at least 0.2 ml aliquot of the re - suspended pellet was kept to perform the rapid patho - tb test and the rest was used to seed culture and perform other required diagnostic test . 2- re - suspended pellet treatment : the re - suspended 0.2 ml aliquot was diluted in 1 to 5 volumes of dns . subsequently , an equal volume of negative control and rehydrated positive control were transferred to two other boiling resistant microtubes . from dissolving solution the rapid patho - tb test was performed with those treated samples following the procedure presented in the following section . 3- rapid test on the cartridge : only one pre - filter was placed in the center of each cartridge ( filtering unit ) . this pre - filter was essential to retain large particulate matter that would clog the filter . a funnel was made on the pre - filter by snapping it securely in place on the cartridge without damaging the pre - filter . thereafter , the funnel and the pre - filter were removed and a second wash was done with 2 drops of washing solution . then 3 drops of antibody solution was added . a red - pink color at the filter center , as mentioned earlier , indicated the presence of tuberculous bacilli in the processed sample . 4- result interpretation : the color intensity is proportional to the number of antigens in the sample . it is classified into three categories : ( + ) a visible central pinkish color , lighter than the control ; ( + + ) a pronounced central pinkish red color , similar to the control ; and ( + + + ) a central purple - red color , darker than the control . when the result of a test was compatible with the first category , the test should be repeated . the appearance of a red or pink ring with or without a red - pink central area is an artifact that does not influence the results and should be considered negative . data were analyzed using the spss software for windows , version 11.5 ( spss inc , chicago , il , usa ) . the diagnostic performances of the test characteristics including sensitivity , specificity and positive and negative predictive values were measured . the patho - tb test was performed as the manufacturer brochure 's following stages : 1- sample decontamination and neutralization : the sputum sample was treated for decontamination by the kubica method in which 2 ml of sample was put in a 50 ml conical centrifuge tube . an equal volume of solublization / decontamination solution was added and mixed well by vortexing and incubating for 20 minutes on an orbital or turning agitator . at least 0.2 ml aliquot of the re - suspended pellet was kept to perform the rapid patho - tb test and the rest was used to seed culture and perform other required diagnostic test . 2- re - suspended pellet treatment : the re - suspended 0.2 ml aliquot was diluted in 1 to 5 volumes of dns . subsequently , an equal volume of negative control and rehydrated positive control were transferred to two other boiling resistant microtubes . from dissolving solution the rapid patho - tb test was performed with those treated samples following the procedure presented in the following section . 3- rapid test on the cartridge : only one pre - filter was placed in the center of each cartridge ( filtering unit ) . this pre - filter was essential to retain large particulate matter that would clog the filter . a funnel was made on the pre - filter by snapping it securely in place on the cartridge without damaging the pre - filter . thereafter , the funnel and the pre - filter were removed and a second wash was done with 2 drops of washing solution . then 3 drops of antibody solution was added . a red - pink color at the filter center , as mentioned earlier , indicated the presence of tuberculous bacilli in the processed sample . 4- result interpretation : the color intensity is proportional to the number of antigens in the sample . it is classified into three categories : ( + ) a visible central pinkish color , lighter than the control ; ( + + ) a pronounced central pinkish red color , similar to the control ; and ( + + + ) a central purple - red color , darker than the control . when the result of a test was compatible with the first category , the test should be repeated . the appearance of a red or pink ring with or without a red - pink central area is an artifact that does not influence the results and should be considered negative . data were analyzed using the spss software for windows , version 11.5 ( spss inc , chicago , il , usa ) . the diagnostic performances of the test characteristics including sensitivity , specificity and positive and negative predictive values were measured . a two - sided significance level of 0.05 was used . one hundred and seventy eight patients were enrolled in the study who were finally classified into two groups ; a group of documented pulmonary tuberculosis ( n = 67 ) and a group of non - tuberculous pulmonary infection ( n = 111 ) . the mean age of the tb patients was 58 17 years and 42% of them ( 28 patients ) were men . the mean age in non tb patients was 60 15 and 46% of them ( 51 patients ) were men . twenty ( 11% ) of the patients were from afghanistan and the rest were iranian . anorexia , weight loss , hemoptysis and malaise were significantly higher in the tb group but fever was more presented in the non - tb group ( table 1 ) . positive reactions to ppd skin test ( more than 10 mm ) and presence of bcg scar were seen in 62% and 37% of ptb group comparing to 54% and 45% in non - tb group , respectively ( p > 0.05 ) ( table 1 ) . the sensitivity , specificity , ppv and npv of ppd test versus culture were 63% , 46% , 41% and 67% , respectively . as it was shown in table 1 , 43 out of 67 ptb patients had positive direct smear examination . the sensitivity of sputum smear examination was 64% versus 90% in patho - tb test but the specificity was 100% versus 70% . the most common clinical findings of pulmonary tuberculosis and non - tuberculous patients the results of patho - tb test were positive in 40 of smear positive and 20 of smear negative tuberculous patients and 33 cases of non - tuberculous control group . four specimens in control group were positive which were retested and only one specimen was reported positive at the end . the sensitivity of patho - tb test in sputum smear positive and negative ptb was 93% and 83% , respectively . specificity of the test was 70% and positive predictive value ranged from 38% in sputum smear negative ptb to 65% in the total number of ptb patients . the negative predictive value was more than 90% in each group ( table 2 ) . among communicable diseases , tuberculosis ( tb ) is the second leading cause of death worldwide , killing nearly 2 million people each year . about one - third of the world population is infected with tb in which 10% progress toward active disease . most of these patients are living in underdeveloped countries of the world.10 the incidence of total ptb and sputum smear positive ptb were estimated about 53.4 and 22.4 per 100,000 respectively in the study region during 2006.11 the definite diagnosis of pulmonary tuberculosis is difficult because of low sensitivity of sputum direct examination . despite the low cost and easy performance of direct sputum smear microscopy , it has a sensitivity of 61.3 - 63.4% and specificity of 97.3 - 97.4% and is being used as the primary diagnostic test for the diagnosis of ptb.12 culture requires several weeks to provide results and other complex tests such as pcr are expensive and rarely available in low income countries . world health organization targets 70% of case detections and 85% of successful treatments and they are not likely to be achieved with the existing methods of diagnosis.13 attempts to develop a rapid and inexpensive diagnostic method with high sensitivity and specificity are still highly desirable for the diagnosis of ptb . different methods by using new reagents or purified antibodies try to provide high sensitivity and specificity for the tests comparable to direct smear microscopy or afb culture.14 there are many different free mycobacterial antigens which can be detected in different types of body fluid . the most commonly used antigens are lipoarabinomannan ( lam , mycodot serologic test ) , antigen 5 ( 38 kda , pathozyme - tb elisa test and ict diagnostics ) , lam and 38 kda ( pathozyme - myco elisa test ) , a-60 antigen ( antigen a-60 elisa test ) , 45/47 kda antigen , kp90 antigen , 30 kda antigen , p32 antigen and some other antigens.the standard methods used for those different antigen assays include sandwich elisa , inhibition elisa , latex agglutination and reverse passive hemagglutination tests.1416 dipstick method had a sensitivity and specificity of 93% and 95% , respectively.17 recently it was reported that patho - tb test has a reasonable sensitivity and specificity . among 310 tb patients , the sensitivity of patho - tb was 91.1% comparing to direct examination that was 91.8% and the specificity was 85.5% versus 100%.18 direct examination in that study was done by auramine staining which was confirmed by ziehl - neelsen in positive specimens but in the present study only ziehl - neelsen staining for direct examination was performed . sensitivities in both of these studies revealed approximately similar results but the specificity was lower in the present study . in the present study , the sensitivity of patho - tb test was 90% in contrast with 64.1% for the direct smear method . the higher sensitivity , coupled with prompt response , low cost and easy performance , will enable the identification of a sizeable number of pulmonary tuberculosis . another advantage of the test is using just a small amount of sputum ( 0.2 ml ) . in order to have good sensitivity , the antibodies used in this test are polyclonal . these antibodies are raised and purified against tb bacilli , especially 35 , 65 and 85 kda mycobacterial antigens , but can also react with other mycobacterial species . thus it is necessary to analyze results together with the clinical data of each patient to obtain a complete diagnosis . concerning the high number of false positive , using a confirmatory diagnostic procedure like culture in association with clinical features is mandatory . none of the patients in this study were hiv positive so a study in immune compromised patients with several opportunistic infections is recommended . as mentioned earlier , performance of anti - mycobacterial antibody test revealed contradictory results , regardless of type of tuberculosis . the value of detection of specific igg or igm antibodies against antigenic particles of mycobacterium tuberculosis revealed different results.1923 rapid immunochromatographic test ( ict ) for detection of antibodies directed against mtb antigens and mtb - specific enzyme - linked immunospot assay , with early antigenic target-6 and culture filtrate protein-10 peptides are two other different new diagnostic methods for tb.2425 despite all these different types of rapid diagnostic tests , newer and more accurate methods of diagnosis are still needed . although clinical manifestations in table 1 such as malaise , anorexia , weight loss and hemoptysis showed a significant difference between ptb and control groups , they are not characteristic for tb . positive tuberculin skin test in our study also did not show any significant difference and the results of diagnostic performance characteristics were much lower than patho - tb test results . although sputum smear examination was highly specific ( 100% ) , it was just 64% sensitive . higher sensitivity of this new rapid flow - through method comparing to direct smear microscopy of afb , might enable using patho - tb test for rapid diagnosis of ptb as a screening test along with traditional diagnostic tests . positive predictive values of the test are low therefore patients with positive patho - tb test need a confirmatory test such as sputum culture to confirm the diagnosis of ptb . concerning the high percentage of npvs , it would be possible to rely on them for patients with a negative patho - tb test and clinical manifestations which are not suggestive of ptb . further studies on larger number of sputum smear negative ptb patients are required to clear the characteristic performance of patho - tb test . ran was the main researcher , wrote the manuscript and coordinated in most of the experiments .

background : despite recent technologic improvements in identifying mycobacterium tuberculosis , we are still facing problems in rapid diagnosis of tuberculosis . the objective of this study is to determine the diagnostic value of a new rapid screening test ( patho - tb ) for diagnosis of pulmonary tuberculosis.methods:between september 2006 to august 2007 , 178 patients were enrolled in the study who were finally classified into two groups ; a group of documented pulmonary tuberculosis ( n = 67 ) and a group of non - tuberculous pulmonary infection ( n = 111 ) . patho - tb test , ziehl - neelsen staining and culture were done on all specimens.results:of all , 43 patients with pulmonary tuberculosis were sputum smear positive for acid fast bacilli and the rest were smear negative . mean age of the patients was 59.8 16.1 years and 44% of them were men . the results of patho - tb test were positive in 40 of smear positive and 20 of smear negative tuberculous patients and 33 cases of non - tuberculous control group . the sensitivity , specificity , positive and negative predictive values and accuracy of patho - tb test were estimated 89.5% , 70.2% , 64.5% , 91.7% and 77.5% , respectively.conclusions:according to the present study it would be suggested that patho - tb test could be a rapid and inexpensive method for diagnosis of pulmonary tuberculosis , given by its high sensitivity and negative predictive value . concerning the high number of false positive results , using a confirmatory diagnostic procedure is mandatory .

Methods None Laboratory Technique Statistical Analysis Results Discussion Conclusions Authors Contributions

a 14-year - old , domestic shorthair cat was treated for transient diabetes mellitus for 3 months with glargine insulin , which was discontinued when the diabetes mellitus resolved . approximately 36 months later the diabetes mellitus recurred and glargine insulin was restarted . within 23 mins of the first injection the cat collapsed , developed profuse vomiting and diarrhea , as well as facial swelling and diffuse erythema . a hypersensitivity reaction was suspected and the cat was treated with antihistamines , aggressive fluid therapy and gastrointestinal support . six months later the cat re - presented for relapse of its diabetes mellitus and an intradermal skin challenge with 1:20 diluted insulin was performed confirming a hypersensitivity to glargine . the cat continues to be well regulated on porcine zinc insulin without any hypersensitivity reactions noted . this is the first reported case of a hypersensitivity reaction secondary to glargine insulin in a cat . clinicians should be aware of this potential complication , particularly in animals with a previous history of insulin administration and the potential to utilize intradermal testing with insulin . hypersensitivity reactions ( hrs ) most commonly occur after an allergen binds to ige on the surface of mast cells and basophils precipitating release of vasoactive substances such as histamine , chymase , tryptase and other chemokines . it is usually associated with prior exposure to the antigen causing sensitization of the immune system . clinical manifestations can range from facial pruritis and urticaria , to cardiovascular collapse and death . previous reports of suspected hrs to pharmaceuticals in veterinary medicine include dexamethasone , ophthalmic medication and human albumin among others . the purpose of this case report is to describe the clinical manifestation and management of a cat with an hr to subcutaneous glargine insulin ( gi ) administration . a 14-year - old , female , spayed , domestic shorthair cat weighing 5.68 kg presented on referral to our hospital for evaluation of acute collapse . the cat had a history of diabetes mellitus ( dm ) , which was first treated with gi in 2013 . the cat was treated for diabetes , which became transient after 3 months of treatment . one week prior to presentation at our institution , the dm had recurred and re - initiation of gi was recommended . the bottle had not been opened prior to use and it was stored in a refrigerator . the evening of presentation the cat had ingested all of its meal ( which had not been changed in over a year ) , according to the owner , and received 1 unit of gi subcutaneously for the first time since 2013 . approximately 23 mins after administration , the cat collapsed and started panting . the cat also demonstrated projectile vomiting and diarrhea . on presentation to the primary veterinarian , the cat was noted to be laterally recumbent and was exhibiting horizontal nystagmus . a blood glucose ( bg ) was not obtained , but the cat was given dextrose submucosally as there were concerns about a hypoglycemic crisis . the cat was then referred to our facility . on presentation to our institution , the cat was noted to be obtunded , was tachycardic ( heart rate of 220 beats per minute ) and tachypneic ( respiration of 50 breaths per minute ) . the respiratory effort was increased and infrequent stridor noted . the face and muzzle were swollen and edematous . the cat again produced a large amount of vomiting and diarrhea ; at this time hematochezia was also noted . though mentation was dull , the nystagmus had resolved and no cranial nerve deficits were noted . bg was measured on presentation with a handheld glucometer ( alphatrak ; abbott animal health ) and was noted to be 517 mg / dl ( reference interval [ ri ] 75116 mg / dl ) ; this was approximately 2 h after the gi had been administered . the cat had no measurable systolic blood pressure on doppler measurement ( parks doppler machine ) and a 10 ml / kg bolus of crystalloids was administered . owing to the concerns about an hr to the insulin , diphenhydramine ( 2 mg / kg ) was administered intramuscularly . within an hour , the muzzle swelling and erythema had improved after antihistamine administration . the cat continued to experience recurrent systemic hypotension in the subsequent hours and after receiving a 3 ml / kg synthetic colloid bolus , was started on a continuous crystalloid ( 75 ml / kg / day ) and colloid infusion ( 8 ml / kg / day ) and the systemic hypotension resolved . blood work performed on admission revealed hyperglycemia ( 666 mg / dl ; ri 75116 mg / dl ) , hypokalemia ( 3.36 meq / l ; ri 3.624.60 treatment for her gastrointestinal signs consisted of maropitant ( 1 mg / kg sc q24h ) , dolestron ( 0.6 mg / kg iv q24h ) and ampicillin ( 20 mg / kg iv q8h).the cat continued to experience a large volume of diarrhea and hematochezia over the first 24 h after admission , but the vomiting resolved . an abdominal ultrasound was performed 1 day after presentation and revealed an enlarged and hyperechoic liver with few small hypoechoic nodules throughout the parenchyma , assessed as consistent with a diabetic hepatopathy . both kidneys had a mild decrease in corticomedullary definition , and the left and right adrenal glands were noted to have normal shape and size . blood work was also repeated at this time and showed normalization of the lactate and potassium levels . during hospitalization , the bg continued to be monitored and ranged from 107355 mg / dl ; the cat s appetite remained poor . no insulin was administered while hospitalized , owing to the poor appetite , persistent diarrhea and the concerns about an hr to gi . the cat was discharged 3 days after presentation on metronidazole ( 10 mg / kg po q12h ) and famotidine ( 1 mg / kg po q24h ) . the owners continued to monitor the cat s bg at home with a handheld glucometer and it ranged between 88 and 380 mg / dl . the owners tended to measure the bg if they felt the cat appeared lethargic ; they did not have a set protocol of when and how frequently to measure its bg . at this time the cat was still not consuming its full caloric needs and we recommended that the owners continue to monitor the cat s bg at home to determine when it would require insulin again . the owners infrequently measured the cat s bg 12 times weekly and it was never greater than 200 mg / dl . six months later , the cat re - presented for recurrence of polyuria / polydipsia and being lethargic at home . the owners were continuing to measure the cat s bg at this time and were intermittently obtaining values greater than 250 mg / dl . the cat was sedated with ketamine ( 0.5 mg / kg iv ) and valium ( 0.3 mg / kg iv ) , and the fur over the lateral left thorax was clipped . one unit of gi and porcine zinc insulin ( pzi ) were each diluted into 20 units/0.2 ml of 0.9% nacl and from each of these mixtures , 0.05 ml/5 units was injected intradermally . as a negative control , within 30 mins of the procedure , a palpable swelling was noted over the gi site . the cat also developed erythema and swelling of its muzzle ( figure 1 ) and vomited ; diphenhydramine ( 2 mg / kg i m ) was administered and the clinical signs resolved . two hours after the procedure , the cat s bg was 410 mg / dl ( ri 90180 mg / dl ) and it was elected to administer 0.5 units pzi subcutaneously , owing to the hyperglycemia . the cat was kept overnight for monitoring and was noted to be painful and intermittently pruritic over the injection site . the cat was discharged the following day and continues to be on subcutaneous pzi every 12 h and has not exhibited any recurrent signs of systemic or cutaneous hr . evidence of cutaneous anaphylaxis after the administration of glargine insulin ; note the generalized erythema and swelling over the muzzle and periocular regions type i hrs , as previously noted , can vary in severity from urticaria to anaphylaxis . this can occur as a biphasic reaction , with immediate itching and burning at the injection site , followed by a more sustained generalized reaction 48 h later with a combination of cutaneous , gastrointestinal and/or respiratory symptoms reported . this presents as small , localized tenderness and painful non - erythematous nodules and central hematomas at injection sites , occurring around 68 h after insulin injection and lasting for 48 h. clinical examples of type iii hrs include serum sickness , adenopathy and insulin resistance . the cutaneous nodules associated with a type iv hr can be distinguished from a type iii hr as the former usually occurs 24 h or more after the insulin injection and last for 47 days . in humans , type iv hrs it appears , given the acute presentation and history of prior exposure to insulin , the cat in this case demonstrated a type i hr , but this was not conclusively proven . severe anaphylactic reactions in cats can cause a multitude of signs , but dyspnea is most common as the lungs are believed to be the shock organ of cats . in the case previous case reports of anaphylactic reactions in cats have also shown that some will also exhibit simultaneous gastrointestinal and respiratory signs . while a gastrointestinal infection or toxin can not be fully ruled out , the cat s diet was strictly controlled by its owners , owing to its dm , and an infection also seemed less likely as it was kept indoors only and there were no other pets in the house . also , the association of the signs with the administration of the gi is also indicative of an hr . confirmation of a cutaneous hr to insulin administration is performed by intradermal testing using a 1:20 dilution of insulin preparation . appearance of a cutaneous lesion within 60 mins of injection indicates a type i hr , as exhibited by the cat in this case , whereas a type iii or iv hr would be suggested by a response seen between 2 and 24 h. human protocols were used as intradermal testing with insulin had never been performed in a cat prior to this . there was subjectively more swelling and discomfort noted at the gi injection site ; the gi site also appeared more erythematous . the increased swelling and erythema over the gi site also provides more evidence that the cat was more sensitive to the gi than the pzi . the cat in this case exhibited more severe cutaneous and gastrointestinal signs than the typical human patients who undergo intradermal testing . should this protocol be utilized in the future in small animal patients , the insulin may need to be further diluted to reduce the severity of adverse effects . other diagnostic modalities for hrs reported in dogs include identification of an elevated alanine transaminase and increased gallbladder wall thickness and a striated wall pattern on abdominal ultrasound . it is unknown if such abnormalities are identified in cases of feline insulin hr ; the abdominal ultrasound performed in the cat reported here failed to identify any gall bladder abnormalities . management of insulin hr involves exclusion of poor injection technique , use of alternative forms of insulin , splitting the dose and injection into separate sites , and addition of antihistamines . if these methods fail , a local injection of dexamethasone in combination with insulin is sometimes used . steroid therapy was not pursued in this cat as we were concerned it would further complicate the management of the dm . similarly , we also did not attempt to divide the dose or inject it in separate sites as the cat had exhibited such severe clincial signs of hypersensitivity initially . poor injection technique or improper storage of insulin was not considered likely as the owners had successfully managed the cat s dm for 3 years . desensitization , using gradually increasing dilutions of insulin intradermally in a controlled setting , can be successful in type i hr , but can require long periods of time to be beneficial . desensitization in people appears to be less successful in patients with the biphasic type i hr . use of newer insulins such as aspart or gi has also been recommended , but hrs to these insulins have also been recently recognized in human medicine . in the cat corticosteroids and epinephrine have been recommended for hrs in veterinary patients but were considered potentially contraindicated given the pre - existing dm , severe hyperglycemia and potential to cause ketosis . the cat appears to be well managed on pzi without any reported signs of an hr . insulin hrs were much more common in human patients prior to the use of human insulin analogues when bovine and porcine insulin were more commonly administered . it is believed that humans that have an hr to insulin are reacting to the type of protein in the insulin given that there are molecular differences between insulin from different species . we elected to use pzi , a protamine - containing recombinant human insulin , in this cat rather than gi , a recombinant human insulin analogue , as this contains a different form of protein . both gi and pzi also contain different adjuvants , with pzi mostly containing zinc and gi mainly containing metacresol . as such , it is unclear if the hr developed secondary to the adjuvants rather than the different insulin protein molecules . in human medicine it is much more common to react to the insulin molecule rather than the adjuvants . furthermore , while we can not completely rule out improper storage or poor administration , the bottle of gi was newly purchased , stored in a refrigerator and had never been used prior to the suspect hr event . the owners had also successfully managed the cat s dm for 3 years and were well versed in subcutaneous administration of insulin , so poor technique does not seem likely . we can not rule out that cats do not naturally develop pruritis and erythema from intradermal gi injections and further studies should evaluate intradermal gi injections in healthy cats . clinicians should be aware of the possibility of hrs to insulin administration in cats , particularly in cats with a previous history of insulin administration . the incidence of hrs to insulin and use of intradermal insulin testing for confirmation in veterinary medicine requires further evaluation .

case summarya 14-year - old , domestic shorthair cat was treated for transient diabetes mellitus for 3 months with glargine insulin , which was discontinued when the diabetes mellitus resolved . approximately 36 months later the diabetes mellitus recurred and glargine insulin was restarted . within 23 mins of the first injection the cat collapsed , developed profuse vomiting and diarrhea , as well as facial swelling and diffuse erythema . a hypersensitivity reaction was suspected and the cat was treated with antihistamines , aggressive fluid therapy and gastrointestinal support . the cat made a full recovery and was discharged 3 days later . six months later the cat re - presented for relapse of its diabetes mellitus and an intradermal skin challenge with 1:20 diluted insulin was performed confirming a hypersensitivity to glargine . the cat continues to be well regulated on porcine zinc insulin without any hypersensitivity reactions noted.relevance and novel informationhypersensitivity reactions to insulin administration are rarely described in human medicine . this is the first reported case of a hypersensitivity reaction secondary to glargine insulin in a cat . clinicians should be aware of this potential complication , particularly in animals with a previous history of insulin administration and the potential to utilize intradermal testing with insulin .

Case summary Relevance and novel information Introduction Case description Discussion Conclusions

furthermore , it is the most common cause of antenatal hospital admission.1 the incidence of preterm birth rate in us was 12% in 2011.2,3 at siriraj hospital , a tertiary care in central part of thailand , the preterm birth rate was 9.4% in 2004 and 13.7% in 2010.4 srinagarind hospital is another tertiary care center in northeastern part of thailand ; the preterm birth rate was as high as 11.5% in 2013 ( unpublished data ) . the complications of preterm births arise from immature organ systems that are not yet ready to support life in the extrauterine environment . for instance , respiratory distress syndrome is caused by a lack of surfactants in the lung epithelium . antenatal administration of corticosteroids to women who were expected to give preterm birth was associated with a significant reduction of neonatal morbidity and mortality , respiratory distress syndrome , intraventricular hemorrhage , and necrotizing enterocolitis of preterm infants . national institute of health and the american college of obstetrician and gynecologists recommended a single course of corticosteroids for pregnant women between 24 and 34 weeks of gestation who are at risk of preterm delivery within 7 days.5,6 the use of tocolytic agents to suppress preterm uterine contraction can delay delivery for at least 48 hours , and this prolongation enable the complete administration of corticosteroids to obtain their maximum effect for gestational age of 2434 weeks.6 despite of the available modalities to prevent preterm birth and its consequences , the proportions of preterm birth are not reduced.7 therefore , we planned to evaluate the success rate of preterm uterine contraction inhibition with tocolytic agents and factors associated with inhibition failure to aware the possibility of preterm delivery . this observational retrospective study was approved by the khon kaen university ethics committee for human research based on the declaration of helsinki and the ich good clinical practice guidelines ( he571337 ) . after reviewing the data collection forms from the medical records of the project , the ethics committee deemed patient written informed consent was not required , as this was a retrospective study . we have reviewed the medical records of all singleton pregnant women between 24 and 33 weeks of gestation admitted to the labor room of srinagarind hospital , khon kaen university , thailand , between january 2013 and july 2014 , and selected the patients who received uterine contraction inhibition with tocolytic drugs under diagnosis of preterm labor or threatened preterm labor . multiple pregnancies , women with diagnosis of dead fetus in utero , and lethal fetal anomalies were excluded . preterm labor was defined as regular uterine contractions accompanied by a change in cervical dilatation , effacement , or both.6 threatened preterm labor was defined as regular uterine contractions occurring at the frequency of at least once in every 10 minutes during 30 minutes of monitoring , with no dilatation and effacement of cervix.4 the high risk of preterm delivery was defined as group of women with any history of obstetric , medical , or surgical complication during pregnancy . the gestational age determination was based on the certain date of the last menstrual period or first trimester ultrasound examination . their medical records were assessed for the baseline characteristics including age , weight , gravidity , parity , gestational age on admission , numbers of antenatal care visit , characteristics of uterine contraction , and cervical status to classify studied women to preterm labor or threatened preterm labor . the types , modes , duration of administration , and the adverse effects of the tocolytic agents used for uterine contraction inhibition were also retrieved . the pregnancy prolongation was measured in terms of the duration ( days ) and the gestational age ( complete weeks ) whether it was beyond 34 and 37 weeks or not . additionally , we also reviewed all of neonatal outcomes including gestational age , route of delivery , appearance , pulse , grimace , activity , respiration ( apgar ) scores , and birth weight . the tocolytic agents commonly used in srinagarind hospital are adalat ( nifedipine ) , bricanyl ( terbutaline ) , and ventolin ( salbutamol ) . the course of oral nifedipine ( 10 mg ) started with the initial load every 15 minutes with the maximum dose of 40 mg until no uterine contraction was observed , followed by the maintenance dose of 10 mg every 6 hours for 24 hours , an then tapered every 8 hours for 24 hours , every 12 hours for 24 hours , and once daily , and discontinue if there was no more contraction . for terbutaline , 4 ampules ( 2 mg ) were added to 5% d / w 100 ml and intravenously dripped beginning with the rate of 5 microdrops / min , then increased 5 microdrops / min every 30 minutes until there was no uterine contraction or getting the maximum rate of 45 microdrops / min . if there was no uterine contraction for 24 hours , the dripping rate would be reduced to 5 microdrops / min until off . in case of salbutamol , 1020 mg of salbutamol if the preterm pregnant women still have uterine contraction while using these drug regimens or have some intolerable side effects , they were classified as inhibition failure and the attending physicians would change to other drugs or none . doses of these drugs could be modified in some cases , depending on the associated maternal or fetal conditions and the judgment of attending physicians . since in our setting , only preterm pregnant women of 24 to 33 weeks of gestation would receive a course of antenatal corticosteroid administration for accelerate fetal lung maturation . so the success of preterm uterine contraction inhibition was assessed as the delay in delivery for at least 48 hours after tocolytics administration between 24 and 33 week of gestation while the inhibition failure was defined as the delay in delivery for < 48 hours . the risk factors of the inhibition failure were also analyzed in this study . the sample size calculation based on the objective to assess the success rate of preterm uterine contraction inhibition with the maximal proportion of defined women was 0.5% and 10% of allowance error . the study required at least 96 singleton pregnant women who admitted to the labor room with preterm uterine contraction and received tocolytic agents for inhibition . for statistical analysis , mean , standard deviation ( sd ) , number , and percentage were calculated . the chi - square and fisher s exact test were used to evaluate the risk factors of labor inhibition failure . these variables were then included if p - value < 0.20 in a stepwise logistic regression analysis to determine which factors , if any , were independently associated with the risk of labor inhibition failure . adjusted odds ratio with a 95% confidence interval ( ci ) , which did not include unity were considered statistically significant . during the studied period , a total of 424 pregnant women met the diagnosis of preterm labor or threatened preterm labor . after exclusion of 39 multiple pregnancies , a women with dead fetus in utero , 2 women with lethal fetal anomalies , 3 women whose medical records were lost , 262 women between 34 and 36 weeks of gestation and also 14 women between 24 and 33 weeks of gestation whose uterine contractions were not inhibited , 103 singleton pregnant women at 24 to 33 weeks of gestation who received tocolytic agents were reviewed . around three - fourth of them ( 77.7% ) met the clinical criteria of threatened preterm labor , while 22.3% met the diagnosis of preterm labor . there were histories of obstetrical , medical , and/or surgical complications during pregnancy among studied women . anemia was the most frequently associated condition ( 34% ) , followed by previous abortion ( 29.1% ) , preterm pre - labor rupture membranes ( 17.5% ) , urinary tract infection or asymptomatic bacteriuria ( 15.5% ) , and history of preterm labor / delivery ( 11.7% ) and vaginal bleeding ( 9.7% ) . few cases of other complications were autoimmune diseases ( 3 cases ) , genital tract infection ( 3 cases ) , infective diarrhea / food poisoning ( 3 cases ) , polyhydramnios ( 3 cases ) , and 1 case each of chorioamnionitis , sepsis , uterine leiomyoma , abdominal surgery during pregnancy , and uterine cervical surgery . in table 2 , the overall success rate of preterm uterine contraction inhibition for prolongation of pregnancy at least for 48 hours was 86.4% . the success rate among who were diagnosed as threatened preterm labor on admission was high ( 93.8% ) , while the success rate among who were diagnosed as preterm labor on admission was low ( 60.9% ) . approximately half of the studied women ( 55.3% ) had prolonged pregnancy beyond 34 weeks . one - third of them ( 38.8% ) gave birth after 37 weeks of gestation . regarding the type of tocolytic agents , terbutaline was the most frequently selected tocolytic agent , followed by nifedipine . intolerable side effects of tocolytic agents were observed in 13 cases , whose medication was discontinued . among these 13 cases , 5 cases developed hypotension after nifedipine treatment and 8 cases showed maternal tachycardia / dyspnea and fetal tachycardia after terbutaline treatment . duration of tocolytic agent administration for labor inhibition was 25 days ( median = 4 days ) . mean baby birth weight was 2,370.9 g ( sd : 833.6 ) , and their mean gestational age at birth was 34.4 weeks ( sd : 3.5 ) . almost half of them ( 49.5% ) were low birth weight ( < 2,500 g ) babies . there were 4 cases of severe birth asphyxia whose apgar score was < 4 at 5 minutes after birth . in the univariate analysis , risk factors including preeclampsia with severe feature , history of abortion , more than 24 hours of uterine contraction , and history of cervical surgery were not significantly associated with inhibition failure ( p - value > 0.20 ) . when using multivariate logistic regression analysis , only preterm labor had statistical significant associated with inhibition failure . estimated worldwide incidence of preterm birth was 9.6% in 2005 , mostly occurred in africa and asia.8 in 2010 , low birth weight babies worldwide were estimated to be around 18 million , and 59% of them were small - for - gestational age term and 41% were preterm . two - third of small - for - gestational age infants were born in asia.9 the understanding of the causes of premature delivery , low birth weight babies and improvement of the effectiveness of available modality of intervention are needed to improve birth outcomes . the results of this study from a tertiary care hospital in northeastern thailand showed the same tendency of worldwide burden of preterm pregnancy complications . women with uterine contraction before 34 weeks of gestation were admitted to the hospital to prolong pregnancy with tocolytic agents . the majority of them ( 86.4% ) could continue pregnancy at least 48 hours after receiving the tocolytic agents and receive completed corticosteroids course to accelerate fetal lung maturity . however , 77.7% of these studied women presented with threatened preterm labor , which is defined as uterine contraction without cervical dilatation . the significant risk factor of inhibition failure was preterm uterine contraction with cervical dilatation ( preterm labor ) . similar to the authors present results , motazedian et al reported that a 48-hour prolongation of pregnancy was achieved in 87% patients receiving terbutaline and 84% patients receiving salbutamol.10 likewise , the study at siriraj hospital , bangkok , thailand , revealed that the success rate in threatened preterm labor inhibition for 12 hours with nifedipine was about 80%.11 however , in cases of women with advanced cervical dilatation ( 48 cm ) who received one or more tocolytics ( magnesium sulfate , indomethacin , or nifedipine ) , only 23% could prolong pregnancy at least 48 hours after admission.12 similarly , in cases of women with the diagnosis of preterm labor based on the presence of uterine contraction with documented cervical change , success rate by nifedipine was 70%.13 in these two studies with the lower success rates , the patients presented as preterm labor with apparent cervical changes . we noticed that , regardless of number and the type of tocolysis , the success rate was higher when uterine contraction inhibition was applied to women with preterm uterine contraction without cervical changes . in the present study , the success rate to prolong pregnancy at least 48 hours was 93.8% in threatened preterm labor group and that of preterm labor group was 60.9% . furthermore , the authors data revealed that preterm labor ( uterine contraction with cervical change ) was the significant risk factor of labor inhibition failure . the main rationale of tocolysis for preterm labor are to delay delivery for at least 48 hours to allow completed course of corticosteroids to accelerate fetal lung surfactant production and allow to transfer mother to a tertiary facility . betamimetics and calcium channel blockers reduce the number of preterm labor women who will give birth within 48 hours . nevertheless , tocolysis does not appear to significantly lengthen the gestational age beyond 7 days.14 it is known that nifedipine is easy to administer and have fewer side effects relative to betamimetics.15 the authors results also support this notion as intolerable side effects occurred more in cases of betamimetics treatment . in spite of the success rate of preterm uterine contraction inhibition was as high as 86.4% in this study , for example , the mean birth weight of babies was approximately 2,300 g and a half of them had low birth weight ( < 2,500 g ) . severe birth asphyxia was seen in 3.9% and more than half ( 58.3% ) of the delivery required surgical intervention . thus , we should provide advanced neonatal care and prepare for complicated delivery for the pregnancy complicated with preterm uterine contraction with or without cervical dilatation . limitations of this study were those inherent to any retrospective study which made it difficult to gain the complete data . fortunately , most of the outcomes of interest were available in the retrieved medical records . another was the limited number of studied women , in that the majority of women enrolled in this study were those with threatened preterm labor that would lead to overestimation of the overall success rate of uterine contraction inhibition . however , we tried to minimize such effects by separating the patients into threatened preterm and preterm labor groups for outcome analysis . regarding the evaluation of pregnancy outcomes of uterine contraction inhibition with tocolytic agents , further research on threatened preterm labor with or without tocolysis would be necessary . also , whether preterm uterine contraction with cervical change should get high success rate as preterm uterine contraction without cervical change if the tocolytics could attenuate uterine contraction should be explored in future . the success rate of preterm uterine contraction inhibition with tocolytic agents to delay delivery for at least 48 hours was high in threatened preterm labor and low in preterm labor . a significant risk factor for inhibition failure was the preterm uterine contraction with cervical change .

objectivethis study aims to assess the success rate of inhibiting preterm uterine contraction with tocolytic agents to delay delivery for at least 48 hours and risk factors of failure inhibition.materials and methodsbetween january 2013 and july 2014 , medical records of all singleton pregnant women between 24 0/7 and 33 6/7 weeks of gestation with the diagnosis of preterm labor ( with cervical dilatation ) or threatened preterm labor ( without cervical dilatation ) who received tocolytic agents were reviewed . the success rate of preterm uterine contraction inhibition was accounted in patients with 48 hours delayed delivery . the risk factors of the inhibition failure and neonatal outcomes were also investigated in this study.resultsamong 424 pregnant women diagnosed of preterm labor or threatened preterm labor , 103 singleton pregnant women met the study criteria . overall success rate of preterm uterine contraction inhibition to prolong pregnancy for at least 48 hours was 86.4% ( 95% confidence interval [ ci ] : 78.3 , 92.3 ) . however , the success rate among the threatened preterm labor group was 93.8% ( 95% ci : 88.3 , 99.1 ) while the preterm labor group was 60.9% ( 95% ci : 39.3 , 82.4 ) . the significant factor associated with inhibition failure was preterm labor ( adjusted odds ratio 7.22 ; 95% ci : 1.99 , 26.20).conclusionthe success rate of preterm uterine contraction inhibition with tocolytic agents to delay delivery for at least 48 hours was high in threatened preterm labor and low in preterm labor . a significant risk factor for inhibition failure was the preterm uterine contraction with cervical change .

Introduction Materials and methods Results Discussion Conclusion

achalasia is a primary esophageal motor disorder of unknown etiology characterized by selective loss of inhibitory neurons in the esophageal wall , resulting in insufficient relaxation of the lower esophageal sphincter and loss of esophageal peristalsis.1,2 jackhammer esophagus is a newly introduced term to describe patients with at least 20% of the swallows with distal contractile integral ( dci ) 8000 mmhg.sec.cm.3 several reports have described patients with diffuse esophageal spasm ( des ) , non - specific esophageal motor disorder ( nsemd ) , nutcracker esophagus , and gastroesophageal reflux disease ( gerd ) progressing to achalasia.49 although no causal relationship has been identified , these reports suggest that the different esophageal motor disorders represent a spectrum rather than unique and stable disorders . we describe , for the first time , a case of a patient who progressed from jackhammer esophagus to type ii achalasia . a 66 year - old woman with a history of atrial fibrillation , hypertension , gastritis , and hypothyroidism presented to our clinic in july 2014 with progressive dysphagia for solids and liquids over 18 months . her symptoms occurred daily and resulted in an 11-kg weight loss over this period of time . she denied heartburn , regurgitation , choking or coughing during eating , chest pain , vomiting , abdominal pain , or change in bowel habits . the barium swallow showed a mildly dilated esophagus with tertiary contractions , delayed emptying of the esophagus , and a narrowed gastroesophageal junction ( fig . 1 ) . the upper endoscopy demonstrated mild - moderate antral gastritis , normal appearing esophageal mucosa , and it was noted that the patient might have a tight gastroesophageal junction . unfortunately , after completing these tests she missed 3 follow - up appointments and was seen in our clinic only one year later . given her progressive dysphagia symptoms and weight loss , a high resolution esophageal manometry ( hrem ) was performed ( fig . the study showed a normal median integrated residual pressure of 8 mmhg , normal mean resting pressure of 21 mmhg , normal mean residual pressure of 7 mmhg , and mean dci of 10 770 mmhg sec cm . of the 10 swallows , 30% were hypercontractile ( > 8000 mmhg sec cm ) , 50% were normal and 20% simultaneous . overall , the findings were consistent with jackhammer esophagus based on the chicago classification of motility disorders.3 pantoprazole was increased to 40 mg twice daily with plan to add a pain modulator if her symptoms recurred . unfortunately , she was lost to follow - up again until she was seen in our clinic a year later in june 2015 . she continued to have worsening dysphagia , lost an additional 7 kg , and reported new onset of occasional post - prandial chest tightness . at this point , 3 ) . this study demonstrated diffuse esophageal dilatation with retained secretions and barium with only 020% change in esophageal volume after 5 minutes . in addition , tertiary peristaltic waves within the distal esophagus , limited emptying of the contrast into the stomach and an air - fluid level were also noted . 4 ) revealing a median integrated residual pressure of 71.5 mmhg , mean resting pressure of 97 mmhg , and mean residual pressure of 90 mmhg . there was panesophageal pressurization with every swallow and 80% of the swallows demonstrated prolonged pan - pressurization , all consistent with type ii achalasia . therapeutic options were discussed with the patient , and she elected to undergo heller myotomy . the patient was referred to general surgery but presented urgently to gi clinic 2 weeks later for worsening dysphagia , dehydration , weakness , inability to tolerate oral intake , and an additional 7-kg weight loss . the patient was admitted to the hospital and was noted to develop acute kidney injury . in addition , a nasogastric tube was placed and she was started on tube feeds to optimize her nutritional status . a week after discharge the patient underwent heller myotomy with dor fundoplication . on a follow - up visit with her general surgeon , she reported that she was tolerating liquids without any difficulties . to our knowledge , this is the first case that describes progression of jackhammer esophagus to achalasia over a period of about one year . smart et al5 reported 5 patients with gerd who subsequently developed achalasia over a period of 210 years . robson et al4 reported a patient with gerd who developed des one year after initial diagnosis and achalasia one year later . several reports describe the progression of des to achalasia.68 khatami et al10 conducted the first prospective cohort study with 12 patients . they observed the progression of des to achalasia in only one patient after 10.6 years . they also noted that low esophageal body amplitude contractions was a predictor of this progression . fontes et al11 conducted the largest prospective study to date to assess the progression of des to achalasia . patients with gerd confirmed by ph monitoring or systemic diseases that may affect the esophagus were excluded . five ( 14% ) of the patients progressed to achalasia at a mean follow - up of 2.1 years . although demographic characteristics were not predictive of the transition to achalasia , the authors observed that amplitude contractions of less than 50 mmhg was a predictive factor ( p = 0.002 ) . anggianash et al9 described the progression of nutcracker esophagus to achalasia in one patient after 3 years . vantrappen et al13 described six patients with nsemd who progressed to achalasia . while our patient presented with symptoms that could be considered classic for achalasia , her initial conventional manometry was consistent with nsemd . only the second hrem as well as the timed barium esophagram were consistent with achalasia . this progression within one year of an esophageal motor disorder was faster than what has been documented in previous reports . however , this case suggests that all esophageal motility disorders represent a spectrum of disorders , where patients may progress over time from one to another . this case also demonstrates the importance of following patients who were diagnosed with non - achalasia motility disorders with a repeat hrem in one year to assess for progression or a shift to a different esophageal motility disorder . because most of the studies documenting progression of a motility disorder to achalasia were case reports using conventional manometry , long - term studies evaluating this progression with hrem are needed . perhaps with the current availability of hrem , researchers may be able to identify predictive factors associated with progression of certain esophageal motility disorders to achalasia .

it has been suggested that patients with certain motility disorders may progress overtime to develop achalasia . we describe a 66 year - old woman who presented with dysphagia for solids and liquids for a period of 18 months . her initial workup showed normal endoscopy and non - specific esophageal motility disorder on conventional manometry . six months later , due to persistence of symptoms , the patient underwent a high resolution esophageal manometry ( hrem ) demonstrating jackhammer esophagus . the patient was treated with a high dose proton pump inhibitor but without resolution of her symptoms . during the last year , the patient reported repeated episodes of food regurgitation and a significant weight loss . a repeat hrem revealed type ii achalasia . multiple case reports , and only a few prospective studies have demonstrated progression from certain esophageal motility disorders to achalasia . however , this report is the first to describe a case of jackhammer esophagus progressing to type ii achalasia .

Introduction Case Report Discussion

the international cancer genome consortium ( icgc ) is a multidisciplinary , multi - institutional collaborative effort aiming to systematically and comprehensively characterize somatic mutations in 50 different cancer types and subtypes ( 1 ) . five hundred tumor genomes , as well as matched normal control genomes for each cancer type , will be analyzed using high - throughput next - generation sequencing technologies to detect a wide range of somatic mutations , including single nucleotide mutations , small insertions / deletions , copy number alterations , translocations and other chromosomal structural rearrangements . genome - wide methylation state analysis and whole - transcriptome sequencing have also been planned to provide additional molecular - level characterizations . to make the effort more scalable each member institution specializes in generating data for a particular tumor type . at the time of writing , figure 1.international cancer genome consortium ( icgc ) projects ( march 2011 ) . international cancer genome consortium ( icgc ) projects ( march 2011 ) . one of the major goals of icgc is to rapidly bring these data to the cancer research community in order to accelerate studies on the discovery of cancer causes , to enhance the accuracy of diagnoses and to improve treatments . in order to achieve this task , the data generated by the consortium members have to be managed efficiently . amongst the most important data management challenges faced by the consortium is the high complexity and heterogeneity of the data types involved , the necessity to link different data types , and the need to protect controlled data . furthermore , the high volume of data and the distributed nature of the sources make traditional centralized approaches to data management impractical . consequently , the icgc has adopted federated data architecture to address their data management needs . the scalability of the system is improved by having each member institution store and process data locally ; the data federation software then presents these separate sources as a single access point for remote data access . biomart , an open source data federation system ( 2 ) , has been chosen as the icgc data management platform . biomart s flexible data model makes it generally applicable to a wide range of biological data types and built - in query optimizations make it suitable for large data sets . in addition , biomart supports an industry - standard security framework that is needed to provide secure access to the controlled data . finally , a large number of existing expert - maintained public annotation databases can be readily federated , adding value to the interpretation of the icgc experimental data . the architecture of the icgc data management system has been modeled on the biomart central portal ( 3 , 4 ) . for a number of years , the portal has been successfully providing a single point of access to a large number of biological databases distributed across the world . each biomart database federated in this portal is maintained independently , released and updated on its own schedule . similarly , each icgc member maintains their local biomart database where clinical annotations and the data produced from genomic analyses are deposited . however , in order to make these data comparable across different cancer projects and create a unified icgc data set , a mechanism for enforcing uniformity is required . thus , the same set of data models , controlled vocabularies , ontologies and reference data sets are used in all of the icgc member databases . furthermore , a gene - centric data model adopted from ensembl mart ( 5 ) is used to reference experimental data to the same set of annotated genes . in order to link individual biomart databases into a unified system , these servers can each access data in three ways : they can access their own local databases directly ; they can communicate with the other icgc biomart servers , in order to retrieve data from remote databases ; and they can communicate with non - icgc biomart servers , such as cosmic ( 6 ) and reactome ( 7 ) , to retrieve publicly - available annotation data . in this manner , the biomart server maintained by each icgc member acts as a fully featured icgc data portal , providing unified access to all the consortium data . the users select a combination of different data sets and specify query criteria using a variety of graphical user interfaces and analysis tools that are available from the portal . application programming interfaces ( apis ) for java , rest , soap and sparql are also available for programmatic access . behind the scenes , biomart software breaks down this query into smaller parts that are distributed to the remote data sources . the results are collected and compiled into a single unified results set that is presented to the user . some of the data in the portal has access restrictions in order to preserve patient privacy . to protect this sensitive data , communication between biomart servers when a user logs into an icgc data portal server using his / her openid , the server will consult the central registry to ensure that the user is authorized to access the data ( figure 2 ) . at present the portal contains data from 24 cancer projects , consisting of 3478 genomes and 13 cancer types and subtypes . this includes the data generated from seven studies performed by five icgc participating institutions located in four countries . the data portal also hosts data from other large - scale cancer genome projects including the cancer genome atlas ( tcga ) ( 8 , 9 ) , tumor sequencing project ( tsp)(10 ) and johns hopkins university ( 1114 ) . open access data sets include ( i ) simple somatic mutations , ( ii ) copy number alterations , ( iii ) structural rearrangements , ( iv ) gene expression , ( v ) mirna , ( vi ) dna methylation and ( vii ) exon junctions . secure access to controlled data sets , such as germline variations , is available to authorized users . a summary of data currently available on the data portal is shown in table 1 . in addition to cancer genomic data , the data portal also federates several public databases ( table 2 ) . currently this includes ensembl genome database ( 5 ) , kyoto encyclopedia of genes and genomes ( kegg ) ( 1517 ) , reactome ( 7 ) , cosmic ( 6 ) , pancreatic expression database ( 18 ) and the breast cancer campaign tissue bank ( 19 ) , and the number of resources is continuously growing . table 1.the summary of data available on the icgc data portal divided by cancer projectsourcecancer projectdata setsimple mutationscopy number alterationsstructural rearrangementsgene expressionmirna expressionexon junctionsdna methylationgermline variationsicgcbreast carcinoma ( wtsi , uk)liver cancer ( ncc , jp)liver cancer ( riken , jp)malignant melanoma ( wtsi , uk)pancreatic cancer ( oicr , ca)pancreatic cancer ( qcmg , au)small cell lung carcinoma ( wtsi , uk)tcgaacute myeloid leukemiabreast invasive carcinomacolon adenocarcinomaglioblastomamultiformekidney renal clear cell carcinomakidney renal papillary cell carcinomalung adenocarcinomalung squamous cell carcinomaovarian serous cystadenocarcinomarectum adenocarcinomastomach adenocarcinomauterine corpus endometrioid carcinomaotherbreast cancer ( jhu , us)colorectal cancer ( jhu , us)glioblastomamultiforme ( jhu , us)lung adenocarcinoma ( tsp , us)pancreatic cancer ( jhu , us) table 2.public databases federated with the icgc data portalsourceurldescription of contentsensemblwww.ensembl.orggenome annotationreactomewww.reactome.orgpathway annotationkeggwww.genome.jp/keggpathway annotationcosmicwww.sanger.ac.uk/genetics/cgp/cosmic/somatic mutations in cancerpancreatic expression databasewww.pancreasexpression.orgpancreatic cancer expression databreast cancer campaign tissue bankwww.breastcancertissuebank.org/bio-informatics.phpbreast cancer expression data the summary of data available on the icgc data portal divided by cancer project public databases federated with the icgc data portal the data portal provides three major interactive entry points : identifier search , analysis and database search ( figure 3 ) . identifier search ( figure 3a ) lets users input identifiers that are commonly used in public annotation databases ( e.g. hgnc gene symbol , ensembl i d , refseq i d , uniprot i d and other accessions ) and returns links to the corresponding gene report page , which displays basic gene description , pathway annotation , mutations found in the cosmic database , as well as pancreas and breast cancer expression data ( figure 4 ) . in addition , the gene report displays a summary of mutation frequencies in the selected gene across all cancer projects ( figure 4d ) . records in the gene report ( i.e. genomic coordinates , pathway name , publication ) are linked to appropriate resources when available , allowing users to easily retrieve additional information on their data of interest . three main entry points are available : ( a ) identifier search , ( b ) analysis and ( c ) database search . figure 4.gene report for kras includes : ( a ) gene annotation data from ensembl , pathway annotation from ( b ) kegg and ( c ) reactome , ( d ) summary of mutation frequencies in each cancer project , ( e ) mutation data from cosmic , expression data from ( f ) pancreatic expression database and ( g ) breast cancer campaign tissue bank ( bcctb ) . different sections of this page come from federated biomart sources . three main entry points are available : ( a ) identifier search , ( b ) analysis and ( c ) database search . gene report for kras includes : ( a ) gene annotation data from ensembl , pathway annotation from ( b ) kegg and ( c ) reactome , ( d ) summary of mutation frequencies in each cancer project , ( e ) mutation data from cosmic , expression data from ( f ) pancreatic expression database and ( g ) breast cancer campaign tissue bank ( bcctb ) . different sections of this page come from federated biomart sources . to help with the interpretation of cancer data , gene and pathway analysis tools are available in the analysis section of the data portal ( figure 3b ) . these tools enable users to view the most commonly affected genes or pathways in one or more cancer projects . results are presented in an easy - to - follow chart that can be exported as an image file , and the numerical data can also be downloaded for further processing ( figure 5 ) . ( a ) affected pathways are shown in a chart , with bars representing the number of affected genes in each pathway . ( b ) by clicking on the bar , users are able to view and download the genes that were mutated in each pathway . ( a ) affected pathways are shown in a chart , with bars representing the number of affected genes in each pathway . ( b ) by clicking on the bar , users are able to view and download the genes that were mutated in each pathway . using the database search entry point ( figure 3c ) users can interactively query the database by several data types : genes , samples , simple mutations , copy number alterations , structural rearrangements , gene expression , mirna , dna methylation and exon junctions . the quick interface contains a pre - selected set of the most commonly used filters such as gene type , mutation type and chromosome , and outputs a fixed set of attributes . the flexible interface contains additional filters and a selection of attributes , allowing the user to choose which data are displayed in the output . the advanced interface contains the complete set of filters and attributes , including technological platforms used for sequencing , and clinical parameters such as patient gender and tumor histopathology . to demonstrate the utility of the icgc data portal we present several queries that can be performed using different query interfaces . search for genes affected by copy number loss and also detected as deletion from structural rearrangement analysis. ( figure 6 ) data setscancer projectsfiltersattributesgenespancreatic cancer ( qcmg , au)rearrangement type : deletionensembl gene idcopy number alteration type : lossgene symbolgene description figure 6.a screenshot of the quickquery interface . query # 2 ( flexible search ) : retrieve clinical staging data for colorectal cancer patients with non - synonymous simple mutations in genes that are involved in wnt signaling pathway. ( figure 7 ) data setscancer projectsfiltersattributesgenescolorectal cancer ( jhu , us)pathway : signaling by wnttumor sample idconsequence type : non_synonymous _ codingmutation idmutation typedonor iddiagnosis idclinical staging ( who)ensembl gene idgene symbol figure 7.a screenshot of the flexiblequery interface . a screenshot of the flexiblequery interface . query # 3 ( advanced search ) : in pancreatic cancer data set , retrieve all rna - seq expression data for genes that are affected by copy number gains. ( figure 8) data setscancer projectsfiltersattributesgenespancreatic cancer ( qcmg , au)copy number alteration type : gainensembl gene idplatform : solid sequencinggene expression : sample idnormalized read countraw read count figure 8.a screenshot of the advancedquery interface . the icgc data portal is the first project to successfully federate large amounts of cancer genomics data and rich annotation data in a single access point . it presents a scalable approach , not only in the traditional sense of parallelizing data processing and storage , but also in a more general sense of outsourcing the external annotation expertise , by federating annotations from independently maintained databases . this approach has proved to be successful in addressing icgc data management needs and can be useful for similar , large - scale collaborative projects . the icgc data portal will continue to expand by adding more data , both from within the project in the form of new cancer genomics data from icgc members , and by integrating other public annotation databases to further the depth of analysis possible through the data portal . additionally , new tools will be developed to increase the flexibility and utility of the system . this includes tools for icgc deployers , to further streamline the data processing , transformation and loading processes , and tools for users , to add new methods of data visualization and analysis to the portal .

the international cancer genome consortium ( icgc ) is a collaborative effort to characterize genomic abnormalities in 50 different cancer types . to make this data available , the icgc has created the icgc data portal . powered by the biomart software , the data portal allows each icgc member institution to manage and maintain its own databases locally , while seamlessly presenting all the data in a single access point for users . the data portal currently contains data from 24 cancer projects , including icgc , the cancer genome atlas ( tcga ) , johns hopkins university , and the tumor sequencing project . it consists of 3478 genomes and 13 cancer types and subtypes . available open access data types include simple somatic mutations , copy number alterations , structural rearrangements , gene expression , micrornas , dna methylation and exon junctions . additionally , simple germline variations are available as controlled access data . the data portal uses a web - based graphical user interface ( gui ) to offer researchers multiple ways to quickly and easily search and analyze the available data . the web interface can assist in constructing complicated queries across multiple data sets . several application programming interfaces are also available for programmatic access . here we describe the organization , functionality , and capabilities of the icgc data portal.database url : http://dcc.icgc.org

Project description Data content Query examples Conclusion and future directions Funding

a 64-year - old male patient who had been on diabetes medication with no other particular medical history visited the clinic for symptoms of chillness , and his temperature was measured to be 38.8. an abdomical ct revealed a 6-cm abscess surrounding a pseudoaneurysm of about 4 cm near the right internal iliac artery , and mycotic aneurysm was diagnosed . although no particular strain was identified from the blood culture test conducted at the time of the clinic visit , based on the week - old abdominal ct results , which clearly showed the growing aspect of the aneurysm , risk of death due to rupture was judged to be high . before conducting a femoral artery bypass , a distal part of the right internal iliac artery was occluded using a 10-mm amplatzer vascular plug . the right common iliac artery and external iliac artery were also occluded in succession using 14-mm and 12-mm amplatzer vascular plugs , respectively . the patient was immediately moved to the or and a left fermoral artery - right femoral artery bypass was performed using a 10-mm ringed gore - tex graft . on the third day post - operation , a pigtail catheter was inserted into the abscess near the mycotic aneurysm of the mycotic aneurysm of the right iliac artery and the surrounding abscess was drained . postoperative abdominal ct showed complete occlusion of the right iliac artery and internal and external iliac arteries . when compared to the preoperative abdominal ct , the mycotic aneurysm and abscess were found to be completely removed ( fig . the patient was discharged after 4 weeks of antibiotic treatment with no symptomatic exacerbation found . currently , he has been off antibiotics for one year , and no complications have been detected in outpatient monitoring . a 56-year - old female visited the clinic with symptoms of fever , coughing , and chillness for 5 days . her temperature at the time of the visit was 38.2. she complained of pain in her left leg . two years and 5 months before her visit to the clinic , the patient had been diagnosed with perforated appendicitis and had received an appendectomy . however , postoperative complications including intraperitoneal abscess and enterocutaneous fistula resulted in treatments including right hemicolectomy , oophorectomy , and colostomy . in addition , the histopathology tests of the patient at the time resulted in the diagnosis of stage iia cecal cancer , and she received adjuvant concurrent chemo - radiotherapy for 12 months thereafter . since then , a pet scan for post - treatment evaluation showed a lump on the soft tissue of the right anterior abdominal wall . upon removal the patient received chemotherapy for an additional 11 months and was discharged . a ct scan to determine the cause of pain in the left lower limb at the time of the visit revealed a deep vein thrombosis . in addition , the part of small intestine creating an enterocutaneous fistula was removed following the previous operation . when detaching a severe adhesion on the abdominal wall , the right external iliac artery was damaged and sutured . on the tenth day post operation , lower right abdominal pain and wound infection were found . in a ct scan conducted during conservative treatment , an 8-cm lump suggestive of acute hematoma in the right external iliac fossa and a false aneurysm connected to the right external iliac fossa were found . an s&g stent graft of 10-mm diameter and 40-mm length was promptly inserted into the right external iliac fossa . although no strains were identified from blood culture testing , a joining of the infected area in the lower right abdomen and the false aneurysm was observed , resulting in the diagnosis of mycotic aneurysm . a 10-mm long amplatzer vascular plug was inserted into the right proximal external iliac artery . the patient was then immediately moved to the or and the right distal external iliac artery was ligated . a 12-mm ringed gore - tex graft was used to perform a left femoral artery - right femoral artery bypass . a postopeative lower extremity vascular ct scan showed complete withdrawal of the mycotic aneurysm previously present at the right external iliac artery ( fig . the patient was discharged 18 days after the operation without specific symptoms , but remote metastasis of the cecal cancer aggravated and she died after 4 months . a 64-year - old male patient who had been on diabetes medication with no other particular medical history visited the clinic for symptoms of chillness , and his temperature was measured to be 38.8. an abdomical ct revealed a 6-cm abscess surrounding a pseudoaneurysm of about 4 cm near the right internal iliac artery , and mycotic aneurysm was diagnosed . although no particular strain was identified from the blood culture test conducted at the time of the clinic visit , based on the week - old abdominal ct results , which clearly showed the growing aspect of the aneurysm , risk of death due to rupture was judged to be high . before conducting a femoral artery bypass , a distal part of the right internal iliac artery was occluded using a 10-mm amplatzer vascular plug . the right common iliac artery and external iliac artery were also occluded in succession using 14-mm and 12-mm amplatzer vascular plugs , respectively . the patient was immediately moved to the or and a left fermoral artery - right femoral artery bypass was performed using a 10-mm ringed gore - tex graft . on the third day post - operation , a pigtail catheter was inserted into the abscess near the mycotic aneurysm of the mycotic aneurysm of the right iliac artery and the surrounding abscess was drained . postoperative abdominal ct showed complete occlusion of the right iliac artery and internal and external iliac arteries . when compared to the preoperative abdominal ct , the mycotic aneurysm and abscess were found to be completely removed ( fig . the patient was discharged after 4 weeks of antibiotic treatment with no symptomatic exacerbation found . currently , he has been off antibiotics for one year , and no complications have been detected in outpatient monitoring . a 56-year - old female visited the clinic with symptoms of fever , coughing , and chillness for 5 days . her temperature at the time of the visit was 38.2. she complained of pain in her left leg . two years and 5 months before her visit to the clinic , the patient had been diagnosed with perforated appendicitis and had received an appendectomy . however , postoperative complications including intraperitoneal abscess and enterocutaneous fistula resulted in treatments including right hemicolectomy , oophorectomy , and colostomy . in addition , the histopathology tests of the patient at the time resulted in the diagnosis of stage iia cecal cancer , and she received adjuvant concurrent chemo - radiotherapy for 12 months thereafter . since then , a pet scan for post - treatment evaluation showed a lump on the soft tissue of the right anterior abdominal wall . upon removal the patient received chemotherapy for an additional 11 months and was discharged . a ct scan to determine the cause of pain in the left lower limb at the time of the visit revealed a deep vein thrombosis . in addition , the part of small intestine creating an enterocutaneous fistula was removed following the previous operation . when detaching a severe adhesion on the abdominal wall , the right external iliac artery was damaged and sutured . on the tenth day post operation , lower right abdominal pain and wound infection were found . in a ct scan conducted during conservative treatment , an 8-cm lump suggestive of acute hematoma in the right external iliac fossa and a false aneurysm connected to the right external iliac fossa were found . an s&g stent graft of 10-mm diameter and 40-mm length was promptly inserted into the right external iliac fossa . thereafter , the false aneurysm was found to have disappeared . however , a profound amount of hematoma was present in the affected area . although no strains were identified from blood culture testing , a joining of the infected area in the lower right abdomen and the false aneurysm was observed , resulting in the diagnosis of mycotic aneurysm . a 10-mm long amplatzer vascular plug was inserted into the right proximal external iliac artery . the patient was then immediately moved to the or and the right distal external iliac artery was ligated . a 12-mm ringed gore - tex graft was used to perform a left femoral artery - right femoral artery bypass . a postopeative lower extremity vascular ct scan showed complete withdrawal of the mycotic aneurysm previously present at the right external iliac artery ( fig . the patient was discharged 18 days after the operation without specific symptoms , but remote metastasis of the cecal cancer aggravated and she died after 4 months . mycotic aneurysm constitutes about 1% of all aneurysms , and its high risk of rupture requires immediate treatment . it tends to occur in a wide range of arteries , including in the lesser curve of the aortic arch and on the opposite side of the visceral branch vessel of the abdominal aorta . although the condition is often thought to be related to structural characteristics of the artery or the turbulent blood flow that occurs in the opposite side of abdominal branching blood vessels , the actual cause of the condition is still unknown . the major bacterial strains known to cause mycotic aneurysms are e. coli , staphylococcus , salmonella , and streptococcal species . these strains are related to atherosclerotic ulcers in the inner arterial membrane , and they act as a nidus for invading a secondarily infected arterial wall , triggering a false aneurysm or rupture of the artery . although the onset rate of mycotic aneurysm is comparatively low , the difficulty of diagnosis often results in late discovery of the disease after serious advancement of the condition . the mortality rate is fairly high , since it often develops in patients with lowered immunity or other underlying diseases . diagnosis of mycotic aneurysm can be done by identifying three typical symptoms : abdominal pain , fever , and a pulsating lump . however , it is unusual to find all the symptoms at once and symptoms are often nonspecific . a ct scan of a subject with a suspected mycotic aneurysm can reveal detailed information of the lesion and diagnosis can be done through blood culture testing . however , only 10~27% of postoperative mycotic aneurysms are found to be benign in tissue culture testing , and there have been no reports of any relationship to prognosis , such as a postoperative infection of the graft . treatment of mycotic aneurysm includes early diagnosis as well as broad removal of the lesion surgically following artificial blood vessel placement and long - term use of antibiotics . although treatment plays a critical role in a patient 's prognosis , several studies have reported high postoperative death rates ( 16~44% ) despite ongoing technical advancement . unlike the general aneurysm , mycotic aneurysm is often located near the upper portion of renal arteries . in that case , in situ graft placement is preferred , since the range of the lesion is broad and reconstruction of blood vessels that reach surrounding organs is required . on the other hand , in cases of mycotic aneurysm in the lower portion of renal arteries , extra - anatomic artificial blood vessel bypass has been recognized as the appropriate treatment . in both cases , if staphylococcus aureus , salmonella species , or other pyogenic infection is grossly suspected , in situ graft placement is forbidden , since the chance of a postoperative graft infection is high . a variety of vascular embolizations have been reported recently for extra - anatomic artificial vessel bypass on mycotic aneurysms in the inferior renal artery . vascular embolization is known to be suitable for the treatment of aneurysms in blood vessels that have below - average size , since the operational approach can be difficult . however , it has been controversial to place a foreign material in the infected artery in treatment of mycotic aneurysm and the risk of post - treatment complications such as rupture can increase due to a weakening of the arterial wall . therefore , these treatments can only be applied in patients with a low chance of continuous exacerbation of infection . in this case report , the authors have planned treatments after comprehensive consideration of the patients ' clinical symptoms as well as the location of lesions . we would like to introduce the bypass surgery in between bilateral femoral arteries following vascular embolization as a successful treatment option for mycotic aneurysm .

mycotic aneurysm is a disease requiring immediate treatment because of the high risk of rupture . a difficult surgical approach , especially in the case of occurrence on the iliac artery , involving endovascular embolization and extra - anatomic bypass grafting , is known to be a suitable treatment . we performed extra - anatomic bypass grafting after endovascular embolization successfully in two patients . the postoperative computed tomography of both patients showed complete exclusion of the mycotic aneurysm .

CASE REPORT 1) Case 1 2) Case 2 DISCUSSION

microarray data is deposited at the ncbi gene expression omnibus ( geo ) database under geo series accession number gse52552 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse52552 ) . to gain insight in the mechanisms of complement resistance in m. catarrhalis , the transcriptional response of m. catarrhalis strain bbh18 , a complement resistant isolate , , upon exposure to 10% pooled normal human serum ( nhs ) was analyzed by microarray expression profiling . this concentration was chosen because test experiments demonstrated that a m. catarrhalis bbh18 gene deletion mutant lacking the key complement resistance factor uspa2h was rapidly killed in 10% nhs ( data not shown ) . to reduce the effect of day to day variation , two fully independent microarray expression profiling experiments were performed , indicated hereafter as experiments a and b ( table 1 ) . m. catarrhalis bbh18 was pre - cultured overnight on brain heart infusion ( bhi ) agar plates at 37 c in an atmosphere containing 5% co2 . bacteria were harvested from plates and resuspended in pbs supplemented with 0.15% gelatin ( pbs - g ) . next , 5 ml bhi medium was inoculated to an od600 nm of ~ 0.075 and cultured until mid - log phase ( od600 nm of ~ 1.0 ) , obtaining cultures with a density of 6 10 cfu / ml . of this culture , 4 ml was harvested by centrifugation , washed in 16 ml pbs - g , and resuspended in 20 ml pbs - g with 0.2 mm mgcl2 and 1 mm cacl2 ( ca and mg ) . 5.5 ml of this suspension was mixed with 5.5 ml of either pbs - g ( ca and mg ) ( control ; experiment a ; n = 3 , experiment b ; n = 2 ) or with 20% pooled nhs ( gti diagnostics ) in pbs - g ( ca and mg ) ( experiment a ; n = 3 , experiment b ; n = 3 ) and incubated at 37 c with agitation ( 200 rpm ) . after 0 , 30 ( experiment a only ) , and 60 min , a 3.5 ml aliquot was taken , harvested by centrifugation , and immediately treated with 1 ml of rnaprotect bacteria reagent ( qiagen ) . total rna was isolated using the rneasy mini kit ( qiagen ) according to manufacturer 's instructions . co - purified genomic dna was removed by a double dnase treatment using the dna - free kit ( ambion ) . five micrograms of total rna obtained from samples that were exposed for 60 min to nhs or control conditions was used for microarray expression profiling . the rna was reverse transcribed and labeled for microarray hybridization according to standard nimblegen gene expression array protocols . two micrograms of cy3-labeled cdna probes was hybridized overnight at 42 c to custom - designed nimblegen m. catarrhalis bbh18 expression arrays ( 4 plex arrays ; 72 k ) and subsequently washed ; all procedures were performed according to manufacturer 's instructions . array images were acquired with a nimblegen ms200 scanner , and images were first processed with nimblescan software . raw expression data ( .pair files ) were processed using arraystar ( dnastar ) and normalized using the quantile rma method yielding an average signal intensity per cds . to identify genes differentially expressed in 10% nhs compared to the control condition , statistical analysis was executed using cybert unpaired two - condition data analysis ( http://cybert.ics.uci.edu/ ) . genes were considered to be differentially expressed when expression was increased or decreased by > 2.5-fold and at a bonferroni corrected p < 0.05 . to eliminate genes with no or very low expression from our analysis , we generated frequency plots of log2 average probe signal intensity per gene ; two distributions could be observed ( data not shown ) . based on these frequency plots we eliminated genes with an average normalized probe signal intensity of log2 < 6 in all tested conditions from further analysis . using these criteria we identified 84 genes with a > 2.5 fold increased expression and 134 genes that showed a > 2.5 fold reduced expression in 10% nhs compared to incubation in pbs - g ( nhs dilution buffer ) . to determine if exposure to 10% nhs resulted in the increased expression of genes grouped in the same functional classes , potential enrichment was analyzed with the fishers exact ( one - tail ) test and corrected for multiple testing according to storey and tibshirani . to validate microarray expression data , real - time quantitative pcr ( rt - qpcr ) analysis was performed for a selection of 18 genes on samples obtained after 60 min exposure to nhs or pbs - g ( experiment b ) . a sub - selection of 11 genes was tested on samples obtained after 30 min ( experiment a ) ( table 1 ) . dna - free total rna ( 500 ng ) was reverse transcribed into cdna using the iscript cdna synthesis kit ( bio - rad ) . rt - qpcr was performed in duplicate for each sample using ssoadvanced sybr green supermix ( bio - rad ) and analyzed on a cfx96 real - time pcr machine ( bio - rad ) . expression fold - changes ( nhs versus pbs - g ) between the different groupings were calculated using the ct method and the gyra gene was used for normalization . correlation between microarray and rt - qpcr data was assessed with a two - tailed pearson correlation test using graphpad prism software . to gain insight in the mechanisms of complement resistance in m. catarrhalis , the transcriptional response of m. catarrhalis strain bbh18 , a complement resistant isolate , , upon exposure to 10% pooled normal human serum ( nhs ) was analyzed by microarray expression profiling . this concentration was chosen because test experiments demonstrated that a m. catarrhalis bbh18 gene deletion mutant lacking the key complement resistance factor uspa2h was rapidly killed in 10% nhs ( data not shown ) . to reduce the effect of day to day variation , two fully independent microarray expression profiling experiments were performed , indicated hereafter as experiments a and b ( table 1 ) . m. catarrhalis bbh18 was pre - cultured overnight on brain heart infusion ( bhi ) agar plates at 37 c in an atmosphere containing 5% co2 . bacteria were harvested from plates and resuspended in pbs supplemented with 0.15% gelatin ( pbs - g ) . next , 5 ml bhi medium was inoculated to an od600 nm of ~ 0.075 and cultured until mid - log phase ( od600 nm of ~ 1.0 ) , obtaining cultures with a density of 6 10 cfu / ml . of this culture , 4 ml was harvested by centrifugation , washed in 16 ml pbs - g , and resuspended in 20 ml pbs - g with 0.2 mm mgcl2 and 1 mm cacl2 ( ca and mg ) . 5.5 ml of this suspension was mixed with 5.5 ml of either pbs - g ( ca and mg ) ( control ; experiment a ; n = 3 , experiment b ; n = 2 ) or with 20% pooled nhs ( gti diagnostics ) in pbs - g ( ca and mg ) ( experiment a ; n = 3 , experiment b ; n = 3 ) and incubated at 37 c with agitation ( 200 rpm ) . after 0 , 30 ( experiment a only ) , and 60 min , a 3.5 ml aliquot was taken , harvested by centrifugation , and immediately treated with 1 ml of rnaprotect bacteria reagent ( qiagen ) . total rna was isolated using the rneasy mini kit ( qiagen ) according to manufacturer 's instructions . co - purified genomic dna was removed by a double dnase treatment using the dna - free kit ( ambion ) . five micrograms of total rna obtained from samples that were exposed for 60 min to nhs or control conditions was used for microarray expression profiling . the rna was reverse transcribed and labeled for microarray hybridization according to standard nimblegen gene expression array protocols . two micrograms of cy3-labeled cdna probes was hybridized overnight at 42 c to custom - designed nimblegen m. catarrhalis bbh18 expression arrays ( 4 plex arrays ; 72 k ) and subsequently washed ; all procedures were performed according to manufacturer 's instructions . array images were acquired with a nimblegen ms200 scanner , and images were first processed with nimblescan software . raw expression data ( .pair files ) were processed using arraystar ( dnastar ) and normalized using the quantile rma method yielding an average signal intensity per cds . to identify genes differentially expressed in 10% nhs compared to the control condition , statistical analysis was executed using cybert unpaired two - condition data analysis ( http://cybert.ics.uci.edu/ ) . genes were considered to be differentially expressed when expression was increased or decreased by > 2.5-fold and at a bonferroni corrected p < 0.05 . to eliminate genes with no or very low expression from our analysis , we generated frequency plots of log2 average probe signal intensity per gene ; based on these frequency plots we eliminated genes with an average normalized probe signal intensity of log2 < 6 in all tested conditions from further analysis . using these criteria we identified 84 genes with a > 2.5 fold increased expression and 134 genes that showed a > 2.5 fold reduced expression in 10% nhs compared to incubation in pbs - g ( nhs dilution buffer ) . to determine if exposure to 10% nhs resulted in the increased expression of genes grouped in the same functional classes , potential enrichment was analyzed with the fishers exact ( one - tail ) test and corrected for multiple testing according to storey and tibshirani . to validate microarray expression data , real - time quantitative pcr ( rt - qpcr ) analysis was performed for a selection of 18 genes on samples obtained after 60 min exposure to nhs or pbs - g ( experiment b ) . a sub - selection of 11 genes was tested on samples obtained after 30 min ( experiment a ) ( table 1 ) . dna - free total rna ( 500 ng ) was reverse transcribed into cdna using the iscript cdna synthesis kit ( bio - rad ) . rt - qpcr was performed in duplicate for each sample using ssoadvanced sybr green supermix ( bio - rad ) and analyzed on a cfx96 real - time pcr machine ( bio - rad ) . expression fold - changes ( nhs versus pbs - g ) between the different groupings were calculated using the ct method and the gyra gene was used for normalization . correlation between microarray and rt - qpcr data was assessed with a two - tailed pearson correlation test using graphpad prism software .

the complement system is an important part of the innate defense against invading pathogens ( blom et al . , 2009 ; [ 1 ] ) . the ability to resist complement - mediated killing is considered to be an important virulence trait for the human - restricted respiratory tract pathogen moraxella catarrhalis , as most disease - associated m. catarrhalis isolates are complement - resistant ( wirth et al . , 2007 ; [ 2 ] ) . here we provide a detailed overview of the experimental methods that we have used to study the molecular basis of m. catarrhalis complement - resistance by transcriptome profiling of the bacterium upon exposure to 10% normal human serum ( nhs ) , associated with the study of de vries et al . published in molecular microbiology in 2014 [ 3 ] .

Direct link to deposited data Experimental design, materials and methods Exposure of Microarray gene expression analysis Validation of microarray data by RT-qPCR analysis

rectal varices are not an uncommon manifestation in cirrhosis with portal hypertension . however , serious bleeding from rectal varices is uncommon . because of its rarity , several authors have reported the utility of transjugular intrahepatic portosystemic shunt ( tips ) placement to control such bleeding when local therapy , such as endoscopic sclerotherapy or banding , fails [ 2 , 3 , 4 , 5 ] . however , we experienced a case of massive bleeding from large rectal varices that could not be controlled by tips placement , despite normalization of the portosystemic pressure gradient . furthermore , we observed rapid decompensation of the cirrhosis that led to severe encephalopathy and death after the tips placement . we report this case to highlight and discuss potential pitfalls of tips placement when using this technique to treat rectal variceal bleeding . a 59-year - old man with a history of alcoholic cirrhosis was hospitalized at an outside facility for massive hematochezia and fainting . he underwent upper endoscopy and tagged red blood cell scan , both of which were negative for active bleeding . a computed tomography of the abdomen was performed and did not reveal the source of the gastrointestinal bleeding ; a nodular liver with splenomegaly consistent with liver cirrhosis was seen , and a markedly dilated inferior mesenteric vein was also noted . during his 5-day hospitalization , he had approximately 1015 bloody bowel movements and received a total of 6 units of packed red blood cells and 2 units of fresh frozen plasma . the patient was then transferred to our institution for further management of his persistent hematochezia . his admission laboratory tests showed a hemoglobin level of 8.3 g / dl , a serum ammonia level of 45 g / dl , a total bilirubin level of 2.8 mg / dl and a model for end - stage liver disease ( meld ) score of 18 . however , he had another episode of massive hematochezia on the following day , with a decrease in hemoglobin level to 5.0 g / dl . therefore , the decision was made to proceed with emergent tips placement . a tips was successfully created with a 10 90 mm viatorr stent - graft ( gore & associates , flagstaff , ariz . this resulted in a decrease in the portosystemic pressure gradient from 12 to 6 mm hg . after the tips placement , brisk hepatopetal flow through the tips was seen on the portogram . despite the successful normalization of the portosystemic pressure gradient , he had another episode of massive bleeding on the following day . an angiogram of the superior rectal vein was performed , which showed tortuous large rectal varices with brisk hepatofugal flow ( fig . 2 ) . flow into the internal iliac vein branches was observed , confirming portosystemic shunting . embolization of the rectal varices was then performed , which necessitated multiple coils , 1 ml of n - butyl cyanoacrylate glue , 5,000 units of thrombin and several sheets of gelfoam ( fig . 3 ) . after the procedure , the patient did not have any further bleeding episodes , and the serial hemoglobin levels remained stable . the remaining hospital course was significant for rapid decompensation of cirrhosis , with an increase in the total bilirubin level to 8.7 mg / dl , the ammonia level to 121 g / dl and the meld score to 28 . he developed acute hypoxic respiratory failure secondary to multiple causes , including progressive liver failure . this case highlights two potential pitfalls of tips placement when using this technique in the treatment of rectal variceal bleeding . first , tipss may not be always successful in controlling serious bleeding from rectal varices , despite optimal portosystemic pressure reduction . we observed rebleeding after the tips placement despite a 50% reduction in the pressure gradient , with a final pressure gradient of 6 mm hg . some authors have also reported that a tips failed to control bleeding from rectal varices [ 6 , 7 ] . recurrent bleeding from rectal varices after tips placement may be related to the large size of the varices . rectal varices , unlike esophageal varices , are true veins and are likely to have larger diameters . according to laplace 's equation , the tension in the varix wall is proportional to the radius of the vessel for any given transmural pressure . , it is recommended to decrease the portosystemic pressure gradient to < 12 mm hg in esophageal variceal bleeding . however , this targeted pressure gradient may not be adequate for controlling bleeding from rectal varices . some authors advocate embolization of the rectal varices at the time of tips placement , even if normalization of portal hypertension is achieved [ 7 , 10 ] . given the successful control of bleeding following embolization in our case , concomitant variceal embolization at the time of tips placement may be necessary to control massive bleeding , especially when the rectal varices are large . second , tipss can be associated with life - threatening complications that can possibly lead to early mortality . since the first application of a tips in the treatment of recurrent bleeding from anorectal varices in 1993 , a tips alone or a tips combined with variceal embolization has been reported to be useful in controlling such bleeding without significant morbidity or mortality [ 2 , 3 , 4 , 5 , 10 ] . however , tipss are inherently associated with risks of procedure - related complications , hepatic encephalopathy and progressive liver failure . godil and mccracken reported a case of rapid liver function decompensation and encephalopathy following tips placement in a 73-year - old woman with rectal variceal bleeding . although no recurrent bleeding was seen , the patient died 4 weeks after the tips procedure . according to the american association for the study of liver diseases practice guidelines , tips is recommended only in the absence of other options for patients with 30-day predicted mortality ( meld > 1518 or serum bilirubin > 4.0 mg / dl ) . although emergent tips placement can be the last resort to control massive bleeding from rectal varices , even in high - risk patients it must be used cautiously because it can lead to life - threatening complications , as seen in our case . despite its minimally invasive nature , tips placement has been associated with a 30-day mortality rate as high as 36% when it is used to control acute gastroesophageal variceal bleeding . one alternative to tips placement in high - risk patients may be the percutaneous embolization of rectal varices via a transhepatic approach . although reports on the use of this technique for bleeding rectal varices are limited , it could be useful in controlling an initial acute bleeding and in stabilizing a patient without affecting liver function . however , collaterals can redevelop after embolization , and recurrent bleeding requiring repeated intervention has been reported . some authors have reported several cases of obliteration of bleeding large rectal varices with the use of balloon - occluded antegrade transvenous sclerotherapy with or without coil embolization [ 14 , 15 ] . in this technique , a sclerosing agent , such as ethanolamine oleate , was injected into the varices while the superior rectal vein was occluded using a balloon catheter . further research is needed to determine its efficacy . in conclusion , although tipss have been reported to be useful in controlling bleeding from rectal varices , there are potential pitfalls in using this technique to treat rectal variceal bleeding . a tips may not always be successful in controlling massive bleeding from large rectal varices even after normalization of portal hypertension , and concomitant variceal embolization may be necessary . furthermore , tipss can be associated with life - threatening complications that may lead to early mortality .

in patients with portal hypertension , bleeding from rectal varices is rare . however , it can be life - threatening . we report a case of massive bleeding from large rectal varices in a 59-year - old man with alcoholic cirrhosis . emergent transjugular intrahepatic portosystemic shunt ( tips ) placement was performed following failed local endoscopic therapy . despite normalization of the portosystemic pressure gradient , the patient had another episode of massive bleeding on the following day . embolization of the rectal varices via tips successfully stopped the bleeding . after the procedure , rapid decompensation of the cirrhosis led to severe encephalopathy , and death was observed . although tipss have been reported to be useful in controlling bleeding from rectal varices , our case illustrates the potential pitfalls in using this technique in the treatment of rectal variceal bleeding . tipss may not be always successful in controlling massive bleeding from large rectal varices , even after normalization of portal hypertension . tipss can also be associated with life - threatening complications that may lead to early mortality .

Introduction Case Presentation Discussion

with an ageing population , alzheimer 's disease ( ad ) and related neurodegenerative disorders represent a serious social and economic threat to most societies . the cost of caring for an increasing number of people with dementia continues to rise . at present , there are an estimated 30 million people with dementia worldwide , with numbers expected to further increase to 80 million by 2040 . the countries or regions with the largest numbers of affected individuals are china and the developing western pacific , western europe , and the unites states . the two core histopathological hallmarks of ad are the amyloid- ( a ) plaques and the tau - containing neurofibrillary tangles ( nfts ) , both of which have been described by alois alzheimer more than a century ago . disease - causing mutations in familial cases of ad have been described two decades ago ; yet there is still no cure for this debilitating disease . the current treatment is limited to acetylcholine esterase ( ache ) inhibitors ( donepezil , rivastigmine , and galantamine ) and the nmda receptor antagonist , memantine ; yet neither of these strategies halts the degenerative process that characterizes ad . what are the characteristics of the major component of the two hallmark lesions in ad and what causes the familial cases ? the 3942 amino acid a peptide in the plaques is fibrillar , as is tau , a microtubule - associated protein that becomes hyperphosphorylated in the disease process and eventually aggregates to form nfts , neuropil threads , and the abnormal neurites that are associated with some amyloid- plaques . although tau has been generally perceived as a neuronal protein , with mainly an axonal localization , we have recently challenged this view by demonstrating an essential dendritic function under physiological conditions and in mediating a toxicity [ 5 , 6 ] . in addition to plaques and nfts , the ad brain is characterized by massive neuronal cell and synapse loss . tau pathology , in the absence of overt a plaques , characterizes a series of diseases that are collectively known as tauopathies . this includes ftld - tau ( frontotemporal lobar degeneration with tau inclusions ) with an inherited form known as ftdp-17 ( frontotemporal dementia with parkinsonism linked to chromosome 17 ) . there is a second , major subset of ftld characterized by tau - negative yet ubiquitin - positive lesions . in this subset , the transcription and splicing factor tdp-43 ( tar dna - binding protein 43 ) has been identified as the aggregating protein , and consequently , this form of ftld has been named ftld - tdp . similar to tau , tdp-43 in the aggregates is hyperphosphorylated and fragmented , a process believed to be linked to toxicity [ 811 ] . finally , in a third subset of ftld , ftld - fus , the nuclear protein fus ( fused - in - sarcoma ) has been identified as aggregated protein . while both proteins form also aggregates in subsets of amyotrophic lateral sclerosis ( als ) , the majority of ad cases are sporadic ( sad ) , with familial ( fad ) cases likely accounting for less than 1% . in three genes , autosomal dominant mutations have been identified : amyloid precursor protein ( app ) , presenilin 1 ( psen1 ) as well as presenilin 2 ( psen2 ) . it is the amyloid precursor protein , app , from which the a peptide is derived by proteolytic cleavage . presenilins are components of the multimeric -secretase complex that generates a. in addition to the three fad genes , apolipoprotein e ( apoe ) has been unanimously confirmed as a susceptibility gene in sporadic ad ( sad ) . more recently , additional risk factor genes have been identified , with clu encoding clusterin , picalm the phosphatidylinositol - binding clathrin assembly protein , cr1 the complement component ( 3b/4b ) receptor 1 , and tomm40 a channel - forming subunit of the translocase of the mitochondrial outer membrane ( tom ) complex [ 1517 ] . at present , a total of ten genes , mostly with roles in the immune system and in cholesterol metabolism , are established risk genes for ad . compared to ad , ftld is a more heterogeneous disorder , with variants that are , for example , characterized by language or behavioural problems . ad and ftld both present with a progressive decline of memory function leading to dementia , although in ftld this is often preceded by motor symptoms such as parkinsonism . it was several years after the first mutations had been identified in the app and psen genes in ad , that the first mutations were identified in a subset of ftld - tau , ftdp-17 , where they were found in exonic and intronic sequences of the mapt gene that encodes tau . another subset of familial ftld is characterized by mutations in the pgrn gene that encodes the pleiotropic protein progranulin , in the vcp gene that encodes valosin - containing protein , and also in tardp encoding tdp-43 ; these ftld - tdp cases are characterized by inclusions of tdp-43 . finally , mutations in chmp2b encoding chromatin modifying protein 2b lead to ftld , in the absence of either tau or tdp-43 inclusions . in this paper , we will be discussing a few selected tau transgenic mouse models with memory and motor phenotypes and how one of these , the k369i mutant human tau expressing k3 strain , has been employed in an enu modifier screen , outlining the strategy from breeding to gene identification . as the focus of this review article is the application of enu mutagenesis to one of our mutant tau transgenic mouse strains , k3 , we will be comparing this particular strain with a few other tau transgenic strains we have established in the past . mouse strains that are otherwise available in the field including those characterized by a plaque formation have been discussed by us in detail elsewhere [ 2427 ] . what has the ambition been in developing transgenic mouse strains for the tau pathology of ad in the first place ? the aim has always been to authentically model the human pathology and to gain a deeper understanding of what causes tau pathology , how this is related to neurodegeneration and functional impairment , and ultimately , how pathogenic processes can be prevented or delayed . when we generated the very first tau transgenic mouse model in 1995 by expressing the longest human tau isoform in mice , we reproduced a major aspect of the human pathology in a subset of neurons : somatodendritic accumulation of hyperphosphorylated forms of tau , a feature of pathological tau in ad brains . we failed however to reproduce nft formation , neurodegeneration , and functional impairment . even by using a stronger promoter to drive human tau expression , this goal of the early days was not achieved , although the mice developed a motor phenotype and amyotrophy [ 29 , 30 ] . only with the identification of pathogenic mutations in ftld - tau in the mapt gene encoding tau , by expressing mutant forms such as g272v or p301l tau , we achieved nft formation , as determined by gallyas silver impregnations and electron microscopy [ 31 , 32 ] . while we discontinued the g272v mutant mice , the p301l tau transgenic pr5 strain was employed to provide support for the amyloid cascade hypothesis that places a upstream of tau , to reveal behavioural impairments in amygdala- and hippocampus - dependent tasks [ 34 , 35 ] , to assess the role of site - specific phosphorylation and in particular of protein phosphatase 2a ( pp2a ) in this process [ 36 , 37 ] , to determine the degree of the cholinergic pathology , and to reveal an impaired stress - related unfolded protein response and a mitochondrial dysfunction caused by pathological tau [ 4042 ] . assessing the role of mirnas in ad adds an additional level of complexity [ 43 , 44 ] . while the pr5 mice display memory impairment as a major clinical feature of ad , another feature , parkinsonism , that characterizes a significant subset of ftld cases , has been modelled in k369i mutant tau transgenic k3 mice . we established this strain based on the identification of the k369i mutation of tau in a single patient with pick 's disease ( pid ) , a form of ftld . as is known for pid in general , the tau lesions in the mutation carrier 's brain showed a remarkable feature when analysed histologically : silver impregnation revealed that the tau lesions were bielschowsky - positive and gallyas - negative ; moreover , tau was phosphorylated at many epitopes but not 12e8 ( ser262/ser356 ) . we succeeded in reproducing this distinct pathology fully in the k3 mice that express k369i mutant tau . owing to a unique expression pattern of the k369i transgene that includes the substantia nigra , the k3 transgenic mice also modelled early - onset parkinsonism , that is , resting tremor , bradykinesia , postural instability , and gait anomalies . they showed an increased cataleptic response to haloperidol and an early , but not late , response to l - dopa , indicating that the dopaminergic system is impaired in these mice . in recent years , impaired axonal transport has been identified as a central pathomechanism in ad [ 4651 ] . conceptually , impaired axonal transport is linked to oxidative stress and mitochondrial dysfunction : mitochondria need to be efficiently transported along the long axonal processes for neurons to function ; furthermore they are a major source of reactive oxygen species ( ros ) [ 53 , 54 ] . we found a selectively impaired axonal transport of distinct cargos including mitochondria and th- ( tyrosine hydroxylase- ) containing vesicles . at the molecular level , we found that this impaired transport phenotype is due to trapping of a component of the kinesin motor machinery , the adapter protein jip1 , by elevated levels of phosphorylated tau , preventing jip1 from executing its physiological function in the axon . a pathological interaction between tau and jip1 was further revealed in ad and not , control brain , highlighting the validity of transgenic animal models in dissecting pathomechanisms in ad . as tau only traps jip1 when it is hyperphosphorylated , this study presents inhibition of abnormal hyperphosphorylation of tau as a promising therapeutic strategy in ad . with the pr5 and the k3 mice we do have two complementary models at hand that present themselves for therapeutic intervention : both strains are characterized by hyperphosphorylation and increased insolubility of tau as well as silver - positive inclusions , gallyas - positive nfts in the case of r5 mice , and bielschowsky - positive inclusions predominantly resembling pick bodies in k3 mice . the pr5 mice show an accelerated extinction in the conditioned taste aversion ( cta ) paradigm , while the k3 mice have a memory phenotype as determined by the novel object recognition test , as well as a motor phenotype that can be easily monitored either by scoring of clasping , gait , and tremor ( table 1 ) or on the rota - rod [ 22 , 57 ] . in addition , as the k3 mice age , they lose substantial numbers of neurons both in the substantia nigra ( th - positive neurons ) and in the cerebellum ( basket cells ) [ 22 , 57 ] . experimental diabetes exacerbates the tau pathology as shown for pr5 mice . as therapeutic strategies , different approaches can be envisaged such as the transplantation of cells with the potential to differentiate in situ either into neuronal or glial cell types . this strategy has been successfully applied to mice with a combined tau and a pathology in which neuronal stem cell transplantation has been shown to improve cognition via brain - derived neurotrophic factor ( bdnf ) . we used both active and passive immunization and monitored efficacy of this approach biochemically , histologically , and behaviourally . we have also been testing a small bioavailable compound , sodium selenate that can be supplied with the drinking water . chronic treatment over a period of four months significantly improved the pathology in both young and old k3 mice . this was associated with reduced tau hyperphosphorylation and decreased numbers of tau inclusions including spheroids . in fact , spheroids were completely absent from frontal cortex , suggesting that selenate reverts the functional impairment leading to spheroids . k3 mice are characterized by a substantial , age - dependent degeneration of cerebellar basket neurons , resulting in the absence of pinceau terminals formed by clustered axons surrounding purkinje cells . these findings show that selenate reduces tau phosphorylation and deposition , mitigates pathological spheroid formation , and prevents axonal degeneration of distinct neuronal populations in k3 mice . we also treated 8-month - old pr5 mice with sodium selenate and found that treatment caused a significant impairment in the cta paradigm . as for the k3 mice , selenate both caused reduced hyperphosphorylation and numbers in tau deposits ( nfts ) as well as increased tau solubility . nft numbers in 12-month - old selenate - treated pr5 mice were similar to those in 8-month - old - untreated pr5 mice , suggesting that treatment had halted disease progression . we further showed that the selenate - induced improvements require protein phosphatase 2a ( pp2a ) , by performing in vitro experiments and showing inefficacy of selenate in mice with a combined tau pathology and reductions in pp2a activity [ 66 , 67 ] . while in the ad and ageing field mouse strains are available with traits that arose spontaneously and have been further established by specific breeding programs such as the senescence - accelerated sam mice , the rate at which new models arise spontaneously means that , even with a concerted effort , discovery is on an asymptomatic trajectory towards a phenotype gap . the concept of genomewide mutagenesis is well established in invertebrates such as the fruit fly drosophila melanogaster and in the nematode worm caenorhabditis elegans , with enu mutagenesis mainly producing a to t and t to a transversions . this mutagenesis strategy was used to create the first genetic maps of mendelian inheritance in drosophila , revealing many of the key genes controlling early embryonic patterning and development . what makes it possible to carry out comparable efforts in mice is the extraordinary mutagenic efficiency of enu in mouse spermatogonial stem cells . by expanding the library of alleles with chemical mutagenesis using n - ethyl - n - nitosourea ( enu ) , identification of each of the single nucleotide changes that result in a particular phenotype is feasible in mice and is now providing novel insights into not only immunity , where this approach has been used widely , but also the functioning of the brain . genomewide , random mutagenesis with the ethylating chemical enu provides a way to produce and screen genetic variants in the mouse . intraperitoneal injection of male founder mice ( g0 ) causes random base pair substitutions in the dna of spermatogonial stem cells . informative mutations are identified by screening members of the pedigrees propagated from these founders for phenotypes of interest . introducing point mutations has the advantage that it most often inactivates or alters the function of individual protein domains rather than eliminating the protein altogether ( as is the case for complete knock - outs ) , making it harder for related proteins to compensate , better mimicking the effects of drugs or natural genetic variants , illuminating active sites in proteins , and revealing specific functions of alternatively spliced forms . we have been investigating a phenotype modulation of our k3 mice by crossing enu male founder mice with k3 females as discussed hereinafter . here , and in general , dominant mutations are expected to manifest in the first generation offspring ( g1 ) , while recessive mutations are brought to homozygosity in g3 mice by either g1 . the rate of recessive mutations has been established based on genotype - driven projects using libraries of g1 gametes generated from an enu - treated founder . sequencing of sentinel regions of the genome of g1 sperm provides quantitative and qualitative characteristics of enu mutagenesis and information about the probable yield of mutations from phenotype - driven screens . the consensus is a mutation rate of 0.51.0 mutations / mbp ( 7.5 107 mutations / bp / g0 gamete , or 15003000 mutations / g1 ) from a standard regimen ( 100 mg / kg / week 3 for a c57bl/6 founder ) . assuming a 1600 cm 2.6 gb haploid mouse genome , of which approximately 1.5% is coding , this comes down to approximately 50 functional mutations in each g1 . the efficiency of enu - induced mutation means that the generation of allelic series is highly feasible . based on a conservative estimate of 20 point mutations per g1 gamete , a survey of 17400 g3s would yield a series of 5 functional alleles per gene with > 99% probability . limiting factors of the enu mutagenesis approach are infertility and lethality , with the standard enu regimen resulting in azoospermia ( absence of motile sperm in semen ) for up to 10 weeks , with fertility being resumed when the testes are repopulated by spermatogonia . the presence of mutations that confer lethality before weaning sets the workable upper limit of the enu dose even more stringently . for example , one study found that 1/13 g1 mice contained lethal mutations in the balancer chromosome 11 region that contains 5% of the genome , with the consequence that 18% of the g3 population will not survive because of lethal mutations . for phenotype - driven strategies ( such as a motor phenotype , see the following ) , f1 and f3 mice are examined for dominant - acting and recessive - acting mutations , respectively [ 23 , 82 ] . the recovery rate of dominant mutations is low ( 2% ) ; on the other hand however , costs for identifying recessive mutations are much higher , due to much higher mouse numbers and sample size to be screened and sequenced . in an ideal screen , the phenotypes are qualtitative ( such as having a motor phenotype ) or binomial ( rejection of transplanted tissue or not ) ; they are free of false positives and are sufficiently penetrant to permit meiotic mapping . the latter is typically achieved first by outcrossing and then an f1 intercrossing to yield f2 mice ; 50 f2 mice are required to obtain a 10 mb mapping resolution with a 95% confidence , although haplotype mapping might enable an estimation of the chromosomal location using as few as 10 affected f2 mice . for a more detailed discussion of the likelihood of a confounding mutation cosegregating with the phenotype the reader is referred to two excellent reviews on the topic [ 23 , 69 ] . what makes the approach feasible is a simple read - out , and in this respect , motor phenotypes are much easier to monitor than memory phenotypes . as in our k3 mice , k369i mutant tau expression causes both a motor and memory impairment , changes to the motor phenotype by any intervention although interesting in itself can also serve as a surrogate marker for changes in memory functions . we initiated an enu mutagenesis program in k3 mice that generated transgene - dependent and -independent pedigrees . in the following we will be discussing five pedigrees , enu164 ( partial rescue , transgene - related ) , enu67 ( slow weight gain , transgene - related ) , enu37 ( circling , not transgene - related ) , enu12/30a ( motor phenotype , not transgene - related ) , and enu16/069 ( motor phenotype , not transgene - related ) . to first establish that the k3 phenotype is fully penetrant on the background chosen for enu mutagenesis , we established backcrosses with 20 mice each , using c57bl/10 , c3h / h3h , fvb , c57led , and balb / b mice , all of which are established enu strains at the apf ( australian phenomics facility , canberra ) . we decided to use balb / b as the strain for enu mutagenesis , as the k3 phenotype of parkinsonism is fully penetrant on this background , both with regards to phenotype and age - of - onset . therefore , balb / b males were injected with enu and paired with the k3 mice ( that are on a c57bl/6 background ) , and the f1 offspring was analyzed . the litters born were scored for three read - outs of parkinsonism in mice , tremor ( staging 03 ) , gait anomalies ( staging 03 ) , limb clasping ( staging 05 ) , as well as body weight ( table 1 ) . we had previously phenotyped wild - type balb / b mice for the three parkinsonism characteristics and weight . although none of them displayed abnormal gait or resting tremor , 37% did clasp their hind limbs when stimulated and 5% clasped their hind limbs without stimulation , while their front paws grasped the cage wire . therefore , to ensure accurate identification of the phenotype , the hindlimb clasping alone was not used as sole indicator , but in conjunction with tremor and abnormal gait . phenotyping was done at weaning , and then at one - week intervals until around 8 weeks of age , depending on the scoring outcome . any mice exhibiting all three phenotypes and genotyped k3-tau positive were culled , as were any mice failing to display any of the phenotypes and genotyped k3-tau negative . mice which showed a genotype / phenotype mismatch were genotyped a second time for confirmation and phenotyped for up to 3 months . any mice which did not display the phenotype until after 5 weeks of age were also noted as having delayed onset of the phenotype and were flagged as a putant ( potential mutant ) of interest . this was followed by next generation whole exome sequencing as a means to identifying mutations . we would like to make the comment that while , previously , we have been mapping the enu mutations down to a chromosomal region for most strains these days it is much faster to directly next generation sequence the exomes of one to two affected mice instead . this has greatly accelerated the speed of discovery from years to months and reduced costs of identifying causal mutations . the snps identified with the sequencing were then validated against the phenotype with an amplifluor snp assay for each snp . hereinafter we will be discussing five pedigrees for which we have arrived at different stages of out - crossing and identification of the underlying gene mutations , starting with those pedigrees with which we have the least progressed . enu164 pedigreethis pedigree is characterized by a partial rescue of the parkinsonism phenotype that characterizes the k3 mice , because some transgene - positive mice in the pedigree showed an improvement in our vigorous weekly scorings ; for example , at week 6 they would show a scoring of 1/2/1 for gait / clasp / tremor instead of 3/4/3 as is typical for k3 mice . as mentioned above , consecutive backcrossing onto c57bl/6 reduces the number of functional enu - induced point mutations such that in the 3rd generation ( f3 ) they are reduced to 3 - 4 mutations . as the k3 mice have been generated on a c57bl/6 background , while enu mutagenesis has been on a balb / b inbred genetic background , strain - specific snp detection enabled the genomic mapping of the causative mutation location on the balb / b background . this narrows down the linked mutation - carrying region to less than 20 mb , at which point candidate genes in the region will be sequenced to detect mutant alleles , and eventually , the causative mutation . 6 of these showed the inherited partial rescue phenotype ( ~15% inheritance ) . from f2 # 483 we screened 11 k3-positive mice , with 3 showing partial rescue ( ~27% ) , from f2 # 430 20 k3-positive mice ( ~35% ) . exome ( ngs ) next generation sequencing is ongoing to identify the one single gene mutation that causes the k3 modulating phenotype . this pedigree is characterized by a partial rescue of the parkinsonism phenotype that characterizes the k3 mice , because some transgene - positive mice in the pedigree showed an improvement in our vigorous weekly scorings ; for example , at week 6 they would show a scoring of 1/2/1 for gait / clasp / tremor instead of 3/4/3 as is typical for k3 mice . as mentioned above , consecutive backcrossing onto c57bl/6 reduces the number of functional enu - induced point mutations such that in the 3rd generation ( f3 ) they are reduced to 3 - 4 mutations . as the k3 mice have been generated on a c57bl/6 background , while enu mutagenesis has been on a balb / b inbred genetic background , strain - specific snp detection enabled the genomic mapping of the causative mutation location on the balb / b background . this narrows down the linked mutation - carrying region to less than 20 mb , at which point candidate genes in the region will be sequenced to detect mutant alleles , and eventually , the causative mutation . 6 of these showed the inherited partial rescue phenotype ( ~15% inheritance ) . from f2 # 483 we screened 11 k3-positive mice , with 3 showing partial rescue ( ~27% ) , from f2 # 430 20 k3-positive mice ( ~35% ) . exome ( ngs ) next generation sequencing is ongoing to identify the one single gene mutation that causes the k3 modulating phenotype . enu67 pedigreethis pedigree shows a worsening of the weight phenotype that characterises the k3 mice . for example , while the average weight of transgene - negative mice is 19,0 grams at six weeks of age , this is reduced to 15.8 grams for transgene - positive k3 mice and is even further reduced in the enu67 pedigree ( k3-positive ) that shows an average weight of only 11.7 grams . both k3-positive mice ( # 347 , # 350 ) were identified as slow to gain weight , and we are currently crossing further to determine the underlying gene mutation . for example , while the average weight of transgene - negative mice is 19,0 grams at six weeks of age , this is reduced to 15.8 grams for transgene - positive k3 mice and is even further reduced in the enu67 pedigree ( k3-positive ) that shows an average weight of only 11.7 grams . both k3-positive mice ( # 347 , # 350 ) were identified as slow to gain weight , and we are currently crossing further to determine the underlying gene mutation . the trait shows mendelian inheritance over three generations . following deep sequencing of the exome of 2 affected mice we identified possible causal point mutations in the following genes:5730455p16rik ( riken cdna);tcof1 ( treacher collins franceschetti syndrome 1 ) , responsible for the production of treacle , which plays a role in the formation of tissue in the face . the human disease is also autosomal dominant;tbx1 ( t - box 1 gene ) t - box genes encode transcription factors involved in the regulation of developmental processes . chromosomal deletions in this region have been linked to neural - crest - related defects . a point mutation could potentially cause a mild neural phenotype;senp7 ( sumo1/sentrin specific peptidase 7 gene ) involved in the posttranslational modification of proteins by the addition of ubiquitin - like sumo proteins;ggt7 ( gamma - glutamyltransferase 7 gene ) : this gene is a member of a family that encodes enzymes involved in both the metabolism of glutathione and in the transpeptidation of amino acids ; changes in the activity of -glutamyltransferase may signal preneoplastic or toxic conditions in the liver or kidney in humans;hkdc1 ( hexokinase domain containing 1 gene ) ; chrna4 ( cholinergic receptor , nicotinic , alpha polypeptide 4 gene ) : this gene encodes a nicotinic acetylcholine receptor . polymorphisms in this gene provide protection against nicotine addiction in humans.amplifluor primers were designed to the above snps and affected and unaffected mice genotyped , which led to the confirmation of the causal mutation in the tbx1 gene , with a t to a substitution of bp 857 ( coding position ) resulting in an asn to ile change of amino acid 276 . a complete knock - out of tbx1 is available and is a model of digeorge syndrome ; it is further characterized by perinatal death [ 87 , 88 ] . the digeorge syndrome is caused by a 1.5 to 3.0 mb hemizygous deletion of chromosome 22q11.2 . haploinsufficiency of the tbx1 gene in particular is responsible for most of the physical malformations . there is evidence that point mutations in the tbx1 gene can also cause the disorder . digeorge syndrome is the most frequent microdeletion syndrome in humans , and is characterized by cardiovascular , thymic and parathyroid , and craniofacial anomalies . the underlying cause is disturbed formation of the pharyngeal apparatus , a transient structure present during vertebrate development that gives rise to endocrine glands , craniofacial tissue , and the cardiac outflow tract . the pharyngeal apparatus is composed of derivatives of ectoderm , endoderm , mesoderm , and the neural crest . thus , complex interactions between cell types from different origins have to be orchestrated in the correct spatiotemporal manner to establish proper formation of the pharyngeal system . the analysis of engineered mouse mutants developing a phenotype resembling digeorge syndrome has revealed genes and signalling pathways crucial for this process . intriguingly , these mouse models reveal that interference with either of two distinct phases of pharyngeal apparatus development can contribute to the aetiology of digeorge syndrome . with regards to ad , furthermore , string 9.0 known and predicted protein - protein interactions for tbx1 identify sept5 which is also among 29 genes that are deregulated in pr5 tau transgenic mice ( jg and lim , manuscript submitted ) . the value of the enu37 strain lies in the absence of a perinatal phenotype which allows for studies which are not feasible with the complete knockout . the trait shows mendelian inheritance over three generations . following deep sequencing of the exome of 2 affected mice we identified possible causal point mutations in the following genes:5730455p16rik ( riken cdna);tcof1 ( treacher collins franceschetti syndrome 1 ) , responsible for the production of treacle , which plays a role in the formation of tissue in the face . the human disease is also autosomal dominant;tbx1 ( t - box 1 gene ) t - box genes encode transcription factors involved in the regulation of developmental processes . chromosomal deletions in this region have been linked to neural - crest - related defects . a point mutation could potentially cause a mild neural phenotype;senp7 ( sumo1/sentrin specific peptidase 7 gene ) involved in the posttranslational modification of proteins by the addition of ubiquitin - like sumo proteins;ggt7 ( gamma - glutamyltransferase 7 gene ) : this gene is a member of a family that encodes enzymes involved in both the metabolism of glutathione and in the transpeptidation of amino acids ; changes in the activity of -glutamyltransferase may signal preneoplastic or toxic conditions in the liver or kidney in humans;hkdc1 ( hexokinase domain containing 1 gene ) ; chrna4 ( cholinergic receptor , nicotinic , alpha polypeptide 4 gene ) : this gene encodes a nicotinic acetylcholine receptor . polymorphisms in this gene provide protection against nicotine addiction in humans.amplifluor primers were designed to the above snps and affected and unaffected mice genotyped , which led to the confirmation of the causal mutation in the tbx1 gene , with a t to a substitution of bp 857 ( coding position ) resulting in an asn to ile change of amino acid 276 . a complete knock - out of tbx1 is available and is a model of digeorge syndrome ; it is further characterized by perinatal death [ 87 , 88 ] . the digeorge syndrome is caused by a 1.5 to 3.0 mb hemizygous deletion of chromosome 22q11.2 . haploinsufficiency of the tbx1 gene in particular is responsible for most of the physical malformations . there is evidence that point mutations in the tbx1 gene can also cause the disorder . digeorge syndrome is the most frequent microdeletion syndrome in humans , and is characterized by cardiovascular , thymic and parathyroid , and craniofacial anomalies . the underlying cause is disturbed formation of the pharyngeal apparatus , a transient structure present during vertebrate development that gives rise to endocrine glands , craniofacial tissue , and the cardiac outflow tract . the pharyngeal apparatus is composed of derivatives of ectoderm , endoderm , mesoderm , and the neural crest . thus , complex interactions between cell types from different origins have to be orchestrated in the correct spatiotemporal manner to establish proper formation of the pharyngeal system . the analysis of engineered mouse mutants developing a phenotype resembling digeorge syndrome has revealed genes and signalling pathways crucial for this process . intriguingly , these mouse models reveal that interference with either of two distinct phases of pharyngeal apparatus development can contribute to the aetiology of digeorge syndrome . with regards to ad , furthermore , string 9.0 known and predicted protein - protein interactions for tbx1 identify sept5 which is also among 29 genes that are deregulated in pr5 tau transgenic mice ( jg and lim , manuscript submitted ) . the value of the enu37 strain lies in the absence of a perinatal phenotype which allows for studies which are not feasible with the complete knockout . 5730455p16rik ( riken cdna ) ; tcof1 ( treacher collins franceschetti syndrome 1 ) , responsible for the production of treacle , which plays a role in the formation of tissue in the face . the human disease is also autosomal dominant ; tbx1 ( t - box 1 gene ) t - box genes encode transcription factors involved in the regulation of developmental processes . chromosomal deletions in this region have been linked to neural - crest - related defects . a point mutation could potentially cause a mild neural phenotype ; senp7 ( sumo1/sentrin specific peptidase 7 gene ) involved in the posttranslational modification of proteins by the addition of ubiquitin - like sumo proteins ; ggt7 ( gamma - glutamyltransferase 7 gene ) : this gene is a member of a family that encodes enzymes involved in both the metabolism of glutathione and in the transpeptidation of amino acids ; changes in the activity of -glutamyltransferase may signal preneoplastic or toxic conditions in the liver or kidney in humans ; hkdc1 ( hexokinase domain containing 1 gene ) ; chrna4 ( cholinergic receptor , nicotinic , alpha polypeptide 4 gene ) : this gene encodes a nicotinic acetylcholine receptor . enu12/301 pedigree ( in short : enu12)enu12 mice , under mild stress , show sudden jerky movements when moving around the cage and appear to tremor constantly . some mice show what could be described as opisthotonus ( a type of spasm in which the head and heels arch backwards in extreme hyperextension and the body forms a reverse bow ) . the mice display varying degrees of severity ; even when severely affected , they are able to move freely and quickly around the cage . they occasionally fall to either side and circle around the cage in very fast movements when highly stressed , such as when the cage is changed . the enu12 phenotype can be recognised at weaning.deep sequencing identified an a to t substitution of bp 1408 in exon 10 of the kcnq1 gene that results in a lys to stop change of amino acid 435 . the kcnq1 gene encodes a voltage - gated potassium channel protein required for the repolarisation phase of the cardiac action potential . the gene product can form multimers with two other potassium channel proteins , kcne1 and kcne3 . inherited forms of a human condition known as long qt syndrome ( lqts ) , an abnormality in the cardiac ventricular repolarisation characterised by qt interval prolongation and abnormal t - waves on an electrocardiogram ( ecg ) , are most commonly associated with mutations in kcqn1 . some patients show only a cardiac defect ( romano - ward syndrome , rws , dominant ) ; others may suffer from severe deafness due to a lack of endolymph in the vestibular and auditory compartment ( jervell and lange - nielsen syndrome , jlns , recessive ) .the complete knockout is a model of jnls , but the mice show an additional circling ( shaker / waltzer ) and rapid head blobbing phenotype . introducing the familial a340e point mutation by a knock - in approach models rws ( cardiac phenotype , but no inner ear defect ) . this point mutation is in the amino - terminus of kcnq1 that contains the six transmembrane domains s1-s6 and the pore domain p . a comparative analysis of the strains allowed the authors to draw conclusions with regards to the etiology of the cardiac phenotype . the k434stop mutation in the enu12 mice is in the carboxy - terminus that contains four helical regions , of which two form coiled - coil assemblies . the enu12 strain has a phenotype similar to the complete knock - out demonstrating an essential role for the helical domain in kcnq1 function . enu12 mice , under mild stress , show sudden jerky movements when moving around the cage and appear to tremor constantly . some mice show what could be described as opisthotonus ( a type of spasm in which the head and heels arch backwards in extreme hyperextension and the body forms a reverse bow ) . the mice display varying degrees of severity ; even when severely affected , they are able to move freely and quickly around the cage . they occasionally fall to either side and circle around the cage in very fast movements when highly stressed , such as when the cage is changed . deep sequencing identified an a to t substitution of bp 1408 in exon 10 of the kcnq1 gene that results in a lys to stop change of amino acid 435 . the kcnq1 gene encodes a voltage - gated potassium channel protein required for the repolarisation phase of the cardiac action potential . the gene product can form multimers with two other potassium channel proteins , kcne1 and kcne3 . inherited forms of a human condition known as long qt syndrome ( lqts ) , an abnormality in the cardiac ventricular repolarisation characterised by qt interval prolongation and abnormal t - waves on an electrocardiogram ( ecg ) , are most commonly associated with mutations in kcqn1 . some patients show only a cardiac defect ( romano - ward syndrome , rws , dominant ) ; others may suffer from severe deafness due to a lack of endolymph in the vestibular and auditory compartment ( jervell and lange - nielsen syndrome , jlns , recessive ) . the complete knockout is a model of jnls , but the mice show an additional circling ( shaker / waltzer ) and rapid head blobbing phenotype . introducing the familial a340e point mutation by a knock - in approach models rws ( cardiac phenotype , but no inner ear defect ) . this point mutation is in the amino - terminus of kcnq1 that contains the six transmembrane domains s1-s6 and the pore domain p . a comparative analysis of the strains allowed the authors to draw conclusions with regards to the etiology of the cardiac phenotype . the k434stop mutation in the enu12 mice is in the carboxy - terminus that contains four helical regions , of which two form coiled - coil assemblies . the enu12 strain has a phenotype similar to the complete knock - out demonstrating an essential role for the helical domain in kcnq1 function . enu16/069 pedigree ( in short : enu16)with minimal stress , enu16 mice display a mild action tremor . with moderate stress the tremor worsens and the mice display an unusual , hypermetric gait in their hind limbs . upon restraining of the tail either one or both hind limbs show dramatic clasping , and when returned to the cage , it takes some time for the mice to regain control over the limbs with what looks to be a sporadic and short - term lack of control of the hind limbs . within seconds of being returned to the cage post restraint the gait appears to improve but can persist and be quite unsteady and waddle - like . the phenotype is noticeable from 4 to 5 weeks of age.deep sequencing identified an a to g substitution of bp 530 in exon 5 of the mpz ( myelin protein zero ) gene resulting in an asp to gly change of aa 121 . aliases of mpz are mpp , p - zero , and p0 , among others . schwann cells , the glia of the peripheral nervous system ( pns ) , play pivotal roles in the development and maintenance of the pns . in addition to forming the myelin sheath , schwann cells are involved in neuronal survival where they also provide support to axons during development and throughout adulthood . the mpz gene encodes a transmembrane glycoprotein that is the major structural protein of peripheral myelin . mutations in the mpz gene result in the autosomal dominant form of charcot - marie - tooth disease type 1 ( cmt1b ) . this domain is important for the homotypic interactions that convey the self - adhesive properties that are essential to maintain myelin compaction and integrity .the complete p0 knockout causes a severe demyelinating neuropathy , but the function of p0 is still not fully resolved . axons in their peripheral nerves are severely hypomyelinated , and a subset contain myelin - like figures and axons degenerate . a second strain is available from jackson laboratories : b6.cg-mpz/grsrj mice carry the spontaneous totterer mutation . homozygous mice are poor breeders , reproducing at most once or twice , and as females are poor mothers , use of the strain is limited . besides the totterer strain , for p0 there is only a complete knock - out available , and not a knock - in strain . the enu16 strain has an n121 g point mutation that is juxtaposed to the only known glycosylation site in the protein . we found however by immunohistochemistry and western blot analysis that the mutated protein ( p0 n121 g ) is expressed at a pattern and at levels similar to wild - type . this allows us to employ the enu16 strain in studies ( such as of adhesion ) that are not feasible with the complete knockout . with minimal stress , the tremor worsens and the mice display an unusual , hypermetric gait in their hind limbs . upon restraining of the tail either one or both hind limbs show dramatic clasping , and when returned to the cage , it takes some time for the mice to regain control over the limbs with what looks to be a sporadic and short - term lack of control of the hind limbs . within seconds of being returned to the cage post restraint the gait appears to improve but can persist and be quite unsteady and waddle - like . deep sequencing identified an a to g substitution of bp 530 in exon 5 of the mpz ( myelin protein zero ) gene resulting in an asp to gly change of aa 121 . aliases of mpz are mpp , p - zero , and p0 , among others . schwann cells , the glia of the peripheral nervous system ( pns ) , play pivotal roles in the development and maintenance of the pns . in addition to forming the myelin sheath , schwann cells are involved in neuronal survival where they also provide support to axons during development and throughout adulthood . the mpz gene encodes a transmembrane glycoprotein that is the major structural protein of peripheral myelin . mutations in the mpz gene result in the autosomal dominant form of charcot - marie - tooth disease type 1 ( cmt1b ) . this domain is important for the homotypic interactions that convey the self - adhesive properties that are essential to maintain myelin compaction and integrity . the complete p0 knockout causes a severe demyelinating neuropathy , but the function of p0 is still not fully resolved . axons in their peripheral nerves are severely hypomyelinated , and a subset contain myelin - like figures and axons degenerate . a second strain is available from jackson laboratories : b6.cg-mpz/grsrj mice carry the spontaneous totterer mutation . homozygous mice are poor breeders , reproducing at most once or twice , and as females are poor mothers , use of the strain is limited . besides the totterer strain , for p0 there is only a complete knock - out available , and not a knock - in strain . the enu16 strain has an n121 g point mutation that is juxtaposed to the only known glycosylation site in the protein . we found however by immunohistochemistry and western blot analysis that the mutated protein ( p0 n121 g ) is expressed at a pattern and at levels similar to wild - type . this allows us to employ the enu16 strain in studies ( such as of adhesion ) that are not feasible with the complete knockout . this overview of pedigrees with motor phenotypes reveals that mice are amenable to enu mutagenesis and that it is possible to establish interesting phenotypes . compared to the functional genomics approaches we have pursued in the past [ 99102 ] , enu mutagenesis is more directed as it not only identifies gene mutations that modulate an existing pathology but also has the added advantage of establishing mutant mouse strains at the same time . as outlined above , enu tends to introduce point mutations rather than causing deletions ; this results in the expression of mutant proteins at levels that are high enough to perform functional studies . a limitation in a more wide - spread application of enu mutagenesis though to mice is the high costs associated with establishing the mutants and identifying the underlying gene mutation and the fact that not all phenotypes are amenable to a robust and fast read - out . therefore , using the motor phenotype as a surrogate marker for memory functions ( as for the k3 transgene - dependent pedigrees ) is an avenue that can be pursued in the field of ad mouse models . what are the challenges of enu mutagenesis in transgenic models of neurodegeneration and what issues both practical and conceptual did we encounter while doing these studies ? in hindsight it appears to have been the preferred strategy to start with as many enu - mutagenised males for cross - breeding with k3 mice as possible in order to saturate the genome with mutations . also , we had initially planned to identify both dominant and recessive mutations , but considering the extensive breeding needed , we realised that such an undertaking is not affordable and therefore we concentrated solely on dominantly inherited traits . in principle the question that may be raised is which other transgenic models could be easily screened and used for enu mutagenesis . we believe that any strain with a phenotype that can be easily picked up is amenable to this strategy . in our case we have been using mice that are characterized by a robust tau - dependent motor and memory phenotype , so we assumed that any modification of the motor phenotype would also modify memory functions , a trait , which would be tested after the modification of the motor phenotype has been confirmed . another question is what the expectations are beyond finding possible new targets and how we see the field evolving in the years ahead . we have started to establish caenorhabditis elegans in the laboratory as a system complementary to mice and it seems that overall a mutagenesis screen in the roundworm system is easier . we still believe that there is a potential in applying enu mutagenesis to mouse models . these models might nicely complement ongoing concerted efforts such as those of the international knockout mouse consortium ( http://www.knockoutmouse.org/ ) . the main objectives of this consortium are to generate , archive , and distribute worldwide up to 12.000 conditional mutations across the mouse genome in mouse embryonic stem ( es ) cells . the main mutagenesis strategies pursued are : ( i ) conditional gene trapping a random approach for expressed genes ; ( ii ) conditional - targeted trapping a directed approach , used for expressed genes ; and ( iii ) conditional gene targeting a directed approach , used for non - expressed genes hopefully , these concerted efforts help in developing a cure for a disease for which only symptomatic treatment is available .

modifier screening is a powerful genetic tool . while not widely used in the vertebrate system , we applied these tools to transgenic mouse strains that recapitulate key aspects of alzheimer 's disease ( ad ) , such as tau - expressing mice . these are characterized by a robust pathology including both motor and memory impairment . the phenotype can be modulated by enu mutagenesis , which results in novel mutant mouse strains and allows identifying the underlying gene / mutation . here we discuss this strategy in detail . we firstly obtained pedigrees that modify the tau - related motor phenotype , with mapping ongoing . we further obtained transgene - independent motor pedigrees : ( i ) hyperactive , circling enu 37 mice with a causal mutation in the tbx1 gene the complete knock - out of tbx1 models digeorge syndrome ; ( ii ) enu12/301 mice that show sudden jerky movements and tremor constantly ; they have a causal mutation in the kcnq1 gene , modelling aspects of the romano - ward and jervell and lange - nielsen syndromes ; and ( iii ) enu16/069 mice with tremor and hypermetric gait that have a causal mutation in the mpz ( myelin protein zero ) gene , modelling charcot - marie - tooth disease type 1 ( cmt1b ) . together , we provide evidence for a real potential of an enu mutagenesis to dissect motor functions in wild - type and tau mutant mice .

1. Introduction 2. Tau Transgenic Mouse Models with Memory and Motor Phenotypes 3. Modulation of Transgenic Phenotype by Therapeutic Intervention 4. ENU Mutagenesis Strategies in Mice 5. ENU Mutagenesis Applied to K3 Mice Identifies Transgene-Dependent and -Independent Pedigrees 6. Outlook

fluoro refers to both fluorescent and fluorinated compounds . despite the shared prefix , there are very few fluorescent molecules that are soluble in perfluorinated solvents . this paucity is surprising , given that optical microscopy is a ubiquitous technique throughout the physical sciences and the orthogonality of fluorous materials is a commonly exploited strategy in synthetic chemistry , materials science , and chemical biology . we have addressed this shortage by synthesizing a panel of fluorofluorophores , fluorescent molecules containing high weight percent fluorine with optical properties spanning the visible spectrum . we demonstrate the utility of these fluorofluorophores by preparing fluorescent perfluorocarbon nanoemulsions .

Supplementary Material

as the number of patients with unruptured cerebral aneurysms has increased , more attention has been given to the primary prevention of the aneurysm rupture4 ) . endovascular intervention , one of the major treatment modalities , has begun to catch up with and surpass the surgery in the number of cases treated , based on the favorable outcomes and reduced morbidity / mortality21220 ) . in addition , recently , patients have shown a preference for endovascular intervention over surgery because of the cosmetic and psychological aspects . scalpel surgeons are being asked to enhance their competitiveness and develop novel surgical devices and skills , such as minimally invasive keyhole surgery81519 ) , neuroendoscopic systems7 ) , indocyanine green ( icg ) fluorescence angiography518 ) , intraoperative neurophysiological monitoring16 ) , and intraoperative cerebral angiography1 ) . microscopic icg fluorescence angiography is very helpful for detecting and readjusting unexpected arterial occlusion and incomplete clipping of cerebral aneurysms . however , the usefulness of icg fluorescence angiography is limited when the microscopic view is poorly visualized or when icg can not absorb sufficient light to emit fluorescence . therefore , these limitations can be overcome using the endoscopic systems that provide sufficient light illumination and excellent visualization of the operative fields . thus , icg fluorescence angiography would be very useful to supplement the shortcomings of microscopic icg fluorescence . to date , there are 3 clinical reports regarding endoscopic icg fluorescence angiography from japan and europe51114 ) . we independently developed an endoscopic camera system for icg fluorescence angiography that can show the real - time simultaneous visible and fluorescent imaging . using this system , all procedures were approved by the local institutional animal care and use committee and were conducted according to international guidelines . a male crossbred swine ( landraceyorkshireduroc ) , aged 3 months and with a weight of 38.5 kg , was used in this study . anesthesia was induced intramuscularly with 5 mg / kg of zoletil and 2 mg / kg of xylazine . the swine was intubated and intravenous 0.9% normal saline was administered via the ear vein at a rate of 5 ml / kg / hr . anesthesia was maintained with 2% to 4% isoflurane at a rate of 23 l / min and oxygen / nitrogen at a rate of 3 l / min . vital parameters , such as arterial blood pressure , heart rate , and carbon dioxide levels , were continuously recorded . a 12 cm small craniotomy was performed at a distance of 2 cm from the midline and 3 cm anterior to the bregma . the icg dye was injected into an ear vein at a dose of 0.3 mg / kg ( 25 mg dissolved in 5 ml of sterile normal saline ) . the endoscopic icg system consists of an endoscope , a light source , a camera , a display , and a computer with software ( fig . 1 ) . although all types of endoscopes with the same size of optical adaptor are adjustable , an endoscope 4 mm in diameter and 30 in angle ( mgb endoskopische gerate gmbh berlin , berlin , germany ) was used in this study . the light source is composed of a white light source and a near infrared ( nir ) laser source ( fig . we developed a white light source that is installed with a hxp 120w/45c vis mercury lamp ( osram gmbh , munich , germany ) . we also developed a nir laser source that is installed with an 805 nm wavelength laser diode ( reallight , beijing , china ; fwhm < 2 nm ) . icg as a fluorophore binds to the hydrophobic core of human serum proteins , which shifts the absorption and emission spectra of icg . after intravenous injection of icg , the main peak of the absorption spectrum of icg moves to 805 nm , so we chose an 805 nm laser that has the same wavelength as the icg absorption peak6 ) . to deliver the combined light from the light source to the endoscope , we selected a 3 mm liquid light guide ( newport corporation , irvine , ca , usa ) . to simultaneously obtain the visible and icg fluorescence images , we used a fluorescence navigation surgery ( fns ) camera developed by korea electrotechnology research institute ( keri ) ( fig . this camera has two charge - coupled device ( ccd ) image sensors : one is for visible color imaging , and the other is for nir fluorescence imaging . a cube beam splitter was installed to divide the visible color image and the nir fluorescence image . the reflected visible color image passes through an infrared ( ir ) cut - off filter to the color - image sensor and the penetrated nir image goes to the mono - image sensor . the two images are processed on an image capture board and the data transfer board delivers the real time imaging to the computer by usb 3.0 interface . we used a 1634 mm zoom optical adaptor ( mgb endoskopische gerate gmbh berlin , berlin , germany ) to make the optical connection between the endoscope and the camera . the image monitoring program was developed by keri and can show and record single or simultaneous images of visible light and fluorescence and a visible / fluorescence overlay image . all procedures were approved by the local institutional animal care and use committee and were conducted according to international guidelines . a male crossbred swine ( landraceyorkshireduroc ) , aged 3 months and with a weight of 38.5 kg , was used in this study . anesthesia was induced intramuscularly with 5 mg / kg of zoletil and 2 mg / kg of xylazine . the swine was intubated and intravenous 0.9% normal saline was administered via the ear vein at a rate of 5 ml / kg / hr . anesthesia was maintained with 2% to 4% isoflurane at a rate of 23 l / min and oxygen / nitrogen at a rate of 3 l / min . vital parameters , such as arterial blood pressure , heart rate , and carbon dioxide levels , were continuously recorded . a 12 cm small craniotomy was performed at a distance of 2 cm from the midline and 3 cm anterior to the bregma . the icg dye was injected into an ear vein at a dose of 0.3 mg / kg ( 25 mg dissolved in 5 ml of sterile normal saline ) . the endoscopic icg system consists of an endoscope , a light source , a camera , a display , and a computer with software ( fig . although all types of endoscopes with the same size of optical adaptor are adjustable , an endoscope 4 mm in diameter and 30 in angle ( mgb endoskopische gerate gmbh berlin , berlin , germany ) was used in this study . the light source is composed of a white light source and a near infrared ( nir ) laser source ( fig . we developed a white light source that is installed with a hxp 120w/45c vis mercury lamp ( osram gmbh , munich , germany ) . we also developed a nir laser source that is installed with an 805 nm wavelength laser diode ( reallight , beijing , china ; fwhm < 2 nm ) . icg as a fluorophore binds to the hydrophobic core of human serum proteins , which shifts the absorption and emission spectra of icg . after intravenous injection of icg , the main peak of the absorption spectrum of icg moves to 805 nm , so we chose an 805 nm laser that has the same wavelength as the icg absorption peak6 ) . to deliver the combined light from the light source to the endoscope , we selected a 3 mm liquid light guide ( newport corporation , irvine , ca , usa ) . to simultaneously obtain the visible and icg fluorescence images , we used a fluorescence navigation surgery ( fns ) camera developed by korea electrotechnology research institute ( keri ) ( fig . this camera has two charge - coupled device ( ccd ) image sensors : one is for visible color imaging , and the other is for nir fluorescence imaging . a cube beam splitter was installed to divide the visible color image and the nir fluorescence image . the reflected visible color image passes through an infrared ( ir ) cut - off filter to the color - image sensor and the penetrated nir image goes to the mono - image sensor . the two images are processed on an image capture board and the data transfer board delivers the real time imaging to the computer by usb 3.0 interface . we used a 1634 mm zoom optical adaptor ( mgb endoskopische gerate gmbh berlin , berlin , germany ) to make the optical connection between the endoscope and the camera . the image monitoring program was developed by keri and can show and record single or simultaneous images of visible light and fluorescence and a visible / fluorescence overlay image . icg fluorescence began to appear on the cerebral cortex with endoscopy in approximately 25 seconds after icg dye injection , it became peak in 1 minute , then it became pale in approximately 5 minutes . visible color and icg fluorescent endoscopic images of cortical vessels were simultaneously observed on the display monitor with a high resolution . in addition , real - time merging images of the fluorescent cortical vessels could be made and marked in a purple color . the real - time merging of the visible color and fluorescent images corresponded well ( fig . since raabe et al.17 ) introduced microscopic icg fluorescence angiography for cerebrovascular surgery in 2003 , it has become a ubiquitous piece of equipment for aneurysm surgery as well as micro - anastomosis and surgery for arteriovenous fistulas and malformations . during aneurysm surgery , real - time identification of the blood flow in the vasculature within the operative field helps to improve surgical outcomes and reduce perioperative complications . this is because the repositioning and addition of clips can be promptly determined on the occlusion of parent arteries and perforators or incomplete clipping of the aneurysms . however , it is invasive and has a limitation to visualize the small perforators around the aneurysms in spite of the superiority in the acquisition of 3-dimensional imaging around the clipped aneurysms . in addition , supplementary facilities for intraoperative cerebral angiography are needed , and a time lag between the identification of compromise of the normal arteries and the repositioning of clips can result in an irreversible ischemic complication . on the other hand , microscopic icg angiography also has some shortcomings : vascular structures beyond the line of microscopic view are hard to visualize and icg fluorescence is weakly detected for the deeply located structures because of the limited spatial resolution and light illumination of microscopy . when keyhole surgery is performed , such limitations in microscopic icg angiography become more profound . to overcome the drawbacks of microscopic icg angiography in fact , endoscopic icg angiography has been applied in other surgical fields before it was introduced in the field of neurosurgery , based on the property of icg , which is circulated through the blood vessels , metabolized in the liver , and excreted via the bile duct into the intestines . examples of the surgical applications of this technique include the detection of the sentinel lymph nodes of patients with gastric cancer13 ) , visualization of bleeding points in the gastrointestinal tract9 ) , assessment of tissue perfusion and bile tree in hepatopancreatobiliary surgery22 ) , and evaluation of free flap perfusion3 ) . neuroendoscopy itself has been used mainly in skull base surgery via the transnasal approach and aneurysm surgery71021 ) . meanwhile , the use of endoscopic icg angiography in aneurysm surgery has recently been introduced5614 ) . the authors consider endoscopic icg angiography to be useful for confirming the occlusion of the aneurysms and the patency of blood flow in the parent arteries and perforators , which could not be visualized under the microscopy and its icg angiography . moreover , it is possible in the endoscopic icg angiography to better magnify the areas of interest5 ) , obtain longer icg fluorescence duration , and be more useful in the ruptured aneurysms11 ) , compared to the microscopic system . light illumination from microscopy alone the advantage of our endoscopic icg angiography system is that icg fluorescence and visible color imaging can be simultaneously visualized and merged in a real - time manner . previous microscopic or endoscopic icg angiography systems can not help but to demonstrate the visible color or nir fluorescence imaging one at a time such that surgeons may feel some difficulty in comparing the real structures with fluorescence images during the surgery . however , the size of our prototype camera is larger than the previous one , which is an improvement . first , an endoscope with smaller diameter and flexibility may be more useful because the operative space is very narrow without retracting brain tissue or drilling bony structures . as the diameter becomes smaller , imaging resolution and transmission of the light illumination and emitting fluorescence tend to be diminished . second , distinct from the endonasal approach , which is performed through the bony nasal cavity , endoscopy with icg angiography during aneurysm surgery reaches and stays around the targeted vascular structures intradurally adjacent to the brain under microscopic inspection . separate displays for microscopy and endoscopy may be detrimental and time - consuming because endoscopy can damage the adjacent cerebrovascular structures . a unified display system that can visualize both the microscopic and endoscopic imaging in one piece may be useful in terms of safety and convenience . different from the previous systems , it was possible to simultaneously display visible color and icg fluorescent images , and make a real - time merging images of them . technical supplementation and approval for the clinical implication are expected to support the application of aneurysm surgery as well as surgery for other cerebrovascular diseases .

objectivemicroscopic indocyanine green ( icg ) angiography is useful for identifying the completeness of aneurysm clipping and the preservation of parent arteries and small perforators . neuroendoscopy is helpful for visualizing structures beyond the straight line of the microscopic view . we evaluated our prototype of endoscopic icg fluorescence angiography in swine , which we developed in order to combine the merits of microscopic icg angiography and endoscopy.methodsour endoscopic icg system consists of a camera , a light source , a display and software . this system can simultaneously display real - time visible and near infrared fluorescence imaging on the same monitor . a commercially available endoscope was used , which was 4 mm in diameter and had an angle of 30. a male crossbred swine was used.resultsunder general anesthesia , a small craniotomy was performed and the brain surface of the swine was exposed . icg was injected via the ear vein with a bolus dose of 0.3 mg / kg . visible and icg fluorescence images of cortical vessels were simultaneously observed on the display monitor at high resolution . the real - time merging of the visible and fluorescent images corresponded well.conclusionsimultaneous visible color and icg fluorescent imaging of the cortical vessels in the swine brain was satisfactory . technical improvement and clinical implication are expected .

INTRODUCTION MATERIALS AND METHODS Animal study Endoscopic ICG system RESULTS DISCUSSION CONCLUSION

red eye is a common eye sign / symptom presenting to the general physicians , other eye care workers , and the ophthalmologist . this symptom accounts for approximately 15% of consultations for ophthalmologists and 6% for general medical practitioners in eastern europe . it also accounted for 40% of all outpatients seen in bawku hospital ghana in 2004 and 10 district hospitals in pakistan . in nigeria , lawan reported 14.8% of patients attending their teaching hospital eye clinic presenting with red eye . studies done in port harcourt and ile - ife in nigeria showed that red eye accounted for 19.61% and 54.9% of pediatric ophthalmic eye diseases , respectively . a major cause of concern to health workers is the late presentation of patients with the condition at a standard health facility . oftentimes , in developing countries such as nigeria , individuals commence various forms of medications , both orthodox and traditional , which they consider as first aid measures . the causes of red eye are many including mild to serious conditions that may threaten vision . some even are managed as emergencies when it is being treated by an ophthalmologist . these include conjunctivitis , corneal ulcer , iritis , trauma , acute glaucoma , and use of traditional eye medicine ( tem ) . by far , the most common cause is conjunctivitis which could be infective or allergic . in a study done in ile - ife nigeria , among pediatric age group , ocular trauma was the most common cause of red eye , followed by allergic conjunctivitis and infections of the eye and its adnexa . however , in developing countries , individuals and communities often commence first aid before presenting themselves to the health practitioner . researchers in ghana studied the pattern of eye care services sought outside the orthodox health system . cataracts , eye injuries , and other ocular conditions were reportedly managed by herbalists , who often instilled the herbal mixtures directly into the eyes . the knowledge and attitude of a community regarding a disease often influence members practices including health - seeking behavior . studies carried out in developing countries of sub - saharan africa showed a sub - optimal level of knowledge , attitude , and perception of ocular conditions and eye health in general . in cape coast , ghana , a study of the eye care seeking behavior of the populace , showed 23.3% of respondents having practiced self - medication during the last episode of eye disorder whereas 5.5% visited a traditional healer . only 32.5% reported at a health facility for the management of the last episode of an eye disorder . the main reason for self - medication among respondents was a poor perception of the severity of the ocular condition . abubakar found 62.3% of secondary school students in kano , having poor knowledge and 58.2% with a poor attitude toward ocular disorders , especially blindness and its prevention . in limpopo province of south africa , the perception of ocular manifestation of hiv / aids was assessed among 2659 high school students with results showing a need for better health communication involving this population group . almost two - third ( 65.6% ) of respondents , felt an ophthalmologist or an optometrist should be consulted for ocular problems whereas 16.5% felt traditional healers could be considered . researchers in ile - ife , southwest nigeria , reported 49% of secondary school students participating in a screening exercise , to have allergic conjunctivitis ; 6% had infective conjunctivitis . more females than males had one form of ocular disorder or the other . among those with allergic conjunctivitis , only 12.5% had visited an eye specialist prior to conducting the research . in addition , health habits formed during this period is often carried on to adulthood and passed to the next generation , which can be largely influenced by their perception and attitude toward disease conditions when in school . early detection and quality treatment often positively influence the outcome of an episode of red eye . period of presentation at a health facility is dependent on several factors , including patients knowledge and perception of the symptom involved . no previous study has examined the perception and practice of senior secondary ( ss ) school students in sagamu , concerning the red eye . findings on perception and practice regarding health and related conditions can be tools to provide education and treatment plans , which ultimately improve patient care and quality of life . this study , therefore , planned to determine the attitude of ss school students in sagamu local government to red eye . the result will be useful to health planners in planning interventional programs to reduce blindness and visual impairment from red eye within the local government area ( lga ) , state , and the country at large and in the other developing countries . sagamu is a peri - urban area consisting of 15 wards with a diverse population in terms of ethnicity , occupation , age structure , and socioeconomic status . the town serves as a transit zone between the southwest and south - south regions of the country . ss school students attending public secondary schools in sagamu lga were recruited into the study . ss-3 students were exempted from participating in the study because their final examinations were on - going and were thus unavailable . a cross - sectional , descriptive study was carried out among 1082 ss ( ss ) school students in sagamu lga . using the formula for descriptive studies and assuming a prevalence of 50% ( since no previous study has been carried out on the subject matter in our environment ) as well as a nonresponse rate of 10% , a sample size of 420 was calculated . however , this was exceeded , and 1082 students were recruited into the study . multi - stage sampling technique was used for selection of study participants . the first stage involved the selection of three wards from the 15 existing wards in sagamu lga by simple random sampling . the second stage involved the selection of one ss school in each of the preselected wards also by simple random sampling . the third stage involved the selection of three arms from the five existing arms of ss-1 and ss-2 in each school . all consenting students in the selected arms were recruited into the study . a validated , semi - structured , self - administered questionnaire was used for data collection among the study participants . the questionnaire obtained information on sociodemographic data , perception , and attitude toward red eye from respondents . data were analyzed with ibm statistical package for the social sciences ( spss ) version 20 ( ibm corp . a score of 50% was graded as good whereas that < 50% was graded as poor . attitude scores were also calculated on a scale of 100% , with a good attitude being a score of 50% and above whereas a score of < 50% was graded as poor . chi - square test was used to test the association between categorical variables , with the level of significance ( p ) set at < 0.05 . approval was obtained from the zonal education office of the state ministry of education , science , and technology . this study followed the ethical standards issued by the nuremberg code and the declaration of helsinki on research in humans . participation was fully voluntary , and respondents were free to withdraw from the study if they so wished . sagamu is a peri - urban area consisting of 15 wards with a diverse population in terms of ethnicity , occupation , age structure , and socioeconomic status . the town serves as a transit zone between the southwest and south - south regions of the country . ss school students attending public secondary schools in sagamu lga were recruited into the study . ss-3 students were exempted from participating in the study because their final examinations were on - going and were thus unavailable . a cross - sectional , descriptive study was carried out among 1082 ss ( ss ) school students in sagamu lga . using the formula for descriptive studies and assuming a prevalence of 50% ( since no previous study has been carried out on the subject matter in our environment ) as well as a nonresponse rate of 10% , a sample size of 420 was calculated . the first stage involved the selection of three wards from the 15 existing wards in sagamu lga by simple random sampling . the second stage involved the selection of one ss school in each of the preselected wards also by simple random sampling . the third stage involved the selection of three arms from the five existing arms of ss-1 and ss-2 in each school . a validated , semi - structured , self - administered questionnaire was used for data collection among the study participants . the questionnaire obtained information on sociodemographic data , perception , and attitude toward red eye from respondents . data were analyzed with ibm statistical package for the social sciences ( spss ) version 20 ( ibm corp . , amonk , ny ) . a score of 50% was graded as good whereas that < 50% was graded as poor . attitude scores were also calculated on a scale of 100% , with a good attitude being a score of 50% and above whereas a score of < 50% was graded as poor . chi - square test was used to test the association between categorical variables , with the level of significance ( p ) set at < 0.05 . approval was obtained from the zonal education office of the state ministry of education , science , and technology . this study followed the ethical standards issued by the nuremberg code and the declaration of helsinki on research in humans . participation was fully voluntary , and respondents were free to withdraw from the study if they so wished . the female students were 593 ( 54.8% ) while males were 489 ( 45.2% ) in the study . many of the students were christians ( 66.5% ) and in ss-2 ( 51.1% ) [ table 1 ] . demographic characteristics of respondents majority ( 81% ) had heard of red eye and this was mainly from neighbors [ table 2 ] whereas 216 ( 19% ) had not . four hundred and fifteen ( 38.4% ) did not know the cause of red eye , while 99 ( 9.1% ) thought trauma was a cause , 147 ( 13.6% ) infection , 73 ( 6.7% ) allergy , 21 ( 1.9% ) sunlight whereas other causes such as drugs forms 327 ( 30.2% ) . two hundred and seventy - six ( 25.5% ) knew that they can be infected by someone with red eye , 632 ( 58.4% ) says they can not be infected by someone with red eye whereas 174 ( 16.1% ) did not know whether they could be so infected or not [ figure 1 ] . about 35% would instill onion when they have red eye [ figure 2 ] , followed by urine ( 21.9% ) and breast milk ( 12.9% ) . other things that they were likely to instill are salt water ( 4.6% ) , sugar water ( 4.6% ) , and battery water ( 1.5% ) whereas 19.4% will not use any of the substances . though majority says that a doctor is the one that takes care of red eye , 24 says self , 19 says their parent , 55 says chemist , 58 nurses whereas 12 says a herbalist can care for red eye . source of respondent 's information about red eye can someone with red eye infect you ? traditional medications used in red eye awareness of red eyes was associated with age ( p = 0.005 ) , but not with sex and religion [ table 3 ] . perception grade was good in 49 ( 4.5% ) and poor in 1033 ( 95.5% ) . five hundred and forty - three ( 50.2% ) felt that red eye could lead to blindness [ figure 3 ] . perception of red eye was associated with age ( p = 0.027 ) and class ( p 0.001 ) . the factors associated with attitude toward red eye are shown in table 3 ; this is statically significant for class ( p 0.001 ) . factors associated with awareness , perception , and attitude toward red eyes can red eye lead to blindness ? attitude refers to the feelings toward a subject , as well as any preconceived ideas ; one may have toward it . practice refers to the ways in which people demonstrate their knowledge and attitudes through their actions on the subject matter . it is not surprising that many of the respondents have heard of red eye because epidemic conjunctivitis called apollo is a very common cause of red eye in nigeria and , especially the west africa coast . in this study , majority heard about red eye from neighbors which show the impact of community on people 's health . that only 13.9% heard from health workers shows that there is a need for eye care workers to intensify health education , especially among students . only 13.6% were aware that red eye could be caused by infections which may also influence their attitude to red eye . this might explain why more than half of the students say they can not be infected by someone with red eye . it is comparable to findings from limpopo , south africa , where only 31.4% felt hiv infection and its sequelae could result in red eyes . most ( 96.2% ) respondents attitude toward red eye was poor , a finding quite different from that reported in kano , where 52.8% of students had a poor attitude toward severe ocular disorders . the observed difference might be due to the fact that this study focused solely on red eyes whereas the study carried out in kano assessed knowledge and attitude toward causes of blindness and its prevention . usually in the developing countries , people usually use traditional eye medication and unprescribed medications to treat their red eye . these medications that are being used for red eye as found in this study include onion , urine , breast milk , salt water , sugar water , and battery water . these can cause chemical conjunctivitis , infective conjunctivitis , especially gonococcal conjunctivitis when infected urine is used and if not detected and treated early , it can result in blindness . this finding also confirms the report of other researchers who have shown that people seek alternative eye care treatment instead of orthodox treatment with implications for ocular health . there was a report of a usage of herbal preparation on the eye with resultant blindness . health education done in schools during assembly especially by eye care workers will go a long way to help reduce this among the students and in the communities as these children will , in turn , tell their parents about what they are taught at school . furthermore , incorporation of school eye health program into the existing school health program will be of immense benefit . perception grade and attitude of this population were poor , this may mean that the perception and attitude to red eye , even though very common is low in the general population as the students are actually reflecting what they have learnt from their homes . intensive effort to enlighten the population will therefore help to reduce blindness / visual impairment in our community . these enlightenment programs can be taken to the religious houses , i.e. , mosques and churches for a wider coverage . we therefore conclude that secondary school students in sagamu have poor perception and attitude to red eye . appropriate eye health services and education should be included in school health services and early presentation to eye care centers for its treatment should be encouraged .

background : red eye is a very common presenting complaint in clinical practice among all age groups , including adolescents . health habits formed during adolescence is carried to adulthood and is often a consequence of their perception . this study , therefore , determined the perception of students toward the red eye.aim:to determine the perception of red eye and its associated factors among secondary school students in sagamu.methods:a cross - sectional descriptive study was carried out among 1082 senior secondary school students in sagamu local government area , using a semi - structured self - administered questionnaire . data were analyzed using spss version 20 . relevant descriptive and inferential statistics were calculated.results:the mean age of respondents was 15.27 1.48 years . there were more females ( 54.8% ) than males . majority ( 81% ) had heard of red eye , and this was mainly from neighbors ; 58.4% felt they could not contact red eye from an infected person . about 35% would instill onion if they had a red eye . about 50.2% felt red eye could lead to blindness . awareness of red eye was associated with age ( p = 0.005 ) , but not with sex and religion . among respondents , 95.5% and 96.2% had a poor perception as well as a poor attitude toward red eye , respectively.conclusion:the perception and attitude of senior secondary school students in sagamu to red eye is poor . appropriate eye health education and promotional services , including periodic eye examination of students , should be carried out in school health services . early presentation to eye care centers for its treatment should be encouraged .

Introduction Methods Study location Study population Study design Sample size determination Sampling technique Study instrument Data management Ethical considerations Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

auricular acupuncture is a diagnostic and treatment system based on normalizing the body 's dysfunction through stimulation of points on the ear . resulting amelioration of pain and illness is believed to be through the reticular formation and the sympathetic and parasympathetic nervous systems ( 1 ) . ear acupuncture , is an acupuncture technique similar to reflexology , and is speculated that the technique works because groups of pluripotent cells contain information from the whole organism and create regional organization centers representing different parts of the body , through recruitment of more cortex cells dedicated to specific areas of the body . thus stimulation of a reflex point in the ear can relieve symptoms of distant pathology with a reliable duration . rudimentary forms of acupuncture which probably arose during the stone age have survived in many parts of the world right down to present day . primitive sharp stones and bamboo were replaced by fish bones , bamboo clips and later various shapes of needles made of metal . when stones and arrows were the only tools of war , warriors wounded in war found that some diseases that affected them for many years were gone , as probably testify scars on the skin of the mummified body of similaun , italy . the bantus of south africa scratch certain areas of their skin to allay the symptoms of many illnesses , while in brazil there is a tribe whose method of treating illness is to shoot tiny arrows from a blowpipe to specific areas of the skin . the practice of cauterizing a part of the ear with a hot metal probe has also been reported among certain tribes in arabia . this is probably a vestige of the acupuncture practiced in ancient egypt and saudi arab . ( now in the british museum ) describes a system of channels and vessels in the body which approximates more closely to the chinese system of channels than to any known system of blood vessels , lymph vessels or nerves . the egyptologist alexandre varille ( 19091951 ) has documented that women in ancient egypt who did not want any more children , had their external ear pricked with a needle or cauterized with heat . gold earrings worn by mediterranean sailors were not just used as decorations , but were said to improve vision . hippocrates , the father of of greek medicine , reported that doctors made small openings in the veins situated behind the ear to facilitate ejaculation and reduce impotency problems . the greek physician galen introduced hippocratic medicine to the roman empire in the second century ce , and commented on the healing value of scarification at the outer ear . after the fall of roman empire , the medical records of egyptian , greek and roman medicine were best preserved in ancient persia and arabian world . included in these persian records were specific references to medical treatments for sciatic pains and sexual related disease produced by cauterization of the external ear . during renaissance sporadic clinical reports in europe the dutch east india company actively engaged in trade with china from 1600s to 1800s , and its merchants brought chinese acupuncture practices back to europe . doctors working with the company had become impressed by the effectiveness of needles and moxa , and cauterization of the external ear , or by cutting the veins behind the ears for relieving conditions such as sciatic pains and arthritis of the hip . in 1637 probably for the first time in europe was described by the portugese physician zacatus lusitanus the treatment of sciatic pain by cauterization of the ear after that bloodletting had failed . the italian anatomist and surgeon antonio maria valsalva ( 16661723 ) , who made the first modern anatomical description of the ear ; in 1717 published the aura humanus tractatus , where he describes the treatment of toothache by scarification of antitragus . in 1810 ignazio colla of parma , italy , reported the observation of a man stung by a bee in the antehelix which resulted in dramatic relief of pain in the legs , and in the same year dr cecconi , another italian physician , performed cauterization to help treatment of sciatic pain . in 1850 the french journal des connaissainces medico - chirurgicales reported 13 different cases of sciatic pain that had been treated by cauterization with a hot iron applied to the ear . but it was not until a century later that paul nogier rediscovered this type of treatment . in 1957 , dr paul nogier a physician resident in lyons , france , first presented his observations of the somatotopic correspondences of the ear . dr nogier ( 2 ) originated the concept of an inverted fetus map on the external ear ( fig . he developed this theory after noticing that some patients attending his clinic had a small scar from a burn on part of their ear . on inquiring into this , he was told that a very small area of their ear had been cauterized by a certain madame barrin for treatment of sciatic pain a treatment that they proved very rapid and effective . later his first great insight was the recognition of the homunculus , the man in the ear , the representation and anatomical correlation of the inverted fetus in the ear . points on the body , for example the knee , corresponded precisely with the fetal representation of the knee in the auricle . auriculotherapy following nogier 's theory uses the ear to help determine whether the right and left hemispheres of the brain are functioning as a dynamic whole , whether there are specific neurological , musculo - skeletal or organ systems that are in imbalance , and whether there are any blockages to treatment , such as scar tissue or emotional disorders and it should be a new diagnostic system too ( 2,3 ) . figure 1.this drawing illustrates the concept of an inverted fetus map on the external ear . then dr nogier noticed that there was a distinct change in the amplitude and dimension of the pulse when certain points on the auricle were stimulated . being able to detect the vas on the radial pulse of the patients left hand enables the practitioner to precisely determine the location of a point , whether there is a pathology in the region of the body that relates to specific points , and whether certain substances are indicated . accurate employment of the vas would be essential in diagnosis and treatment following the principles of nogier 's auricolomedicine . nogier collaborated with a group of medical colleagues who , in a spirit of cooperation and discovery , shared their experiences . one of those colleagues , dr jacques niboyet , convinced nogier to introduce his discoveries to the congress of the mediterranean society of acupuncture in february of 1956 . attending that congress was dr grard bachmann who published nogier 's research , translated into german , in a acupuncture journal in 1957 . this journal had an international circulation and it was not long before japanese acupuncturists became familiar with nogiers reflex system . the discovery of the system spread to china and led to intensive research by the chinese medical authorities at a time of renewed interest in traditional chinese medicine . after learning about the nogier ear charts in 1958 , a massive study was initiated by the nanjing army ear acupuncture research team . this chinese medical group verified the clinical effectiveness of the nogier approach and assessed the conditions of over 2000 clinical patients , recording which ear points corresponded to specific diseases . the outcome of that research was very positive and resulted in the utilization of this therapy by the was published an ear chart remarkably similar to that of dr nogier in 1958 ( 4 ) . nogier acknowledged that chinese traditional medicine had been using ear points for acupuncture prior to his discovery , but these had been considered empirical points for particular treatments and were not associated with a somatotopic representation of the homunculus in the ear . this oversight appears to have inhibited awareness of options laid open to recognize and treat other points in the ear following an anatomical relationship to the points already known at the time . later the american physician td oleson has published a very important paper that is a real milestone in ear acupuncture ( 5 ) . to experimentally evaluate the claims by french and chinese ear acupuncture that a somatotopic mapping of the body was represented in the external ear , 40 patients were examined to determine areas of their body where there was musculoskeletal pain . each patient was draped with a sheet and a physician conducting the auricular diagnosis had no prior knowledge of the patient 's medical condition , but simply examined the patient 's ear for areas of elevated skin conductivity or tenderness . the concordance between the established medical diagnosis and the auricular diagnoses was 75.2% ( 5 ) . these results thus supported the hypothesis that there is a somatotopoic organization of the body represented upon the human auricle , but represented following definite areas not meridian lines or other energetic concepts . in the last years modern clinical and basic research is confirming the efficacy of ear acupuncture mostly in the treatment of pain both acute and chronic ( 69 ) , and of anxiety related disorders ( 1012 ) . while the treatment of irritable bowel syndrome , obesity , smoke cessation , alcohol withdrawal and other types of substance abuse disease is still waiting definitive confirmation ( 1317 ) . basic research is trying to explain the effect of therapeutic reflexes induced by ear acupuncutre so behavioral analgesia produced by auricular acupuncture can be blocked by the opiate antagonist naloxone , indicating the role of endorphinergic systems in understanding the underlying mechanisms of auriculotherapy analgesia ( 18 ) ; and ear stimulation in healthy persons is associated with changed activity in the sympathetic and parasympathetic nervous system depending on the site of stimulation and period of observation ( 19 ) . auricolotherapy is a treatment diffusing in all over the world , and its patterns follow the principles of chinese acupuncture , revised and updated , with chinese maps of the ear ; the principles of paul nogier and also the principles of reflexology basing on somatotopic maps that do not recognize energetic - based stimulation , while just the evocation of a reflex stimulating precise areas of the ear ; moreover are used for stimulation of ear skin many different tools : finger acupressure , laser , electricity , different types of needles , magnetic balls , seeds . actually one of the many methodological problems with auricular acupuncture is that there are so many maps of the ear and little agreement exists regarding point location , lacking definitive anatomic study on ear skin and its somatotopic correspondences . another problem is that all correspondence or reflex systems do not correlate to the knowledge of anatomy and physiology based on the patterns of mainstream medicine ( 20 ) .

auricular acupuncture is a diagnostic and treatment system based on normalizing the body 's dysfunction through stimulation of definite points on the ear . rudimentary forms of acupuncture which probably arose during the stone age have survived in many parts of the world right down to present day . it was used in the ancient egypt , rome , greece and all the mediterranean area . it is a microacupuncture technique similar to reflexology , and was first described in france in 1950 by paul nogier who is considered the father of modern ear acupuncture . it was speculated that the technique works because groups of pluripotent cells contain information from the whole organism and create regional organization centers representing different parts of the body . nevertheless stimulation of a reflex point in the ear seems relieve symptoms of distant pathologies . modern research is confirming the efficacy of ear acupuncture for analgesia and anxiety related disease , while tobacco dependence and other substance abuse still need confirmation . actually main methodological problems with auricular acupuncture are that exist too many maps with little agreement regarding point location in the ear , and that the correspondence or reflex systems does not correlated with modern knowledge of anatomy and physiology .

What is Ear Acupuncture? Ancient History of Ear Acupuncture Dr Paul Nogier: The Father of Ear Acupuncture Ear Acupuncture and Evidence Based Medicine

in this dataview , we look at the sponsors of health care , providing statistics on business , household and government health spending . the companion article , national health expenditures , 1995 ( levit et al . , 1996 ) presents health care expenditure data in an accounting structure that describes the size , current growth and historical trends of health care by service , matched against the sources that pay the health care bill , such as private health insurance ( phi ) and government programs like medicaid and medicare . the national health expenditures ( nhe ) structure provides policymakers , researchers , and the public with valuable health care expenditure statistics . however , it does not provide information on the size and impact of rising health costs on the sponsors of health care . the accounting structure used in this dataview breaks apart the nhe to examine the effects of health care expenditures on the sponsoring sectors . business , households and government finance health care bill payers through taxes , premium payments ( for both private and public health insurance ) , and general revenues ; these sectors also make direct payments to providers . for 1990 through 1995 , we will present statistics on how much each sponsor spent on health care and the impact of these expenditures on their ability to pay . since 1993 , a combination of slower health care cost growth and an upswing in the economy has stabilized or eased the burden that business , households and government faced in financing health care . business , especially , is benefiting from the changing health care cost environment . during the same period , spending for health services and supplies ( hss ) ( a subset of nhe ) reached $ 957.8 billion in 1995 . business paid for 26 percent of hss , households paid for 34 percent , and the public sector paid for 38 percent . another component , non - patient revenues , made up the remaining 2 percent . over time , this relationship among the sponsors has changed . in 1965 , households were the primary sponsors of health care . since then households have been gradually paying for a smaller proportion of hss and business and the public sector has been paying for a larger proportion . however , by aggressively controlling their health care expenses , business decreased its share of hss during the 1990s . actions by the public sector were less dramatic with the result that the pubic sector share of health care costs increased significantly during this period after nearly 20 years of relative stability . by 1991 , business spent $ 249.4 billion on health care in 1995 , including $ 183.8 billion for employer - sponsored health insurance . the 1993 to 1995 average annual growth of 4.3 percent was the slowest since we began to measure business health spending in 1965 . the burden placed on business eased over the years 1993 through 1995 . during this time overall health care costs grew at a slow rate . also , enrollment in managed care plans grew . these plans generally charged lower premiums than traditional fee - for - service plans . in highly competitive markets , managed care plans also kept premiums low to increase enrollment and boost market share . this in turn forced traditional indemnity insurance companies competing with managed care plans to develop new low cost products or lose market share . these marketplace changes contributed to health care costs consuming less of the business 's compensation costs and profits . according the bureau of labor statistics ' employment cost index ( eci ) for civilian workers , the primary driver of the slowdown in benefits growth was the decline in the cost of health benefits . although a separate health insurance benefit index is not available , the eci program does publish employer compensation cost levels using current employment weights . based on that eci measure , average civilian employer health insurance costs fell 6.2 percent ( $ 1.29 to $ 1.21 per hour worked ) between march 1994 and 1995 . during the same period , wages and salary costs grew 0.5 percent , from $ 13.06 to $ 13.12 per hour worked . private health insurance is obtained primarily through employer - sponsored health plans and health plans purchased separately by individuals . in 1995 , private health insurance premiums continued to grow at a slow rate , increasing just 2.6 percent from the previous year . however , the employee share of employer - sponsored health insurance increased 8.1 percent from 1994more than twice the growth rate of employer contribution to premiums with the result that the share of health plan premium costs paid by employers inched further downward . in recent years , many employers shifted more of the premium burden to their employees by either requiring workers to contribute to their own health insurance premiums or by raising the required employee premium more rapidly than overall premium cost growth . one factor contributing to the premium cost shift from employers to employees was the migration of covered workers into lower cost managed care plans , such as health maintenance organizations , preferred provider organizations , and point of service plans . although employees generally incurred both reduced out - of - pocket costs and expanded services under a managed care plan , employers typically paid a smaller portion of the total premium for these plans than for traditional indemnity ( fee - for - service ) plans . therefore , it is likely that the rapid expansion of managed care enrollment in the 1990s had the effect of boosting the employee share of total premiums . the majority of spending ( $ 182.6 billion ) was for out - of - pocket health care expenses not covered by insurance and for copayments and deductibles . the remainder was spent on phi premiums ( $ 68.5 billion ) and premiums and contributions to the medicare trust funds ( $ 72.2 billion ) . the switch to managed care affected the level of household out - of - pocket payments . starting with the late 1980s , the growth in out - of - pocket expenses for health care has been the lowest since 1970 . this coincides with the increased enrollment in managed care plans that have lower deductibles and copayments than traditional fee - for - service plans . households have paid approximately 5.5 percent of their income after taxes for health care ( u.s . bureau of labor statistics , 1990 - 95 ) . according to consumer expenditure survey data , elderly households ( households with reference persons 65 years of age or over ) spend three times as much of their income after taxes on health care as non - elderly households . in 1995 , the public sector paid 38 percent , or $ 360.4 billion , of hss . the federal government paid $ 203.4 billion , while state and local government paid $ 157.0 billion . the portion of federal government revenues financing health care costs declined slightly in 1994 and 1995 after several years of significant growth . the decrease from 24.0 percent in 1993 to 22.8 percent in 1995 was primarily due to an increase in the federal revenues rather than a slowdown in federal health spending . medicare incurred expenditures of $ 187.0 billion in 1995 , up 11.6 percent from the previous year . the medicare program is funded through 3 sources : ( 1 ) payroll taxes paid by employers and households ; ( 2 ) premiums and income taxes on social security benefits paid by households ; and ( 3 ) income from the federal government . in this accounting scheme , federal government contributions to the medicare program include trust fund interest income , net changes in the trust fund balances and transfers from the general fund of the treasury ( board of trustees of the federal hospital insurance trust fund , 1996 and board of trustees of the federal supplemental insurance trust fund , 1996 ) . in 1995 , medicare hospital insurance ( hi ) benefit payments and administrative expenses exceeded income by $ 2.6 billion . this financing shortfall was met by redeeming $ 2.6 billion in treasury securities held by the hi trust fund . this necessity contributed to the increase in the government 's share of total expenditures in 1995 . the disparity between income and expenses is expected to grow rapidly , with the assets of the hi trust fund becoming exhausted about 2001 in the absence of corrective legislation .

for the period 1990 - 95 , we will present data on health care spending by business , households , and government . in addition , we will measure the relative impact of these expenditures on each sector 's ability to pay . in 1994 and 1995 , health care costs experienced the slowest growth in 3 decades . combined with healthy revenue growth , slow cost growth helped ease or stabilize the financing burden faced by business , households and government .

Introduction Overall Business Private Health Insurance Premiums Households Government

we developed a sensitive and accurate analytical method for quantifying methyleugenol ( me ) in human serum . our method uses a simple solid - phase extraction followed by a highly specific analysis using isotope dilution gas chromatography - high resolution mass spectrometry . our method is very accurate ; its limit of detection is 3.1 pg / g and its average coefficient of variation is 14% over a 200-pg / g range . we applied this method to measure serum me concentrations in adults in the general u.s . population . me was detected in 98% of our samples , with a mean me concentration of 24 pg / g ( range < 3.1 - 390 pg / g ) . lipid adjustment of the data did not alter the distribution . bivariate and multivariate analyses using selected demographic variables showed only marginal relationships between race / ethnicity and sex / fasting status with serum me concentrations . although no demographic variable was a good predictor of me exposure or dose , our data indicate prevalent exposure of u.s . adults to me . detailed pharmacokinetic studies are required to determine the relationship between me intake and human serum me concentrations.imagesfigure 1figure 2figure 3figure 4figure 5figure 6figure 7

Images

it can be developmental or acquired and rarely may be associated with temporomandibular joint ( tmj ) ankylosis . it has been only occasionally reported since then , probably due to its usually asymptomatic nature . in 1941 , hrdlicka reported the first cases of bmc in 21 specimens from an unspecified number of dried skulls , and in 1948 , sicher first reported this anomaly in a living person . honee and bloem described a case of bifid condyle in the cadaver of a 71-year - old patient . although this type of morphologic change is generally associated with trauma , conditions such as teratogenic drug use , genetic inheritance , infection and exposure to radiation can also cause the development of this anomaly . the first patient was a 14-year - old female with a history of extraction of lower decayed and painful left first molar done about 3 months earlier . the patient continued to have pain and recurrent swelling on and off after extraction and was managed by her treating dentist with medications . the patient presented to the department with dull pain and facial swelling in relation to lower left molar region extending to the angle of mandible . axial and coronal computed tomography ( ct ) images of bilateral tmj and mandible with multiplanar reformatting ( mpr ) were done for evaluating any pathologic fracture and tmj pathosis . these findings are best seen on the axial and sagittal images [ figure 1 ] . coronal and axial ct images demonstrate left bmc oriented anteroposteriorly with articular surface irregularity the second patient was a 12-year - old female patient , referred for ct examination for evaluation of mild facial asymmetry and suspected tmj ankylosis . she had a reduced degree of jaw opening since childhood , and subsequently developed midline deviation to the left along with difficulty in mastication . these images demonstrated sagittal splitting of the left mandibular condyle into medial and lateral condylar head . the first patient was a 14-year - old female with a history of extraction of lower decayed and painful left first molar done about 3 months earlier . the patient continued to have pain and recurrent swelling on and off after extraction and was managed by her treating dentist with medications . the patient presented to the department with dull pain and facial swelling in relation to lower left molar region extending to the angle of mandible . axial and coronal computed tomography ( ct ) images of bilateral tmj and mandible with multiplanar reformatting ( mpr ) were done for evaluating any pathologic fracture and tmj pathosis . these findings are best seen on the axial and sagittal images [ figure 1 ] . the second patient was a 12-year - old female patient , referred for ct examination for evaluation of mild facial asymmetry and suspected tmj ankylosis . she had a reduced degree of jaw opening since childhood , and subsequently developed midline deviation to the left along with difficulty in mastication . these images demonstrated sagittal splitting of the left mandibular condyle into medial and lateral condylar head . szentpetery et al , in their study of 1882 cadaveric skulls , found the incidence of bmc to be 0.48% . a report of four cases of bifid condyles presented by loh and yeo included one involving an edentulous cadaver . in a literature review of reported cases in living patients , subsequently , four cases by stefanou et al and two other cases have been reported . recently , artvinli and kansu ( 2003 ) and antoniades et al ( 2004 ) have reported the first two cases of trifid condyle in patients who also had bifid condyles on the other side . described the first case of bilateral bifid condyles in a living patient . since then 10 more cases have been reported . current literature review in living patients revealed a total of 45 cases of bifid condyle , out of which 11 cases are bilateral , giving a ratio of approximately 3:1 . however , if the survey of the dry skulls and cadaveric reports are included , then the total number is 84 cases , of which 15 are bilateral . they appear to be more common on the left side in unilateral cases by a ratio of 2:1 . most cases ( 67% ) are asymptomatic and are found on routine dental radiographic examination . however , some have been reported in patients presenting with tmj symptoms , swelling , trauma , or ankylosis . the embryologic theory suggests that it is due to the obstruction of the blood supply to the condyle or the persistence of the vascularized fibrous septa . another theory postulates trauma as the cause with disruption or dislocation of joint integrity due to birth trauma , condylar fractures or surgical condylectomy in separate studies conducted by loh and yeo and antoniades et al , it was found that most cases of bifid condyle were asymptomatic and not associated with any history of trauma .. the site of fracture of the mandibular condyle and its relation to the insertion of the lateral pterygoid muscles are factors determining the future development of bifid condyle . other causes that have been proposed include genetics , endocrine disturbances , infection , radiation , nutritional deficiencies , and exposure to teratogenic substances . support for the latter suggestion comes from the work of gundlach et al who experimentally induced bifid condyles in rats by injecting teratogenic substances such as n - methyl - n - nitrosourea and formhydroxamic acid in different concentrations , at various stages of pregnancy . the extent of bifid condyle may range from a shallow groove to discrete condylar heads and the orientation may be anteroposterior or mediolateral . it has been postulated that anteroposterior splitting usually occurs in patients with identifiable antecedent trauma , while mediolateral splitting is usually developmental in origin . but here in our patient we have seen that there was a history of trauma for the mediolaterally splitted condyle and the anteroposterior splitting was because of osteomyelitis and the patient denied any previous trauma in all her life . szentpetery et al . have suggested that when two condylar parts lie in the sagittal plane , trauma is indicated as the cause , and when the parts lie in the coronal plane , the persistence of the fibrous septa at the condylar cartilage is likely to be the cause . while this may be true for the majority of cases , some mediolateral bifid condyles have been reported following sagittal fracture through the condylar head . according to blackwood , two articulating surfaces of the bmc were divided by a groove and could be orientated mediolaterally or anteroposteriorly , characterizing a specific entity . in this case report , as postulated above , groove formation and presence of medial and lateral head of both condyles clearly demonstrated the formation of the bmc . majority of the cases are detected during routine radiographic examination . in most of the cases , however , bmc is reported to be associated with pain , swelling , restricted mouth opening and most commonly tmj clicking . in our case due to the lack of clinical symptoms , diagnosis is made by radiographic findings . ct scan is the best radiograph for detection of bmc because it allows for detailed evaluation of condylar morphology . however , bmc can also be seen on opg , but sometimes the overlapping of the anatomic structures can hide the bifidity . in summary , bmc , an anatomic variation of condyle is a rare anomaly whose etiology is unknown . in the present case , literature says that it is most commonly due to facial trauma in the early stages of development . patients with internal articular derangement should be treated with occlusal splints and arthroscopic surgery , while patients with associated articular ankylosis may need surgical condylectomy or arthroplasty . awareness of this abnormality will help to avoid mistaking it for a fracture or a tumor . our first case also illustrates the point that bmc may be associated with tmj ankylosis . such a case requires detailed clinical examination and evaluation by ct for further management and for prognosticating the outcome .

bifid condyle is a rare anatomic variation of mandibular condyle . it can be symptomatic or diagnosed incidentally on routine radiographic examination . no definite etiologic factor has been identified . it is suggested that bifid condyle could be a developmental anomaly or secondary to trauma . we are reporting two cases of bifid mandibular condyle . both were diagnosed using computed tomography scan , which additionally revealed the associated pathosis in the angle of the mandible in first patient and the ankylosis of temporomandibular joint in the second patient .

I C Case 1 Case 2 D

this retrospective study included 343 glaucoma subjects who were examined at the glaucoma clinic of the asan medical center . patients who met our inclusion criteria of at least three years of follow - up and seven or more reliable vf measurements were consecutively enrolled by medical record review . eyes that underwent intraocular surgery ( including cataract and glaucoma filtering procedures ) during the follow - up period were excluded . at initial diagnosis , each patient received a comprehensive ophthalmologic examination that included a review of medical history for the presence of systemic diseases like systemic hypertension , diabetes , cerebrovascular accident , dyslipidemia , and migraine , measurement of best - corrected visual acuity to confirm that visual acuity was adequate for automated perimetry performance , slit - lamp biomicroscopy , goldmann applanation tonometry , gonioscopy , dilated fundoscopic examination using a 90- or 78-diopter lens , stereoscopic optic disc and retinal nerve fiber layer ( rnfl ) photography , vf examination by standard automated perimetry ( humphrey swedish interactive threshold algorithm standard strategy 24 - 2 ; carl zeiss meditec , dublin , ca , usa ) , and measurement of central corneal thickness ( cct ; dgh-550 instrument , dgh technology inc . , all included patients met the following criteria at the baseline exam : best - corrected visual acuity of 20 / 30 or better , with a spherical equivalent ( se ) within 5 diopters and a cylinder correction within + 3 diopters ; vf mean deviation ( md ) higher than -20 db ; and the presence of a normal anterior chamber and open - angle on slit - lamp and gonioscopic examinations . diagnosis of glaucoma was based on both the presence of typical glaucomatous changes in the optic disc and glaucomatous vf defect . thus , eyes with a glaucomatous optic disc and a normal vf were not defined as having glaucoma . glaucomatous optic disc change included increased cupping ( vertical cup - disc ratio more than 0.7 ) , a difference in the vertical cup - disc ratio ( more than 0.2 between the eyes ) , diffuse or focal neural rim thinning , disc hemorrhage , or rnfl defects . eyes defined as having glaucomatous vf defects met two of the following three criteria ( as confirmed by more than two reliable consecutive tests ) : 1 ) a cluster of three points with a probability of less than 5% on a pattern deviation map in at least one hemifield , including at least one point with a probability of less than 1% or a cluster of two points with a probability of less than 1% ; 2 ) a glaucoma hemifield test result outside the normal limit ; and 3 ) a pattern standard deviation result of less than 5% . reliable vf assessment was defined as a vf test with a false - positive error of less than 15% , a false - negative error of less than 15% , and a fixation loss of less than 20% . if at least one eye showed glaucomatous changes , that patient was included for analysis . iop - lowering medication was prescribed if the eyes showed glaucomatous optic disc changes or had both a glaucomatous optic disc and vf changes . all procedures conformed to the declaration of helsinki , and the study was approved by the institutional review board of the asan medical center . since glaucomatous damage involves both structural and functional changes that may not appear at the same time , glaucoma progression was determined by either structural or functional measures . structural progression was assessed using a stereoscopic optic disc and red - free rnfl photographs . two glaucoma experts ( krs and jhn ) independently assessed all of the photographs in order to estimate the glaucoma progression between the patients ' first and last visits . both graders were blind to each other 's progression assessments and to all clinical and vf information . photographs were presented in chronological order and with masking of patient identification , age , and test date . each grader viewed all photographs of each eye before making an assessment and was asked to determine the possible presence of a glaucomatous optic disc or of rnfl progression , as revealed by an increase in the extent of neuroretinal rim thinning , enhancement of disc excavation , any widening or deepening of a rnfl defect , and/or the appearance of new disc hemorrhage . if the opinions of the two observers differed , a third examiner ( dj ) made the final decision . commercial software ( guided progression analysis , carl zeiss meditec ) was used to determine progressive functional loss of the vf . vf defect progression was defined as a significant deterioration from the baseline pattern of deviation at three or more of the same test points and was evaluated on three consecutive examinations . if only one eye showed progression by either structural or functional assessment during the follow - up period , the patient was included in the upg group . in contrast , if both eyes showed progression , the patient was included in the bpg group . age , gender , and the prevalence of systemic diseases ( systemic hypertension , diabetes , cerebrovascular accident , migraine , and dyslipidema ) were compared between the upg and bpg groups . in the upg group , pre - treatment iop , mean post - treatment iop , cct , se , baseline vf md , vf pattern standard deviation , and progression rates were compared between the pe and the npe within the same patient . the progression rates ( md slope ) were determined by linear regression analysis of md values in serial vf analyses . in the bpg group , the same parameters were compared between the fpe and the spe within the same patient . normally distributed data were compared between the upg and bpg groups using unpaired t - tests . non - normally distributed data were compared using either the mann - whitney or wilcoxon signed rank test . this retrospective study included 343 glaucoma subjects who were examined at the glaucoma clinic of the asan medical center . patients who met our inclusion criteria of at least three years of follow - up and seven or more reliable vf measurements were consecutively enrolled by medical record review . eyes that underwent intraocular surgery ( including cataract and glaucoma filtering procedures ) during the follow - up period were excluded . at initial diagnosis , each patient received a comprehensive ophthalmologic examination that included a review of medical history for the presence of systemic diseases like systemic hypertension , diabetes , cerebrovascular accident , dyslipidemia , and migraine , measurement of best - corrected visual acuity to confirm that visual acuity was adequate for automated perimetry performance , slit - lamp biomicroscopy , goldmann applanation tonometry , gonioscopy , dilated fundoscopic examination using a 90- or 78-diopter lens , stereoscopic optic disc and retinal nerve fiber layer ( rnfl ) photography , vf examination by standard automated perimetry ( humphrey swedish interactive threshold algorithm standard strategy 24 - 2 ; carl zeiss meditec , dublin , ca , usa ) , and measurement of central corneal thickness ( cct ; dgh-550 instrument , dgh technology inc . , all included patients met the following criteria at the baseline exam : best - corrected visual acuity of 20 / 30 or better , with a spherical equivalent ( se ) within 5 diopters and a cylinder correction within + 3 diopters ; vf mean deviation ( md ) higher than -20 db ; and the presence of a normal anterior chamber and open - angle on slit - lamp and gonioscopic examinations . diagnosis of glaucoma was based on both the presence of typical glaucomatous changes in the optic disc and glaucomatous vf defect . thus , eyes with a glaucomatous optic disc and a normal vf were not defined as having glaucoma . glaucomatous optic disc change included increased cupping ( vertical cup - disc ratio more than 0.7 ) , a difference in the vertical cup - disc ratio ( more than 0.2 between the eyes ) , diffuse or focal neural rim thinning , disc hemorrhage , or rnfl defects . eyes defined as having glaucomatous vf defects met two of the following three criteria ( as confirmed by more than two reliable consecutive tests ) : 1 ) a cluster of three points with a probability of less than 5% on a pattern deviation map in at least one hemifield , including at least one point with a probability of less than 1% or a cluster of two points with a probability of less than 1% ; 2 ) a glaucoma hemifield test result outside the normal limit ; and 3 ) a pattern standard deviation result of less than 5% . reliable vf assessment was defined as a vf test with a false - positive error of less than 15% , a false - negative error of less than 15% , and a fixation loss of less than 20% . if at least one eye showed glaucomatous changes , that patient was included for analysis . iop - lowering medication was prescribed if the eyes showed glaucomatous optic disc changes or had both a glaucomatous optic disc and vf changes . all procedures conformed to the declaration of helsinki , and the study was approved by the institutional review board of the asan medical center . since glaucomatous damage involves both structural and functional changes that may not appear at the same time , glaucoma progression was determined by either structural or functional measures . structural progression was assessed using a stereoscopic optic disc and red - free rnfl photographs . two glaucoma experts ( krs and jhn ) independently assessed all of the photographs in order to estimate the glaucoma progression between the patients ' first and last visits . both graders were blind to each other 's progression assessments and to all clinical and vf information . photographs were presented in chronological order and with masking of patient identification , age , and test date . each grader viewed all photographs of each eye before making an assessment and was asked to determine the possible presence of a glaucomatous optic disc or of rnfl progression , as revealed by an increase in the extent of neuroretinal rim thinning , enhancement of disc excavation , any widening or deepening of a rnfl defect , and/or the appearance of new disc hemorrhage . if the opinions of the two observers differed , a third examiner ( dj ) made the final decision . commercial software ( guided progression analysis , carl zeiss meditec ) was used to determine progressive functional loss of the vf . vf defect progression was defined as a significant deterioration from the baseline pattern of deviation at three or more of the same test points and was evaluated on three consecutive examinations . if only one eye showed progression by either structural or functional assessment during the follow - up period , the patient was included in the upg group . in contrast , if both eyes showed progression , the patient was included in the bpg group . age , gender , and the prevalence of systemic diseases ( systemic hypertension , diabetes , cerebrovascular accident , migraine , and dyslipidema ) were compared between the upg and bpg groups . in the upg group , pre - treatment iop , mean post - treatment iop , cct , se , baseline vf md , vf pattern standard deviation , and progression rates were compared between the pe and the npe within the same patient . the progression rates ( md slope ) were determined by linear regression analysis of md values in serial vf analyses . in the bpg group , the same parameters were compared between the fpe and the spe within the same patient . normally distributed data were compared between the upg and bpg groups using unpaired t - tests . non - normally distributed data were compared using either the mann - whitney or wilcoxon signed rank test . a total of 343 korean patients were included in the final analysis , of which 182 were men and 161 were women . the mean follow - up period was 4.2 years . at baseline , 134 patients had unilateral glaucoma . among these 134 patients , 102 showed glaucomatous optic disc changes but a normal vf in the fellow eye , while the remaining 32 patients had a normal appearing optic disc and a normal vf in the fellow eye . among the 134 unilateral glaucoma patients , 25 patients showed unilateral progression during follow - up while 6 patients showed bilateral progression ; thus , 31 patients had either unilateral or bilateral progression while 103 patients were stable . among the 32 patients with unilateral glaucoma and a normal optic disc and vf in the fellow eye , none of the fellow eyes showed progression . at baseline , 209 patients had bilateral glaucoma , of which 43 showed progression with 25 showing bilateral progression . 1 . hence , the bpg group consisted of 31 patients ( 6 from the unilateral glaucoma at baseline group and 25 from the bilateral glaucoma at baseline group ) , while the upg group consisted of 43 patients ( 25 with unilateral glaucoma at baseline and 18 with bilateral glaucoma at baseline ) . the mean age was significantly higher in the upg group than in the bpg group ( 58.7 10.0 vs. 52.6 3.9 years , p = 0.035 ) . however , the prevalence of all analyzed systemic diseases was not different between the two groups ( table 1 ) . all pes in the upg group showed more severe clinical characteristics in terms of baseline vf parameters . however , baseline iop , mean follow - up iop , cct , and se were not significantly different between the pe and the npe ( table 2 ) . the progression rate determined by the md slope was compared between the fpe and the spe within the same patient in the bpg group . as expected , the progression rate was higher in the fpe , but other parameters did not differ significantly ( table 3 ) . when the pe in the upg group was compared to the fpe eye in the bpg group , the progression rate was significantly higher in the fpe in the bpg group , but no other parameters differed ( table 4 ) . according to the data from 343 patients analyzed in this study , 134 ( 39.1% ) presented with unilateral glaucoma at baseline . among these unilateral glaucoma patients , 76.1% showed glaucomatous optic disc changes in the fellow eye , while only 32 patients out of all glaucomatous patients ( 9.3% ) had completely unilateral glaucoma with both a normal optic disc and a normal vf in the fellow eye . among the 134 unilateral glaucoma patients , 23.1% showed progression during the follow - up period . the fellow eye of the 32 patients with unilateral glaucoma did not show progression during follow - up , although iop - lowering treatment was not performed in those eyes . among the 209 bilateral glaucoma patients , 20.6% showed progression . thus , the overall prevalence of progression was similar between patients with unilateral glaucoma and bilateral glaucoma . interestingly , 80.6% of patients with progressing disease in the unilateral glaucoma group demonstrated unilateral progression , while 58.1% of patients with progressing disease in the bilateral glaucoma group showed bilateral progression . chen and park showed that 6.2% of fellow eyes progressed over a period of 8.7 years . among 134 unilateral perimetric glaucoma patients , 6 ( 4.5% ) showed progression in the fellow eye in the present study . the results from the current study indicated no significant differences in the prevalence of any analyzed systemic disease between the upg and bpg groups . overall , the prevalence of systemic disease was very low in both groups , which may affect the statistical analysis and contribute to the lack of significance of the data . in contrast to the present data , a study by kim and kim showed that the prevalence of systemic disease was different between patients with unilateral and bilateral glaucoma . the discrepancy is likely due to the fact that systemic disease can affect the development of bilateral glaucoma , but the progression of disease is modulated by various factors thus reducing the effect of systemic disease on glaucoma progression . for instance , myopia is known to be a risk factor for the development of glaucoma but not for progression of the disease , although this remains under debate . when we compared the pe and the npe in the upg group , the pe showed a more advanced disease status at baseline . however , there was no difference in iop , cct , and se between the fpe and spe in the bpg group . in the comparison between the pe and npe in the upg group , iop similarly , iop did not differ between the fpe and the spe in the bpg group . as shown by mean baseline iop measurements , most of the patients in the current study had low iop and can be categorized as having normal - tension or low - tension glaucoma . whether a correlation exists between iop asymmetry and glaucoma severity in cases of normal - tension glaucoma has been under debate in previous studies . some studies have shown that higher iop is related to more advanced stages of glaucoma . however , the low - pressure glaucoma treatment study reported that iop asymmetry is unrelated to vf asymmetry . the present study revealed no differences in iop between the pe and the npe in the upg group or between the fpe and the spe in the bpg group . while iop reduction substantially decreased the progression rate in the collaborative normal - tension glaucoma study , some treated eyes also showed progression in that randomized clinical trial . thus , taken together , our results and those of previous studies suggest that factors other than iop , which may affect the progression of glaucoma with lower iop readings , should be investigated in future studies . furthermore , no differences were noted in baseline vf defect severity and iop values between the pe in the upg group and the fpe in the bpg group ; however , the progression rate was significantly higher in the fpe in the bpg group . the progression rate of the pe in the upg group was -0.70 db / year , which is approximately two times higher than the usual progression rates reported in previous randomized clinical trials ( -0.36 to -0.41 db / yr ) . the progression rate of the fpe in the bpg group was approximately five times higher ( -3.43 vs. -0.70 db / yr ) than that of the pe in the upg group . thus , we speculate that in simultaneously bilaterally progressing patients , both eyes progress faster than the pe in upg . in contrast , baseline vf defect severity was not different between the pe in the upg group and the fpe in the bpg group . first , the prevalence of systemic disease was determined by self - reported histories from the patients . second , the follow - up period ( mean 4.2 years ) was relatively short . considering the slow - progressing nature of glaucoma , this period may not be sufficient to observe the natural progression of the disease . some cases of unilateral glaucoma may eventually progress to bilateral glaucoma and some cases with a unilaterally progressing eye may show progression in the fellow eye over time . however , during the four years of observation in this study , unilateral glaucoma tended to progress only in the glaucomatous eye , while simultaneously bilaterally progressing patients showed much faster progression rates than those with a unilaterally progressing eye . no systemic factors analyzed in this study significantly differed in prevalence between the upg and bpg groups . therefore , glaucoma patients with simultaneous bilateral progression should be treated with caution in clinical practice because these cases can show relatively rapid progression .

purposeto compare the clinical characteristics of unilaterally progressing glaucoma ( upg ) and simultaneously bilaterally progressing glaucoma ( bpg ) in medically treated cases.methodsprimary open angle glaucoma patients were classified as having upg or bpg according to an assessment of optic disc and retinal nerve fiber layer photographs and visual field analysis . risk factors including the presence of systemic diseases ( hypertension , diabetes , cerebrovascular accident , migraine , and dyslipidema ) were compared between the upg and bpg groups . baseline characteristics and pre- and post - treatment intraocular pressure ( iop ) were compared between the progressing eye ( pe ) and the non - progressing eye ( npe ) within the same patient in the upg group and between the faster progressing eye and the slower progressing eye in the bpg group.resultsamong 343 patients ( average follow - up period of 4.2 years ) , 43 were categorized into the upg group and 31 into the bpg group . the prevalence of all analyzed systemic diseases did not differ between the two groups . pes in the upg group had more severe pathology in terms of baseline visual field parameters than npes ( mean deviation -6.9 5.7 vs. -2.9 3.9 db , respectively ; p < 0.001 ) . however , baseline iop , mean follow - up iop , and other clinical characteristics were not significantly different between the pe and the npe in the upg group . the progression rate was significantly higher in the faster progressing eye in patients with bpg than in the pe for patients with upg ( -3.43 3.27 vs. -0.70 1.26 db / yr , respectively ; p = 0.014).conclusionsthere were no significant differences in the prevalence of systemic diseases between the upg and bpg groups . simultaneously bilaterally progressing patients showed much faster progression rates than those with a unilaterally progressing eye .

Materials and Methods Subjects Glaucoma progression assessment Analysis Results Discussion

diabetes is the most common metabolic disease , with an increasing prevalence that reduces life expectancy to a third ( 1 , 2 ) . there are 200 million people with diabetes worldwide ( about 5% of adult population ) ( 3 ) . according to a nationwide study in 2001 , the prevalence of diabetes in iran was estimated at 4.67% among the population older than 20 years ( 4 ) . diabetes is usually associated with short - term complications such as hypoglycemia , and long - term complications like cardiovascular diseases , neuropathy , nephropathy , and retinopathy . amid patients with diabetes are twice exposed to the risk of psychiatric diseases compared to normal population , and according to some studies , one in every 5 patients with diabetes suffer from depression ( 5 , 6 ) . symptoms of depression were observed in 29% of men and 30.5% of women with diabetes in bangladesh villages ( 7 ) . in khamseh study , 71.8% of patients with diabetes had major depression ( 8) , and in sepehrmanesh study in kashan , this rate was 57.7% among women and 41% among men ( 9 ) . many factors can be affected by treatment of the disease , including physical function and daily activity , social relationships , quality of life , and personal mood . as the disease affects patient s self - confidence and increase his or her fear of worsening complications , the risk of psychiatric diseases increases ( 10 ) . depression is associated with increased risk of high blood glucose , complications of diabetes , cardiovascular diseases , and possibly patient s reduced compliance too ( 11 - 14 ) . furthermore , diabetic patients with depression have greater burden of treatment costs and referrals than those patients without depression ( 15 ) . various studies have shown that unlike normal people , patients with diabetes have lower quality of life , and this is much more pronounced in those suffering from complications of diabetes ; depression also has a significant effect on patients quality of life ( 16 - 18 ) . in a study in yazd , the mean score of quality of life in studied patients was 25.65 out of 60 , and in patients with complications like retinopathy or neuropathy , this score was lower compared to patients without these complications ( 19 ) . comorbidity of depression and diabetes can have a cumulative effect on people s functions ( 20 ) . diagnosis of depression in these patients is particularly important in order to reduce complications and improve quality of life . li et al . reported that depression was not diagnosed in 45% of diabetic patients ( 21 ) . depression leads to patient s noncompliance , blood glucose mismanagement , and other physical complications ( 11 , 19 , 22 ) . ciechanowski et al . reported that depressed patients are unable to comply with dietary regimen or manage blood glucose , and in this respect , they are clearly different from non - depressed patients ( 23 ) . with regard to the effect of diabetes with depression on the quality of life , in 2004 , filakovic et al . demonstrated that comorbidity of depression with diabetes further lowers patients quality of life by many folds ( 24 ) although several studies have been conducted on either quality of life or depression in diabetic patients , there are few studies on the relationship between quality of life and depression in diabetic patients . moreover , given that failure to diagnose depression in diabetic patients is commonplace , the need to identify and screen psychiatric disorders in patients with chronic diseases , especially diabetes , and the impact of depression on the quality of life , this article aimed to investigate the relationship between depression and quality of life in patients with diabetes using medical records at diabetics clinic in gorgan . the current survey was approved in the 46 ethics committee of golestan university of medical sciences ( no . 327123847 ) . in total , 330 eligible patients with diabetes were selected from patients admitted to the only specialized diabetic clinic in city of gorgan , north of iran , at 5th azar hospital , in the first 6 months of 2012 . inclusion criteria were confirmed diabetic record by the clinic specialist , age range of 25 - 75 years , having regular visits to the clinic , being under treatment with insulin and oral medication , not having type i diabetes , and not being treated for psychological health problems . patients were assured of the confidentiality of data and their informed consents were obtained . according to ethics committee guidelines , study ( 25 ) , the correlation between the depression and quality of life ( r = 0.39 ) , 95% confidence interval , and 90% power test , 66 diabetic patients with depression were needed . to achieve that and with respect to 20% estimation of depression in patients with diabetes ( 5 , 7 ) , 330 patients with type 2 diabetes clinic is the only specialized and state referral clinic in gorgan that is supported by 5th azar hospital and is also one of the 3 state hospitals belonged to the golestan university of medical sciences . since more than 80% of attended patients with diabetes to the health centers , had been visited by the aforementioned clinic ( with continuous access ) and according to the all patients with record ( n = 1150 ) , 330 subjects were finally selected using systematic sampling with an interval of 3 ( k = n / n = 3 ) , in the way that the first patient was randomly selected . then , a number was selected from 1 to 3 ( the second patient was selected ) . in other cases and with respect to the sampling interval as 3 , other samples were included ( such as number 5 , 8 to 989 ) . demographic questionnaire was completed in the presence of patients , and related data to the disease and its complications were obtained from their records . beck depression inventory ( bdi ) was used to assess depression , and the short version ( 26 items ) of the world health organization quality of life questionnaire ( whoqol - bref ) was used to assess quality of life . bdi contains 21 items , which evaluates signs of depression , including sadness , guilt , loss of interest , social isolation , and suicidal ideation . each item has rated on a 4-point likert scoring style from 0 for the lowest to 3 for the highest . each option is determined from mild to severe ( 0 for psychological health , and scores 1 , 2 , and 3 for mild , moderate , and severe disorders , respectively ) . overall depression score for each patient is found by summing up scores of all items , ranging from 0 to 63 , and higher scores indicate more severe depression , as follows : 0 to 15 means no depression , 16 to 31 mild depression , 32 to 47 moderate depression , and 48 to 63 severe depression . its reliability was determined through internal consistency using cronbach of 84% , and correlation was obtained 70% using split half method ( 26 ) . in this study , cronbach for the reliability of questionnaire for diabetic patients was found 0.77 , which indicates the questionnaire was suitable for this study . diez - quevedo et al . also studied it in diagnosing mental disorders in 1003 general hospital spanish inpatients in 2001 ( 27 ) . in the 26-item whoqol - bref , the first two items assess general health status and quality of life , and the next 24 items assess quality of life in 4 domains of physical health , psychological health , social relationships , and environment using 3 , 6 , 7 and 8 items , respectively . in this study the 5-point likert scale was applied for items 1 and 15 ( very bad , bad , not bad , good , and very good ) , items 2 , and 16 to 25 ( very unhappy , unhappy , not unhappy , happy , and very happy ) , items 3 to 14 ( not at all , a little , medium , high , and very high ) , and for item 26 ( never , rarely , occasionally , often , and always ) . scoring for items 3 , 4 , and 26 was in reverse . to compare domains and the first two items in depressed and non - depressed groups of patients , quality of life score for each patient ( xi ) was converted into scores between 0 and 100 using the following equation 2 ( 28 - 31 ) : validity and reliability of whoqol - bref have been reported favorable in many native and foreign studies ( 17 , 30 , 32 ) . in the present study , validity of this questionnaire was estimated according to internal consistency for the whole scale of 85% , physical health 78% , psychological health 80% , social relationships 69% , and environment 84% . the normality of qol scores and its dimensions were tested and confirmed by kolmogorov - smirnov test . where an item was missing , we substituted the mean of other items in the domain . also , when more than 2 items were missing from a domain , we did not calculate the domain score . the missing data in bdi questionnaire were replaced using median near points . to describe data , central and dispersion indicators such as mean and standard deviation were calculated . to compare qualitative characteristics of depressed and non - depressed patients , chi - square test was used . mean scores of qol and its sub - domains , age , and duration of disease in two groups were analyzed using independent t test . we conducted multiple linear regression analyses to assess prediction of qol scores and its relationship with other variables . in addition , we used scatter diagrams to visualize the relationship between dependent variable and each metric independent variable to be sure that they are linear . in addition , we also checked whether the relationship between dependent variable and categorical independent variables is homoscedastic . the current survey was approved in the 46 ethics committee of golestan university of medical sciences ( no . 327123847 ) . in total , 330 eligible patients with diabetes were selected from patients admitted to the only specialized diabetic clinic in city of gorgan , north of iran , at 5th azar hospital , in the first 6 months of 2012 . inclusion criteria were confirmed diabetic record by the clinic specialist , age range of 25 - 75 years , having regular visits to the clinic , being under treatment with insulin and oral medication , not having type i diabetes , and not being treated for psychological health problems . patients were assured of the confidentiality of data and their informed consents were obtained . according to ethics committee guidelines , study ( 25 ) , the correlation between the depression and quality of life ( r = 0.39 ) , 95% confidence interval , and 90% power test , 66 diabetic patients with depression were needed . to achieve that and with respect to 20% estimation of depression in patients with diabetes ( 5 , 7 ) , 330 patients with type 2 diabetes clinic is the only specialized and state referral clinic in gorgan that is supported by 5th azar hospital and is also one of the 3 state hospitals belonged to the golestan university of medical sciences . since more than 80% of attended patients with diabetes to the health centers , had been visited by the aforementioned clinic ( with continuous access ) and according to the all patients with record ( n = 1150 ) , 330 subjects were finally selected using systematic sampling with an interval of 3 ( k = n / n = 3 ) , in the way that the first patient was randomly selected . then , a number was selected from 1 to 3 ( the second patient was selected ) . in other cases and with respect to the sampling interval as 3 , other samples were included ( such as number 5 , 8 to 989 ) . demographic questionnaire was completed in the presence of patients , and related data to the disease and its complications were obtained from their records . beck depression inventory ( bdi ) was used to assess depression , and the short version ( 26 items ) of the world health organization quality of life questionnaire ( whoqol - bref ) was used to assess quality of life . bdi contains 21 items , which evaluates signs of depression , including sadness , guilt , loss of interest , social isolation , and suicidal ideation . each item has rated on a 4-point likert scoring style from 0 for the lowest to 3 for the highest . each option is determined from mild to severe ( 0 for psychological health , and scores 1 , 2 , and 3 for mild , moderate , and severe disorders , respectively ) . overall depression score for each patient is found by summing up scores of all items , ranging from 0 to 63 , and higher scores indicate more severe depression , as follows : 0 to 15 means no depression , 16 to 31 mild depression , 32 to 47 moderate depression , and 48 to 63 severe depression . its reliability was determined through internal consistency using cronbach of 84% , and correlation was obtained 70% using split half method ( 26 ) . in this study , cronbach for the reliability of questionnaire for diabetic patients was found 0.77 , which indicates the questionnaire was suitable for this study . diez - quevedo et al . also studied it in diagnosing mental disorders in 1003 general hospital spanish inpatients in 2001 ( 27 ) . in the 26-item whoqol - bref , the first two items assess general health status and quality of life , and the next 24 items assess quality of life in 4 domains of physical health , psychological health , social relationships , and environment using 3 , 6 , 7 and 8 items , respectively . in this study the 5-point likert scale was applied for items 1 and 15 ( very bad , bad , not bad , good , and very good ) , items 2 , and 16 to 25 ( very unhappy , unhappy , not unhappy , happy , and very happy ) , items 3 to 14 ( not at all , a little , medium , high , and very high ) , and for item 26 ( never , rarely , occasionally , often , and always ) . scoring for items 3 , 4 , and 26 was in reverse . to compare domains and the first two items in depressed and non - depressed groups of patients , quality of life score for each patient ( xi ) was converted into scores between 0 and 100 using the following equation 2 ( 28 - 31 ) : validity and reliability of whoqol - bref have been reported favorable in many native and foreign studies ( 17 , 30 , 32 ) . in the present study , validity of this questionnaire was estimated according to internal consistency for the whole scale of 85% , physical health 78% , psychological health 80% , social relationships 69% , and environment 84% . the normality of qol scores and its dimensions were tested and confirmed by kolmogorov - smirnov test . where an item was missing , we substituted the mean of other items in the domain . also , when more than 2 items were missing from a domain , we did not calculate the domain score . the missing data in bdi questionnaire were replaced using median near points . to describe data , central and dispersion indicators such as mean and standard deviation were calculated . to compare qualitative characteristics of depressed and non - depressed patients , chi - square test was used . mean scores of qol and its sub - domains , age , and duration of disease in two groups were analyzed using independent t test . we conducted multiple linear regression analyses to assess prediction of qol scores and its relationship with other variables . in addition , we used scatter diagrams to visualize the relationship between dependent variable and each metric independent variable to be sure that they are linear . in addition , we also checked whether the relationship between dependent variable and categorical independent variables is homoscedastic . a total of 330 diabetic patients ( 35.5% men , and 64.5% women ) were studied , with mean and standard deviation of age and history of disease of 50.6 9 and 5.4 4.5 years , respectively , and age range of 25 to 75 years . the prevalence of depression in all diabetic patients was 58.2% , of whom 124 patients ( 64.6% ) had mild depression , 56 ( 29.2% ) moderate depression , and 12 ( 6.2% ) severe depression . this was in 64.8% of men and 61.5% of women , with insignificant differences between them . depression was observed in 45.1% of the single diabetic patients and in 60.6% of married ones , with significant difference ( p = 0.039 ) . more than 50% of depressed diabetic patients and 62.5% of non - depressed diabetic patients were literate , and the difference between two groups was significant . however , there were insignificant differences between the two groups in terms of employment status ( table 1 ) . ( % ) . mean and standard deviation of depression score according to bdi was 29.4 10.1 in depressed patients , and 8.1 4.1 in non - depressed patients , with a significant difference between them ( p < 0.001 ) . comparison of the relationship between clinical characteristics and depression in patients with diabetes showed insignificant differences between the two groups with respect to dietary regimen , diabetes complications ( retinopathy , neuropathy , and nephropathy ) , and duration of the disease . however , hypertension was 13.9% more ( p < 0.001 ) , and physical activity 24.7% less in diabetic patients with depression , and the difference between groups was significant ( p = 0.01 ) ( table 2 ) . comparing scores of various domains of quality of life between depressed and non - depressed patients with diabetes using independent t test indicated significant lower mean scores in subscales of general health , quality of life in general , psychological health , environment , and overall score of quality of life in depressed diabetic patients at error level of 0.001 . however , in physical health domain , mean score was lower in depressed patients , with a significant difference ( p = 0.009 ) ( table 3 ) . linear regression model of prediction of quality of life score with independent variables of age , gender , duration of disease , hypertension , diabetes complications , education , marital status , occupation , physical activity , and depression score showed significant and inverse relationship between overall score of quality of life and variables of physical activity , diabetes complications or otherwise , and depression score ( p = 0.01 ) ( table 4 ) . the present study results are in line with the results in other studies , and suggest high prevalence of depression among patients with diabetes ( 7 - 9 , 19 ) . according to the present study , 58.2% of participants had moderate to severe depression and 15.5% had mild depression , i.e. , 73.7% of the patients are likely to have depression . in a study by nedjat et al . ( 28 ) , 28% of patients were depressed and 38% had mild depression , making a total of 66% . the rate of depression was 41.9% in larijani , and 53.3% in sepehrmanesh studies ( 9 , 19 , 21 ) . kahn et al . argue that not only depression could result from diabetes ; but also it is a risk factor for the onset of diabetes ( 22 ) . depression is associated with 60% increased risk of type 2 diabetes , while diabetes only causes an increased risk of depression by about 15% ( 23 ) . however , in the present study , there was an insignificant difference between sexes in the prevalence of depression , which is in line with results of pinquart et al . the mean overall quality of life score in depressed patients was 50.68 14.04 , which was significantly lower than non - depressed patients with 60.46 13.27 , and is close to the results found by nedjat et al . ( 28 ) . in psychiatric and habitat areas and overall score , the differences between groups was significant , and among depressed patients , mean score was lower , which is in line with other studies ( 6 , 25 , 28 ) . only in social relationships domain , the difference between the two groups was insignificant . perhaps , it could be attributed to rich relationships between patients and their family and relatives , which often become richer at times of disease . depression can affect physical outcomes such as myocardial infarction , life expectancy in cancer patients , and infections . also , it explains the effect of depression on immune system the most . other reasons for this effect can be due to the fact that depression in diabetic patients affects compliance , proper dietary regimen , smoking , and exercise ( 7 ) . in a study by lin et al . , it was confirmed that depressed patients perform more poorly than other patients in compliance with personal care , including proper nutrition , exercise , compliance with treatment and prevention ( 34 ) . in another study it was found that regarding incident disability and mortality , the effects of interaction between diabetes and depression seemed to be synergistic ( that is , greater than the additive main effects of diabetes and depression ) . compared depressed patients and 8 groups of patients with chronic diseases , and found that depressed patients showed greater reduction in the quality of life than the other 8 groups , and this effect remained , even after eliminating factors such as age , gender , and chronic diseases ( 35 ) . in the present study , high prevalence of comorbidity of depression with diabetes in patients indicates that effect of depression comorbidity on the quality of life can significantly affect outcomes of the disease , and early diagnosis of depression can improve quality of life ( 25 ) . study limitations included lack of measurement of hba1c , which could help assess better the patients blood glucose , and its effect on disease complications . it is , therefore , suggested that its measurement be taken into consideration in future similar studies . given the results of this and other studies , it seems that there is high prevalence of depression among patients with diabetes , and it has a significant effect on diabetes outcomes and quality of life . it is recommended that besides regular doctors visits in terms of physical problems , the psychiatric problems and quality of life of these patients should also be more considered . regular psychiatrist visits , or psychological assessments for screening mental disorders can help early diagnosis of these disorders . our survey weaknesses were as follow : 1 ) we used self - administered questionnaire , which could include patients bias , 2 ) participants may not have fully understood or have misinterpreted questions that might have biased the responses , and 3 ) we conducted a cross - sectional method that provided us with information for that particular issue in time . however , our study strong points can be mentioned as covering more than 80% of patients with diabetes in golestan province who were attended to the only specialized diabetes clinic supported by golestan university of medical sciences .

background : frequency of mood disorders in patients with chronic diseases , especially diabetes and its effects on life quality are dramatically increasing.objectives:this study aimed to investigate the relation between depression and quality of life in patients with diabetes.patients and methods : this is a cross sectional survey . subjects were selected from 330 eligible people referred to the only diabetes clinic in gorgan city during 6 months , using systematic random sampling . beak depression questionnaire and the brief questioner with 26 questions recommended by the world health organization ( whoqol - bref ) were used to measure depression and quality of life , respectively . data were analyzed through descriptive methods , chi - square , independent t test and linear regression model using spss16 ; moreover , p value < 0.05 was considered as significant.results:in total , 330 patients with diabetes ( 35.5 % male and 64.5% women ) were studied . the mean and standard deviation of their age and years involved with diabetes were 50.6 9.0 and 5.4 4.5 years , respectively . range of age was 25 - 75 years , as well . the prevalence of depression in all patients with diabetes was 58.2% ( 124 mild , 56 medium , and 12 with severe depression ) . hypertension was 13.9% more in diabetic patients with depression ( p value < 0.001 ) and physical activity in 24.7% of the cases was less with a meaningful difference ( p value = 0.01 ) . the mean and standard deviation of quality of life in diabetic patients with and without depression was 50.7 14 and 60.5 13.3 , respectively that was significant in two groups ( p < 0.0001).conclusions : the prevalence of depression is high in patients with diabetes and has a considerable impact on the consequences of diabetes and quality of life too .

1. Background 2. Objectives 3. Patients and Methods 3.1. Study Design 3.2. Participants 3.3. Sample Size 3.4. Sample Frame 3.5. Instrument 3.6. Data Analysis 4. Results 5. Discussion

we first tested the proposed strategy by designing a symmetric rna dna hybrid dx motif ( fig . , there are two parallel hetero - duplexes that are joined together at two points where strands cross over from one duplex to the other . the dx motif is composed of five nucleic acid strands : one central , long , two times repetitive dna strand ( l2 ) , two identical , short , peripheral dna strands ( s ) and two rna strands ( r ) . although strands l and s cross between duplexes , each r strand continuously extends through one duplex from one end to the other . a twofold rotational axis passes through the centre of the motif and is perpendicular to the tile plane . because of this symmetry , the two s strands are identical , as are the two r strands . each dx tile only needs three unique , component strands : l , r and s. strand r is an rna molecule , and strands l and s are both dna molecules . the dx tiles have single - stranded overhangs ( sticky ends ) at each end of the two duplexes . two neighbouring dx tiles can then associate with each other through hybridization of these sticky ends . the distance between the two crossover points in the tile is set as two turns ( 22 bp ) . to allow the dx tiles to tessellate a plane instead of forming one - dimensional arrays , it is essential to keep any two adjacent , interacting dx tiles coplanar , which requires the distance between the two adjacent crossover points of two interacting dx tiles to be n + 0.5 turns ( n is an integer ) . in the current design the self - assembly of rna dna hybrid nanostructures is a one - pot process . an aqueous - solution mixture of the component dna and rna strands at the designated ratio is slowly cooled from 70 c to 25 c over 24 h. in the process , the dna rna strands recognize and associate with each other , first into individual dx tiles and then into large assemblies . the assemblies were analysed by native polyacrylamide gel electrophoresis ( page ) and visualized by atomic force microscopy ( afm ) . in native page ( supplementary fig . s2 ) , all dna rna complexes appeared as sharp bands with the expected mobility , indicating that the hybrid complexes were formed and stable . large two - dimensional crystals and tubular structures were clearly visible ( fig . 1 ; supplementary fig . the apparent height of the single - layered two - dimensional crystals was 2.1 nm , a reasonable value for the diameter ( 2.5 nm ) of a - form duplexes as rna the two - dimensional crystals showed clear periodicities both along and perpendicular to their helical axes . the distance between two adjacent crossover points of two interacting dx tiles ( 2.5 turns or 29 bp , as indicated in fig . 1b , inset ) is important for ensuring that all dx tiles are coplanar in the final assemblies . if this distance deviates from this value , the helical nature of the duplexes would cause any two interacting tiles to be on different planes , and promote the formation of tubular structures . when this distance is decreased by 1 or 2 bp from 29 bp , the hybrid dx tiles are still able to assemble into large structures , which are predominantly tubular ( their heights are much higher than 2.5 nm , the diameter of an a - form duplex ) , and sometimes extended two - dimensional crystals . the tubular structures are occasionally ripped into monolayers , the regular patterns of which can be observed . when the distance increases by 1 bp or decreases by 3 bp , the hybrid dx tiles do not form any large , regular assemblies . overall , the formation of tubules is preferred over two - dimensional arrays in all cases , and rna dna hybrid dx tiles are more prone than pure dna dx tiles to form tubular structures . the strategy of using dna to program rna self - assembly is a general approach and can be used for the construction of a variety of structures . to demonstrate this capability , we engineered two rna dna hybrid star motifs . symmetric dna star motifs are a family of related nanostructures that have different numbers of branches ( 3 , 4 , 5 and 6 ) . each motif is assembled from three different strands : a long repetitive strand ( l ) , a medium strand ( m ) and a short peripheral strand ( s ) . all star motifs share the same strands m and s , but differ from one another in strand l. the sequence repeating time of the l strand determines the branch number of the motif . in the current work , the m strand is replaced by an rna strand ( r ) , resulting in rna dna hybrid star motifs . rna residues comprise half of the nanomotifs . when appropriate sticky ends are present at the peripheral ends of the duplexes , the hybrid motifs can further associate with each other into two - dimensional arrays ( figs 2 and 3 ) . the l strands contain unpaired , single - stranded loops ( shown in orange in figs 2 and 3 ; three or four bases long for the three- or four - pointed star , respectively ) at the centre . the short loops are long enough to prevent the branches from stacking with one another at the centre , and short enough to prevent the tiles from being too flexible . to prevent the intrinsic curvatures of the tiles from accumulating in one direction , a corrugation strategy was also applied12,33,34 . any two interacting tiles are separated by 4.5 turns , so the two adjacent tiles are related by a twofold rotational axis that is perpendicular to the molecular plane . this arrangement cancels the intrinsic curvatures of the tiles and promotes the tiles to form extended two - dimensional arrays . during slow cooling , the rna dna hybrid star tiles readily assemble into two - dimensional crystals , which can be visualized by means of afm imaging . the four - point star tiles assemble into periodic two - dimensional arrays ( fig . 2 ) with an apparent height of 2.2 nm . the cavities in the resulting two - dimensional crystals , to our surprise , are not squares . instead their dimensions are ( 27.2 1.6 nm ) ( 19 1.4 nm ) . this observation suggests that the overall geometry of the individual rna dna hybrid tile is a skewed four - point star or a four - leafed pinwheel ( fig . any two opposite leafs of the hybrid star are shifted away by 4 nm from one another in the direction perpendicular to the helical axes . the overall tile structure is quite different from that of its homo - dna counterpart . it is unlikely that this is due to a surface phenomenon of differential adsorption onto the mica substrate . the two faces of the two - dimensional arrays are identical to each other , as many twofold rotational symmetry axes ( for example , the twofold rotational axes indicated by the bold black arrows shown in figs 2b and 3b ) exist in the plane . a similar phenomenon , although less obvious , can be observed for the rna dna hybrid three - point star motif ( fig . its overall geometry is a skewed three - point star or three - leafed pinwheel with a threefold rotational symmetry . consequently , two - dimensional arrays of such tiles only have threefold , instead of sixfold , rotational symmetries . the strategy of dna - programmed rna self - assembly is not limited to two - dimensional self - assembly ; it can also be applied to three - dimensional self - assembly . to demonstrate the three - dimensional assembly each vertex has a connectivity of 3 and can be represented by a three - point star tile . the hybrid three - point star is similar to the one used for assembling trigonal two - dimensional arrays . however , the three - point star tiles in dodecahedra are significantly bent away from a planar structure . to provide enough flexibility for the tile to bend , the central single - stranded loops on the l strand are elongated from 3 to 11 bases . in addition , any two interacting tiles are designed to be separated by four turns ( an integral number of turns ) . at such a separation , any two interacting tiles will face the same side of the tile plane , and any intrinsic tile curvatures will accumulate in the same direction . in turn , this will promote the tiles to assemble into closed , discrete structures17 . similarly to two - dimensional self - assembly , the assembly process for three - dimensional structures is a simple one - pot process . dna hybrid dodecahedra have been characterized by dynamic light scattering ( dls ) and cryogenic electron microscopy ( cryoem ) imaging . the hydrodynamic radius of the hybrid complex is 20.7 1.3 nm as measured by dls . the value is consistent with the radius ( 20.5 nm ) of the circumscribed sphere of the hybrid dodecahedron model , assuming a pitch of 0.26 nm / bp and a diameter of 2.3 nm for the rna 4 ) . in the raw images , randomly distributed dodecahedral particles are observed , which are of the expected size . some small fragments and large aggregates also exist it is not clear whether those undesired structures are formed during the assembly process or result from damage accompanying sample preparation . the yield of the correctly formed particle is 40% , based on visual observation of the cryoem images ( this is significantly lower than the assembly yield of pure dna dodecahedrons ) . from the experimentally observed particles , a three - dimensional map of the rna dna hybrid dodecahedron is generated by single - particle three - dimensional reconstruction , a powerful technique routinely used by structural virologists35 . the map resolution was determined to be at 2.5 nm using the fourier shell correlation ( 0.5 threshold criterion ) of two three - dimensional maps independently built from half data sets . there is clear similarity between the computer - generated two - dimensional projections from the reconstructed structural model and the raw , individual particle images ( fig . 4e ) , and the class averages of raw particle images with similar views ( supplementary fig . the radius of the reconstructed dodecahedron model is 20.5 nm , consistent with both the design and the dls data . the skewness of the star motif is not observed from the reconstructed structural model , as it was in the two - dimensional arrays , presumably due to the low resolution of our cryoem data . when considering which strand of the overall assembly should be rna , our guideline was to make sure that every helical domain was composed of one dna strand and one rna strand . this ensures that all helical domains will be uniform , hybrid a - form duplexes . red strands in the tiles , but not for the green or black strands ( according to the colour code of the figures ) . theoretically , it is also possible to simultaneously use rna for the green and black strands and dna only for the red strand . it is also possible to use rna only for all three strands ( green , red and black ) . however , we have not experimentally explored this possibility , and it is not our intention , either . in general , rna strands are more difficult to prepare , much less stable , and much more expensive than dna strands with exactly the same sequence . thus , we decided to use the minimum number ( here only one ) of rna strands in the current study . for two - dimensional self - assembly of star motifs of either homo - dna or rna dna hybrid , the symmetry remains the same : three- or fourfold rotational symmetry ( figs 2 and 3 ) . in the design of the star motifs , each branch is an identical crossover structure and three- or fourfold rotational symmetries are expected . in the previous homo - dna structures , the star motif exhibits near d3 or d4 symmetry . those higher - degree symmetries are a surprising finding , and not the results expected from the design . dna structures is likely due to the subtle difference between a- and b - form duplexes at the central region of the tile ( supplementary fig . the base pairs stack directly onto one another and go through the centre of the dna helix . in contrast , base pairs in an a - form duplex are shifted from one another , and a cavity appears in the centre of the duplex when looking down the duplex . in addition , the negative charges on the phosphate groups are distributed differently along the different forms of the duplexes . electrostatic interactions would affect the homo- or hybrid duplexes differently , causing a structural adjustment to minimize the electrostatic repulsion . single - stranded loops ( 11 bases long ; the orange segments in strand l3 ) were used at the centre to provide enough flexibility for the motif to bend , but a dodecahedron formed instead of a tetrahedron . dna hybrid star motifs are more rigid than their corresponding homo - dna star motifs . the most important challenge for rna self - assembly is to rationally design and construct well - defined , stiff , branching motifs , the basic building blocks for nanoconstruction . to appreciate the level of challenge , a four - arm dna junction always adopts a right - handed , x - shaped structure . dna hybrid junction can be either right- or left - handed36,37 . compared with this challenge , it is simple and straightforward to adjust the repeating length for the different duplex forms . in the present study , we have also observed numerous different behaviours of the a- and b - form duplexes in nanoconstruction , including the strong tendency of hybrid dx to form tubular structures , the skewed appearance of the star motifs , and the surprising three - dimensional assembly results . star motifs ( with four - base - long sticky ends ) associate with one another and form low - mobility , high - molecular - weight species ( smears ) in the absence of divalent cations ( supplementary fig . under the same conditions , the corresponding homo - dna motifs will not associate with each other . dna hybrid structures in the current study were chemically less stable than the corresponding homo - dna structures . for example , homo - dna structures do not change noticeably under afm imaging after being stored for one week at 4 c , but the rna dna hybrid structures will degrade , and no well - defined nanoarray can be found under afm imaging after such storage . as well as the current approach for introducing rna into dna nanostructures , a more straightforward method is simply to create dna nanostructures with appended , single - stranded oligonucleotides with which rna strands can hybridize . furthermore , the rna moieties will be fully accessible to the surrounding solvent , harsh chemicals and degrading enzymes ( rnases ) , and are therefore prone to degradation . with the strategy reported here , it is possible to tightly fold rna deep inside the nanostructures to prevent such rnase accessibility ( this is in our study plan ) . in summary the presented strategy of using dna to program rna self - assembly comprises a versatile method for assembling rna molecules into a range of nanostructures . we believe that this strategy can be applied to all established dna nanomotifs and dna origami . some unexpected phenomena ( such as the fact that the star motifs are not straight ) have also emerged . to fully understand such phenomena and achieve tight structural controls , a systematic , theoretical modelling and experimental studies are necessary . we expect this strategy to provide a unified platform for assembling both rna and dna into large nanostructures with multifunctional rna modalities : sirnas , micrornas , antisense rnas , ribozymes and aptamers . dna nanoparticles for cellular deliveries of small rnas : aptamers for cellular targeting and other small rna rna molecules were transcribed with t7 rna polymerase from the corresponding dna templates , which were designed by the sequin38 computer program and purified with page . to form the designed nanostructures , rna and dna strands were combined according to the correct molecular ratios in a tris acetic edta mg ( tae / mg ) buffer and slowly cooled from 70 c to room temperature . after annealing , the samples were visualized by tapping - mode afm on a multimode afm with nanoscope iiia controller ( veeco ) in air at 22 c . for cryoem imaging , rna dna sample solutions were concentrated to 3 m , spread onto quantifoil grids , then plunge - frozen . data were recorded using a gatan 4k 4k charge - coupled device ( ccd ) camera in a philips cm200 transmission electron microscope . three - dimensional reconstructions of the dna dodecahedra used the single - particle image - processing software eman39 . an icosahedral symmetry for the polyhedra was established by processing the images assuming different symmetries and finding the symmetry that yielded a three - dimensional reconstruction consistent with the particle images . rna molecules were transcribed with t7 rna polymerase from the corresponding dna templates , which were designed by the sequin38 computer program and purified with page . to form the designed nanostructures , rna and dna strands were combined according to the correct molecular ratios in a tris acetic edta mg ( tae / mg ) buffer and slowly cooled from 70 c to room temperature . after annealing , the samples were visualized by tapping - mode afm on a multimode afm with nanoscope iiia controller ( veeco ) in air at 22 c . for cryoem imaging , rna dna sample solutions were concentrated to 3 m , spread onto quantifoil grids , then plunge - frozen . data were recorded using a gatan 4k 4k charge - coupled device ( ccd ) camera in a philips cm200 transmission electron microscope . three - dimensional reconstructions of the dna dodecahedra used the single - particle image - processing software eman39 . an icosahedral symmetry for the polyhedra was established by processing the images assuming different symmetries and finding the symmetry that yielded a three - dimensional reconstruction consistent with the particle images .

dna has recently been used as a programmable smart building block for the assembly of a wide range of nanostructures . it remains difficult , however , to construct dna assemblies that are also functional . incorporating rna is a promising strategy to circumvent this issue as rna is structurally related to dna but exhibits rich chemical , structural and functional diversities . however , only a few examples of rationally designed rna structures have been reported . herein , we describe a simple , general strategy for the de novo design of nanostructures in which the self - assembly of rna strands is programmed by dna strands . to demonstrate the versatility of this approach , we have designed and constructed three different rna dna hybrid branched nanomotifs ( tiles ) , which readily assemble into one - dimensional nanofibres , extended two - dimensional arrays and a discrete three - dimensional object . the current strategy could enable the integration of the precise programmability of dna with the rich functionality of rna .

Results Discussion Conclusions Methods Oligonucleotides, formation of RNADNA complexes and imaging Single particle reconstruction Supplementary Material

consequently , safer alternative anti - inflammatory agents to prevent and treat excessive postoperative inflammation and pain following cataract surgery would be advantageous . cyclo - oxygenase ( cox ) is an important enzyme in the inflammatory process and catalyzes the biosynthesis of prostaglandins and thromboxanes from arachidonic acid.1 two main isoforms , cox-1 and cox-2 , have been well characterized . cox-1 is constitutively expressed in most mammalian cells , including the kidney , gastrointestinal tract , platelets , and vascular endothelium , and plays a pivotal role in normal physiological function . cox-2 , on the other hand , is an inducible enzyme that is thought to be primarily responsible for inflammatory - mediated reactions . nonsteroidal anti - inflammatory drugs ( nsaids ) are potent inhibitors of cox enzymes and thereby the synthesis of prostaglandins . while the anti - inflammatory actions of corticosteroids are in part from the inhibition of phospholipase a2 preventing the release of arachidonic acid from membrane - bound phospholipids , nsaids act more downstream in the cascade and directly inhibit cox-1 and cox-2 enzymes . within the eye , it is firmly established that prostaglandins disrupt the blood ocular barrier , increase vasodilation , facilitate leukocyte migration , and promote pain.1 consequently , their inhibition should have favorable effects on both intraocular inflammation and pain . in support of this , several randomized , prospective , double - masked , and placebo - controlled studies have shown that topically applied indomethacin 1% , flurbiprofen 0.03% , ketorolac 0.4% and 0.5% , diclofenac 0.1% , nepafenac 0.1% , and bromfenac 0.09% reduce postoperative inflammation following cataract surgery.1 similarly , prospective randomized studies have demonstrated that diclofenac 0.1% , ketorolac 0.4% , nepafenac 0.1% , and bromfenac 0.09% reduce ocular discomfort after cataract surgery.1 of all the commercially available ophthalmic nsaids , ketorolac tromethamine ( ketorolac ) possesses the greatest number of studies supporting its efficacy in preventing and treating postoperative inflammation and pain after cataract surgery ( table 1).521 ketorolac 0.5% ( acular , allergan inc , irvine , ca ) is approved by the food and drug administration ( fda ) for seasonal allergic conjunctivitis , inflammation following cataract surgery , and ocular discomfort after refractive surgery.22 to reduce the incidence of burning and stinging , a 0.4% concentration of ketorolac ( acular ls , allergan inc ) was formulated , and appears to have a similar therapeutic effect.18,23 ketorolac 0.4% is approved by the fda for the reduction of ocular pain and burning following corneal refractive surgery . more recently , a preservative - free 0.45% preparation of ketorolac ( acuvail , allergan inc ) in carboxymethylcellulose was approved by the fda in 2009 for the treatment of pain and inflammation following cataract surgery and is dosed twice daily.24 the specific intent of this review is to assess the available evidence supporting the efficacy of acuvail in the treatment of pain and inflammation after cataract surgery . nsaids are a chemically heterogenous group of compounds that inhibit the formation of prostaglandins and lack a steroid nucleus biosynthetically derived from cholesterol . there are six major classes , but topical formulations are limited to the relatively water - soluble classes , ie , indole acetic , aryl acetic , and aryl propionic acids.25 ketorolac tromethamine is an aryl acetic acid derivative . the chemical name is ( )-5-benzoyl-2,3- dihydro-1h - pyrrolizine-1-carboxylic acid , compounded with 2-amino-2-(hydroxymethyl)-1,3-propanediol ( 1:1 ) . current preparations consist of a racemic mixture of r-(+ ) and s-( ) ketorolac tromethamine . when administered systemically , ketorolac has proven analgesic , anti - inflammatory , and antipyretic activity.1 its mechanism of action , as with all nsaids , is presumed to be due to inhibition of prostaglandin biosynthesis . however , systemic administration is not thought to achieve sufficient intraocular drug levels to inhibit prostaglandin synthesis completely in the ciliary body and iris.1 topical administration of ketorolac , on the other hand , reaches adequate levels to inhibit prostaglandin synthesis in these target tissues . in one study , after a single topical application of ketorolac 0.4% , a peak aqueous concentration of 57.5 ng / ml was achieved after 60 minutes.26 another study demonstrated mean aqueous concentration of 1079 ng / ml after a total of 12 doses of ketorolac 0.4% administered over 2 days.27 the original ophthalmic formulation of ketorolac is a 0.5% solution marketed as acular . a 0.4% formulation ( acular ls ) the 0.5% and 0.4% preparations are supplied as an isotonic aqueous mixture with a ph of 7.4 , and have an osmolality of approximately 290 mosmol / kg . however , both the 0.4% and 0.5% solutions contain benzalkonium chloride ( a preservative ) , the surfactant octoxynol-40 , and sodium edetate ( a metal - chelating agent ) , and are associated with a high incidence of burning and stinging on instillation ( as high as 40%).22,23,28 in an effort to increase ocular bioavailability , a new formulation of ketorolac was developed to preserve the efficacy of prior formulations , while enhancing tolerability with a less frequent dosing regimen.21 compared with the ketorolac 0.4% formulation , the concentration of acuvail is 12% greater ( 0.45% ) and carboxymethylcellulose is added to the emulsion to allow greater drug retention on the ocular surface and improve comfort and drug penetration . other key changes include lower ph and absence of surfactant , metal - chelating agents , and preservatives . as such , acuvail is supplied as a sterile isotonic preservative - free solution with a ph and osmolality of approximately 6.8 and 285 mosmol / kg , respectively , and is approved by the fda for treatment of pain and inflammation following cataract surgery . in a study by attar et al evaluating the pharmacokinetics of ketorolac 0.45% versus 0.4% , the addition of carboxymethylcellulose in combination with the 12% increase in concentration resulted in a 35% enhancement of bioavailability in the aqueous humor of the 0.45% preparation.29 furthermore , decreasing the ph from 7.4 to 6.8 in combination with addition of carboxymethylcellulose enhanced bioavailability in the aqueous humor by two - fold and in the iris - ciliary body by three - fold . at the time of writing , the pharmacokinetics of acuvail have not been assessed in humans , but both acular ls and acuvail formulations were directly compared after a single topical application of 35 l.29 peak concentration in the aqueous humor and iris - ciliary body was 389 ng / ml and 450 ng / g , respectively , for acuvail , and 211 ng / ml and 216 ng / g , respectively , for acular ls . this study concluded that acuvail delivered significantly higher concentrations of ketorolac to the aqueous humor and iris - ciliary body . waterbury et al directly compared the peak and trough intraocular levels of acuvail , bromfenac 0.9% ( xibrom , ista pharmaceuticals inc , irvine , ca ) , and placebo after three applications ( 35 l ) every 20 minutes in an animal model of lipopolysaccharide - induced inflammation.30 peak concentrations in the aqueous humor and iris - ciliary body were 738 ng / ml and 556 ng / g , respectively , for acuvail and 94 ng / ml and 46 ng / g , respectively , for bromfenac 0.9% . trough concentrations in the aqueous humor and iris - ciliary body were 127 ng / ml and 59 ng / g , respectively , for acuvail , and 17 ng / ml and 8 ng / g , respectively , for bromfenac 0.9% . the study concluded that acuvail achieved aqueous and iris - ciliary body concentrations that exceeded its inhibitory concentration of 50% ( ic50 ) values for cox-1 and cox-2 at both peak and trough ( table 2).1,26,31,32 in addition , while both nsaids inhibited lipopolysaccharide - induced aqueous prostaglandin e2 elevation , only acuvail significantly prevented vascular leakage , as measured by fluorescein isothiocyanate - dextran leakage at trough levels . although suggestive , the number of animals used in both studies was small , and therefore additional studies with larger sample sizes are needed to confirm these initial results . furthermore , both studies used new zealand white rabbits which lack pigment , blink infrequently , and have an unusually unstable blood aqueous barrier , so these results should be extrapolated to humans with caution . given the favorable pharmacokinetic data of acuvail , approval was granted for twice - daily dosing in contrast with four times daily dosing for acular ls . this reduced dosing regimen offers a distinct therapeutic advantage for acuvail because in addition to patient convenience , several studies have suggested increased patient compliance with less frequent dosing.33 the efficacy of acuvail was assessed in two identical multicenter , double - masked , randomized , placebo - controlled , parallel studies and specifically conducted to evaluate the effects of acuvail on relief of pain and inflammation after cataract surgery.21 the results of these parallel studies were analyzed together and collectively involved 511 patients . patients were randomized in a 2:1 ratio to receive either acuvail or vehicle in the operative eye for 16 days . patients were dosed twice daily the day before surgery . on the day of surgery , patients had one drop upon awakening , three drops each 20 minutes apart starting two hours before surgery , one drop before discharge , and one drop 12 hours after the first dose upon awakening , resulting in a total of six drops of the study medication . patients continued one drop twice daily of the study medication for 14 days after surgery . all patients underwent elective unilateral , uncomplicated , extracapsular cataract extraction with posterior chamber intraocular lens implantation . the primary efficacy endpoint was the percentage of patients with a summed ocular inflammation score ( sois ) of 0 for anterior chamber cell and flare on day 14 . the main secondary efficacy endpoint was the percentage of patients with no pain ( grade = 0 ) on postoperative day 1 . in this study , anterior chamber cell was graded on a six - point scale and anterior chamber flare was graded on a five - point scale . the sois was calculated as the sum of scores for anterior chamber cell and flare . to assess the degree of pain , patients called an interactive voice response system diary twice daily during the two weeks after the day of surgery and were instructed to rate their level of ocular pain on a five - point scale ( 0 = none , 1 = mild , 2 = moderate , 3 = severe , 4 = intolerable ) . the acuvail group had a significantly higher percentage of patients with a sois score of 0 compared with vehicle at days 7 and 14 . at day 7 , acuvail and vehicle had a sois score of 0 for 32% ( 102/318 ) and 17% ( 26/155 ) of patients , respectively ( p < 0.001 ) . at day 14 , acuvail and vehicle had a sois score of 0 for 53% ( 167/318 ) and 27% ( 41/155 ) of patients , respectively ( p < 0.001 ) . a pain score of 0 on day 1 was reported in 72% ( 233/322 ) of acuvail patients versus 40% ( 62/156 ) in vehicle patients ( p < 0.001 ) . the median time to postoperative ocular pain resolution was one day in patients treated with acuvail and two days in patients treated with vehicle ( p < 0.001 ) . although the combined results of these controlled studies demonstrate the efficacy of acuvail for the prevention and treatment of postoperative inflammation and pain after cataract surgery , these results should be interpreted with caution . the importance of achieving a median of one less day of pain with acuvail versus placebo needs to be assessed in the appropriate context of treatment cost , clinical impact , and in the absence of concomitant corticosteroid use . moreover , cell and flare were combined together in contrast with grading each outcome independently , and their summation may have amplified the treatment differences observed between the acuvail and vehicle group . in addition , because corticosteroids were not used concomitantly , no information can be discerned about the additive benefits of acuvail with a corticosteroid in regards to inflammation and pain following cataract surgery . in a study comparing the efficacy of ketorolac 0.5% with prednisolone acetate 1% , simone et al observed that prednisolone acetate was more effective at reducing intraocular inflammation by day 7 after cataract surgery than ketorolac , although this difference resolved by day 28.10 several studies have demonstrated an additive benefit of a topical nsaid with a corticosteroid and their combined use , therefore , is common in clinical practice.1 therefore , the results of this study can not be directly applied in the setting of concomitant corticosteroid use . nevertheless , previous studies have demonstrated an additive benefit of ketorolac 0.5% or 0.4% when used in conjunction with corticosteroids in reducing inflammation , pain , and cme following cataract surgery . thus , given the favorable pharmacokinetics of acuvail in comparison with these older formulations , similar therapeutic benefit may be likely.521 several in vitro studies indicate that ketorolac is the most potent inhibitor of cox-1 , while both amfenac ( active component in nevanac , alcon laboratories inc , fort worth , tx ) and bromfenac have been reported as being the most potent inhibitors of cox-2.1,26,31,32 bromfenac may be a 318 times more potent inhibitor of cox-2 than diclofenac , amfenac , and ketorolac.25,32 another study found that amfenac was a more potent inhibitor of cox-2 than bromfenac . therefore , the anti - inflammatory actions of nsaids are presumed to relate to their ability to inhibit this isoform . however , this paradigm has not been consistently demonstrated in clinical trials , and the possibility exists that cox-1 also plays an important role in inflammation and , in the presence of substrate , may readily convert arachidonic acids into prostaglandins . thus , the clinical importance of selective cox-1 versus cox-2 inhibition for ocular disease remains unproven . although ketorolac is approximately six times more potent as an inhibitor of cox-1 than cox-2 ( table 2 ) , it is nevertheless a potent inhibitor of cox-2 , with an ic50 in the range of 0.090.12 m ( 33.945.2 ng / ml).32 this allows ketorolac to inhibit cox-2 in the iris - ciliary body after topical application , an important fact that can be overlooked if the relative cox-1/cox-2 potencies of ocular nsaids are emphasized . on the other hand , ketorolac is an approximately 550 times more potent inhibitor of cox-1 than diclofenac , bromfenac , and amfenac . the ability to inhibit both isoforms of cox for short periods of time may be advantageous in allowing more rapid and complete inhibition of prostaglandin production , but long - term inhibition of cox-1 may not be desirable because it is involved in normal physiologic function . the combination use of topical nsaids and corticosteroids is sometimes referred to as synergistic in the literature . this clinical impression of synergy remains unproven and would seem unlikely , given the fact that both drug classes inhibit prostaglandin production ( figure 1 ) . synergy is defined , in general , as two or more agents working in combination to produce an effect that could not be obtained by each agent independently . a classic example of synergy involves penicillin and aminoglycoside antibiotics where use of both antibiotics in combination significantly lowers the ic50 of each antibiotic for a given organism . although a large , randomized , prospective study demonstrated that ketorolac 0.5% was more effective than dexamethasone sodium phosphate 0.1% solution in facilitating re - establishment of the blood aqueous barrier after surgery , differences in drug formulation and intraocular concentration preclude any conclusions about synergy.3436 furthermore , although many prospective studies have confirmed that the combination use of an nsaid and corticosteroid is superior to a corticosteroid alone for pain , inflammation , cme , and visual improvement after intraocular surgery , these findings can be explained by an additive effect of a second anti - inflammatory agent.1,17,37 the distinction between a synergistic effect and an additive effect has important implications because synergy implies that an nsaid used in combination with a corticosteroid provides a therapeutic effect that can not be replicated by simply increasing the dosing regimen of the corticosteroid . although topical administration of nsaids provides aqueous humor levels adequate to suppress prostaglandin synthesis in the iris and ciliary body , the ability to suppress prostaglandin synthesis in the retina / choroid is less certain . in an animal model , ketorolac 0.5% could not be detected in the vitreous after topical administration , but in a small prospective comparative study of patients undergoing vitrectomy , ketorolac 0.4% could be detected in the vitreous ( 2.8 ng / ml ) and reduced vitreous prostaglandin e2 levels.38,39 this observed reduction was presumably from inhibition of iris and ciliary body prostaglandin e2 production because the measured vitreous concentration of ketorolac was considerably less than the ic50 for both cox-1 and cox-2 inhibition , and therefore unlikely to have inhibited cox activity in retinal cells . accumulating evidence indicates that cox-2 has important implications for retinal disease.1,40 cox-2 is the predominant isoform in human retinal pigment epithelial cells and is significantly upregulated in response to proinflammatory cytokines.41 cox-2 is also present in choroidal neovascularization , as well as in other highly vascularized lesions , and its expression increases in diabetic retinopathy.1,42 in a variety of experimental systems , cox-2 inhibition suppresses angio - genesis.43 in this regard , both nepafenac 0.1% and bromfenac 0.09% could be detected in the rabbit retina after topical administration , and in one study nepafenac inhibited 55% of retinal prostaglandin synthesis.31,44 in another study , topical ketorolac 0.4% inhibited experimentally induced choroidal neovascularization and reduced both retinal prostaglandin e2 and vascular endothelial growth factor levels by > 30%.45 while interesting , these results were obtained in animal models and can not be directly extrapolated to humans . given the favorable pharmacokinetics of acuvail versus acular ls , it is worth speculating that clinically meaningful retinal cox inhibition may now be achievable . the overall incidence of treatment - related adverse events was significantly higher in the vehicle group ( 14% ) than in the acuvail group ( 6%).21 the most common adverse events reported were increased intraocular pressure , conjunctival hyperemia , and/or hemorrhage , corneal edema , ocular pain , headache , tearing , and blurred vision . the most common adverse events reported at a higher frequency than vehicle included increased intraocular pressure ( 5.8% versus 1.8% ) , conjunctival hemorrhage ( 1.2% versus 0.6% ) , and blurred vision ( 1.2% versus 0.6% ) , and were generally considered by the clinical investigators to be a consequence of the cataract procedure . adverse events of burning and stinging were low , with only 1.5% of patents in the acuvail group and 0.6% in the vehicle group reporting such an occurrence . when ketorolac 0.5% was topically applied in one eye three times daily , only five of 26 subjects had a detectable amount of ketorolac in their plasma ( range 10.722.5 ng / ml ) at day 10 . in contrast , when ketorolac 10 mg was administered systemically every six hours , peak plasma levels were around 960 ng / ml . given the possibility of systemic absorption after topical application , techniques such as lid closure and nasolacrimal occlusion may be used to decrease systemic exposure . as with other nsaids , acuvail may slow or delay healing , and in some susceptible patients , continued use of topical nsaids may result in epithelial breakdown , corneal thinning , corneal erosion , corneal ulceration , or corneal perforation . there exists the potential for cross - sensitivity to acetylsalicylic acid , phenylacetic acid derivatives , and other nsaids , and therefore caution should be used when treating individuals who have previously exhibited sensitivities to these drugs . therefore , it is recommended that topical nsaids be used with caution in patients with known bleeding tendencies or who are receiving anticoagulation . there are no adequate well controlled studies of topical nsaids in pregnant woman and , as such , these medications are classified as category c. because of the known effects of prostaglandin - inhibiting drugs on the fetal cardiovascular system ( closure of the ductus arteriosus ) , the use of topical nsaids should be avoided during late pregnancy . severe corneal toxicity has been reported with diclofenac 0.1% , ketorolac 0.5% , nepafenac 0.1% , and bromfenac 0.09%.1 although uncommon , these dramatic events are referred to as corneal melt . however , a definite link between nsaid use and corneal melt remains unproven , but it is still prudent to avoid nsaid use in patients with severe corneal surface disease . older formulations of ketorolac had an incidence of transient burning and stinging on instillation of approximately 20%40% for the 0.4% concentration and 40% for the 0.5% concentration.22,23 in contrast , acuvail was associated with substantially lower rates ( 1.5% ) of burning and stinging.21 most importantly , a significantly higher percentage of patients randomized to acuvail had a 3-line or more improvement in vision from baseline compared with those treated with vehicle.21 at least two previously published studies using ketorolac 0.4% demonstrated a similar beneficial effect upon visual acuity following cataract and vitreoretinal surgery.17,37 in patients undergoing routine vitreoretinal surgery , patients randomized to ketorolac 0.4% experienced an average 4.3-line improvement from baseline compared with a 2.5-line improvement with placebo.37 this greater improvement in vision in the ketorolac group was observed despite concomitant corticosteroid use in both groups . excessive postoperative inflammation and pain after cataract surgery can delay visual recovery and affect long - term outcomes . there is substantial evidence from well designed studies with sufficient numbers of patients that ketorolac formulations 0.4% and 0.5% effectively treat both inflammation and pain after cataract surgery . given the favorable pharmacokinetics , better tolerance , and reduced dosing requirement of acuvail , additional clinical studies comparing acuvail with other nsaids for the treatment of inflammation and pain after cataract surgery are indicated to confirm these promising results .

background : the purpose of this review was to provide a critical appraisal of the literature supporting the efficacy of ophthalmic ketorolac ( acuvail ) in the treatment of pain and inflammation after cataract surgery.methods:literature search and expert opinion of the authors.results:recent studies indicate greater intraocular drug levels in the anterior chamber and iris - ciliary body after topical application of acuvail in comparison with older formulations of ketorolac . a large randomized , multicenter , placebo - controlled study demonstrated significantly less inflammation and pain after cataract surgery using acuvail.conclusion:acuvail appears to be effective in reducing post - cataract surgery pain and inflammation .

Introduction Pharmacology of ketorolac Efficacy studies Selective COX-1 versus COX-2 inhibition NSAIDs and corticosteroids Ketorolac and posterior segment disease Safety and tolerability Patient satisfaction Conclusion

malaria , which is caused by a protozoan parasite of the plasmodium genus , represents a serious global health concern given that nearly half of the world population is at risk of infection ( who , 2014 ) . although several anti - malarial drugs for treating the symptomatic stage ( or blood stage ) of malarial infection are commercially available ( antony and parija , 2016 ) , their efficacy has declined appreciably in the last few decades owing to widespread drug resistance developed by the parasite ( breman et al . , chloroquine was the first - line malaria treatment for many decades until drug - resistant p. falciparum strains became common . the drug causes a dose - dependent decrease in hemozoin formation ( chou and fitch , 1992 , slater and cerami , 1992 ) and an associated increase in toxic free heme in the food vacuole of the parasite ( combrinck et al . , 2013 , loria et al . , 1999 ) . over the past few decades , researchers have proposed many different mechanisms for chloroquine action , including 1 ) dna intercalation ( meshnick , 1990 ) , 2 ) alteration of digestive food vacuole ph ( yayon et al . , 1985 ) , 3 ) inhibition of heme polymerase ( yayon et al . , 1985 ) , and 4 ) formation of a toxic chloroquine - ferriprotoporphyrin ix complex ( sugioka et al . , yet , there is no consensus on the exact mechanisms by which chloroquine kills the parasite and under what circumstances they operate . shedding light on the possible actions of chloroquine is critical for developing more potent drugs or combination drug treatments against the parasite and for understanding how the parasite can develop resistance against them . here we used systems biology model and transcriptional profiles of p. falciparum to obtain information about metabolic and biological precursors that determine the physiological or pathophysiological state of the parasite . specifically , we used the p. falciparum transcriptomic data obtained by hu and colleagues during the intraerythrocytic development cycle ( idc ) at different concentrations of chloroquine ( hu et al . , 2010 ) . our primary goal was to use these data to investigate the effect of chloroquine on p. falciparum dna replication . we chose to study this effect because it has been proposed as a mechanism of chloroquine action in different plasmodium species ( gutteridge et al . , 1972 , meshnick , 1990 , polet and barr , 1968a , polet and barr , 1968b ) , in bacteria ( ciak and hahn , 1966 ) , and in bioassays examining the effect of chloroquine on rna and dna polymerases ( cohen and yielding , 1965 , o'brien et al . we first developed a method that integrates the information embedded in the transcriptome data with the whole - genome metabolic network model for p. falciparum ( fang et al . , 2014 ) to predict the metabolic phenotype corresponding to those transcription data . we validated the developed method by using a data set from an independent study that jointly capture transcriptomic and metabolomic data from p. falciparum , to correctly predict the metabolic phenotype tied to genetic perturbation of two krebs cycle enzymes ( ke et al . , 2015 ) . we then focused on the inhibition of dna replication in p. falciparum ( as a consequence of chloroquine treatment ) a critical metabolic phenotype ( ciak and hahn , 1966 , cohen and yielding , 1965 , polet and barr , 1968a ) , and used our method to simulate the effect of chloroquine on the metabolic fluxes of p. falciparum during the idc . we identified genes that were substantially altered in response to chloroquine treatment and linked to the inhibition of p. falciparum dna replication . the cohort of identified genes suggests that dna replication is inhibited by the downstream effect of heme accumulation . specifically , our analysis suggests that continuous accumulation of heme inhibits redox metabolism , carbon fixation , and pyrimidine metabolism , which leads to inhibition of dna replication and facilitates death of the parasite . our results provide a mechanistic explanation for why parasites with an efficient redox metabolism may have a lower sensitivity to chloroquine ( kasozi et al . , 2013 , lehane et al . , 2012 , metabolic flux profiles of p. falciparum strains 3d7 ( pf3d7 ) and dd2 ( pfdd2 ) during the idc were estimated by using transcriptome data previously reported by llinas and colleagues ( llinas et al . , 2006 ) , who extracted cdna from parasites cultured in erythrocytes and hybridized them to dna microarrays to yield hourly levels of gene expression for the pf3d7 ( or pfdd2 ) strain during the idc . the temporal resolution of these hourly gene expression profiles allowed us to make high - resolution time - dependent metabolic flux predictions during the idc . we used previously reported gene expression data ( hu et al . , 2010 ) to simulate the effect of chloroquine on the time - dependent metabolic fluxes of pf3d7 and pfdd2 during the idc . briefly , hu and colleagues ( hu et al . , 2010 ) administered chloroquine 18 h after erythrocyte invasion at concentrations of 41 , 72 , and 144 nm for pf3d7 and at 43 nm for pfdd2 , and obtained gene expression profiles every hour for 8 h during the idc following the chloroquine treatment . the model was identical to a previously published model ( fang et al . , 2014 ) , with one minor modification : we added reactions explicitly carried out by pdx2 ( pf11_0169 ) and pdx1 ( mal6p1.215 ) . this was done by 1 ) modifying an old reaction that incorrectly assumed that pyridoxal 5-phosphate ( plp ) was synthesized from ribulose 5-phosphate and 2 ) adding a new ( and separate ) reaction for pdx1 because experiments suggest that it can synthesize plp independent of the ammonia generated by pdx2 ( hanes et al . , 2008 ) . in the updated model , pdx2 hydrolyzes l - glutamine to yield glutamate and ammonia while pdx1 incorporates the ammonia generated by pdx2 to yield plp from the substrates ribose 5-phosphate and glyceraldehyde 3-phosphate ( gengenbacher et al . , 2006 ) , with the isomerization of ribose 5-phosphate to ribulose 5-phosphate as a side reaction . we used gene - to - reaction mappings available in the metabolic network model to obtain reaction expressions rexp , which depend on the expression of a gene ( or genes ) catalyzing a particular metabolic reaction . for a reaction catalyzed by a single enzyme , rexp was equal to the expression of the gene catalyzing that reaction however , if a reaction was associated with more than one gene , then their gene - to - reaction mappings were defined by using the boolean operators and and or . we implemented these operations by taking the minimal ( for the and operator ) and maximal ( for the or operator ) values of the associated gene expression data ( song et al . , 2014 ) . , 2014 ) that integrates hourly gene expression data with a whole - genome metabolic network model to estimate the hourly metabolic flux profiles of p. falciparum during the 48-h long idc period . to take advantage of additional studies that collect gene expression data as a function of genetic or chemical perturbations , we modified the method because such studies usually do not provide the same granularity and density of gene expression data to fully cover the entire idc . therefore , we aimed to develop a method that could utilize a reduced transcriptomic data set to predict alterations in metabolic flux profiles during the idc . briefly , we used the following steps to estimate metabolic fluxes under no - stress conditions : step 1 : estimate nutrient fluxes sufficient for the nominal growth rate ( 40% of the maximum growth rate).step 2 : estimate reaction fluxes vin for metabolite i corresponding to the nominal growth rate constrained by the nutrient fluxes obtained in step 1.step 3 : estimate reaction fluxes vit for reaction i at any time point t during the idc by incorporating time - dependent transcriptomic profiles under stoichiometric and nutrient uptake constraints . step 1 : estimate nutrient fluxes sufficient for the nominal growth rate ( 40% of the maximum growth rate ) . step 2 : estimate reaction fluxes vin for metabolite i corresponding to the nominal growth rate constrained by the nutrient fluxes obtained in step 1 . step 3 : estimate reaction fluxes vit for reaction i at any time point t during the idc by incorporating time - dependent transcriptomic profiles under stoichiometric and nutrient uptake constraints . the last step was achieved by globally minimizing for all reactions and time points the value of t that minimized the weighted absolute difference , ig|vitritvin| , where g represents the set of unidirectional reactions with known gene - to - reaction mapping . the factor rit = rexp , itmin(rexp , it)mean(rexp , it)min(rexp , it ) ; where , rexp , it represents reaction expression of ith reaction at time t obtained from the gene - to - reaction mapping of the p. falciparum metabolic network , according to the approach outlined in section 2.2 . this factor , rit , ensured that the mean of the ith reaction during the idc was equal to vin . , we estimated the organism - level impact of a drug on metabolism by using transcriptomic data obtained after drug treatment . the original formulation outlined above ( steps 13 ) can not be directly translated to stress conditions unless the complete transcriptomic profile for each stress condition during the entire idc is available ( fang et al . , 2014 ) . therefore , we introduced a parameter i to account for up- or down - regulation of individual reactions in response to stress to simulate the effect of exogenous stress on metabolic flux profiles during the idc . we modified the factor rit to iritmin(irit)mean(rit)min(irit ) , where i denotes the median relative change in the reaction expression of the ith reaction following chloroquine treatment , as observed in previous experiments ( hu et al . , 2010 ) . note that the mean of each individual modifying factor rit , i.e. , 1nj=1tnrij , obtained by summing the values across all time points in the idc , tracks with i ; that is , when the mean is less than one , greater than one , or equal to one , i<1 , i>1 , or i=1 , respectively . in other words , i increases or decreases the nominal flux of the ith reaction during the entire idc depending on the relative increase or decrease in the ith reaction expression . in principle , a drug may differentially affect gene transcription at different time points during the idc . here , we assumed that the median change in gene transcription after a stress treatment was representative of the transcriptional response throughout the idc . we used a previously published data set ( ke et al . , 2015 ) to validate our methodology to simulate the effect of external stress on metabolic fluxes during the idc . ke and colleagues ( ke et al . , 2015 ) constructed a p. falciparum mutant with two tricarboxylic acid ( tca ) cycle enzymes deleted ( idh/kdh ) , cultured wild - type and mutant parasites in erythrocytes , and obtained matched transcriptomic and metabolomic data . we used this data set because it describes changes in gene expression ( transcriptomics ) and phenotype ( metabolomics ) under the same experimental stress conditions . if the reaction fluxes estimated from previously reported transcriptome data ( ke et al . , 2015 ) adequately represent the reaction fluxes in vitro , then the predicted changes in reaction fluxes should be sufficient to predict the changes observed in the corresponding metabolomic data . we used a computer model of mitochondrial oxidative phosphorylation and the tca cycle ( wu et al . , 2007 ) to predict changes in tca cycle metabolites in response to changes in these estimated reaction fluxes ( ke et al . the kinetic parameters of this computer model were determined and validated by using extensive experimental data . we simulated wild - type reactions by running this model , with its default parameters , for 2000 s ( time required to reach steady - state condition ) and stored the concentrations of tca cycle metabolites . we performed simulations for the mutant parasite in the same way , albeit after scaling the model parameters that govern tca cycle enzyme activities by the estimated increase or decrease in reaction fluxes of those enzymes . we used a freely available software package called bisen ( vanlier et al . , 2009 ) to implement the tca cycle computer model ( wu et al . , 2007 ) . p. falciparum takes up nutrients to synthesize dna , rna , proteins , and phospholipids . these processes are not constitutively activated , because the parasite will synthesize them as needed throughout the idc . we used a validated in - house developed method ( fang et al . , 2014 ) to simulate time - dependent dna synthesis during the idc . for comprehensiveness , we introduce the constraint governing the rate of dna synthesis in the metabolic network model : cdatpdatp+cdctpdctp+cdgtpdgtp+cdttpdttpdna+(cdatp+cdctp+cdgtp+cdttp)diphosphate . here , datp , dctp , dgtp , and dttp refer to deoxyadenosine , deoxycytidine , deoxyguanosine , and deoxythymidine triphosphates , respectively . we refer the reader to the original article ( fang et al . , 2014 ) for further details on this method for simulating time - dependent p. falciparum macro - molecules during the idc . the criticality of a gene is determined by optimizing the following objective function : idnacimi , subjected to : smxnvnx1=0 and vlbvvub . here , dna is the gene set containing the metabolites that contribute to dna synthesis ( introduced above ) and ci is the contribution of the ith metabolite ( mi ) . in addition , s is the stoichiometry matrix , v is a vector containing all metabolic reactions of the p. falciparum metabolic network model , m is the number of metabolites in the network , n is the number of reactions in the network , and vlb and vub are the upper and lower bounds , respectively , of metabolic reactions . we deemed a reaction as critical if there was no dna synthesis after knocking out the reaction in silico . chloroquine administration inhibits dna replication in malaria parasites ( cohen and yielding , 1965 , polet and barr , 1968a ) . 2010 ) also show that metabolic genes critical for p. falciparum dna synthesis are suppressed by chloroquine treatment . we sought to identify the genes that are substantially altered in response to chloroquine and cause inhibition of p. falciparum dna synthesis . obvious candidates are genes that are down - regulated and critical for p. falciparum dna synthesis . however , not all such genes will have a pronounced effect on dna synthesis , which depends on the extent to which an individual gene is down - regulated and/or the individual contribution of that gene to the dna synthesis of the parasite . this suggests that many combinations of such genes may underlie the metabolic response associated with chloroquine treatment . , 1990 ) , we aimed at identifying the minimal number of genes ( hereinafter referred to as the core genes ) that are sufficient to completely account for the effect of chloroquine on dna synthesis by the parasite . we identified the core genes based on the bayesian information criteria ( bic ) score ( schwarz , 1978 ) , which is given by , nln(ssen)+kln(n ) , where sse=i=1n(victlvicqn)2max(vctl)min(vctl ) and n denotes the number of time points , k the number of genes being tested , vctl the estimated rate of dna synthesis under control conditions , and vcqn the estimated rate of dna synthesis after chloroquine treatment ( with =1 for the k genes being tested ) . we implemented an algorithm that starts with a set of size k=1 and increases the size of the test set as long as a given combination of genes for the new set ( size k+1 ) has a bic score lower than the minimal bic score of the previous size . we exhaustively repeated this process at each chloroquine dose ( 41 , 72 , and 144 nm for pf3d7 , and 43 nm for pfdd2 ) . we modified our previous approach ( fang et al . , 2014 ) to simulate the effect of external stress , as depicted in fig . 1 and described in the materials and methods section . we validated the methodology by demonstrating that reaction fluxes obtained in response to stress were sufficient to predict metabolic phenotypes corresponding to that stress . 2a shows the biochemical pathways incorporated into the computer model used to predict the functional consequences of altered fluxes of tca cycle enzymes in response to previously studied genetic perturbations ( ke et al . , 2015 ) . 2b shows the predicted alterations in tca cycle metabolites in response to genetic perturbations of tca cycle enzymes ( fig . 2a , red circles ) . we calculated the altered metabolite concentrations by scaling the control parameters in the tca cycle model ( wu et al . , 2007 ) by the median of the predicted changes in tca cycle enzyme activities ( fig . 2c ) . 2b , filled bars ) qualitatively captured the trends of relative change in experimentally measured tca cycle metabolites ( fig . 2b , open bars ) . 2b , filled bars ) was not within the margin of error of the experimental measurements ( fig . 2b , open bars ) . we examined all possible enzymes / transporters ( e.g. , citrate synthase , pyruvate dehydrogenase , aspartate aminotransferase , citrate - malate antiporter ) whose perturbation could yield mitochondrial citrate levels higher than those in the wild - type system . in each scenario , either the predicted citrate levels did not increase or the predicted oxaloacetate levels increased . therefore , based on our model analysis , the source of this increase in the experimentally reported citrate level appears to be non - mitochondrial in nature . in agreement with previous experimental observations ( cohen and yielding , 1965 , polet and barr , 1968a ) , our simulations suggested that chloroquine treatment results in dose - dependent inhibition of dna replication ( fig . a , blue diamonds ) and 43 nm ( pfdd2 , panel b , blue diamonds ) , the rate of dna synthesis during the ring stage ( the first 18 h after infection ) was less inhibited than it was during the late trophozoite and early schizont phases ( beyond 30 h after infection ) . these results are in agreement with previous observations ( yayon et al . , 1983 ) . because many studies suggest that pfdd2 is less sensitive to chloroquine than pf3d7 ( moneriz et al . , 2011 ) , dna synthesis might have been expected to be more inhibited in pf3d7 than in pfdd2 . in our simulations , however , a similar concentration of chloroquine resulted in greater dna synthesis inhibition in pfdd2 ( fig . 3b ; blue diamonds versus black circles , paired - sample t - test ; p < 0.0001 ) than in pf3d7 ( fig . 3a ; blue diamonds versus black circles , paired - sample t - test ; p = 0.15 ) . these differences likely stem from differences in the relative gene expression ratios of pfdd2 and pf3d7 . notably , the transcriptional profile for pfdd2 was more down - regulated than that for pf3d7 at a similar concentration of chloroquine . in other words , the percentage of down - regulated genes critical for p. falciparum dna synthesis was greater in pfdd2 than in pf3d7 ( table s1 ) . together with the highly similar ic50 values reported previously ( hu et al . , 2010 ) for pf3d7 ( 41 nm ) and pfdd2 ( 43 nm ) , these results suggest that the pfdd2 strain used here may have diverged from its true ( chloroquine - resistant ) phenotype . we found that 81 metabolic reactions in our network model are critical for p. falciparum dna synthesis . we denoted these reactions as critical because their elimination resulted in abolished dna synthesis ( see section 2.6 ) . of these 81 reactions , 64 had a known gene association whereas 17 did not . these genes belonged to pathways involved in glycolysis , pentose phosphate synthesis , pyruvate metabolism , purine metabolism , pyrimidine metabolism , redox metabolism , and mitochondrial metabolism . in the online supplement ( see spreadsheet ) , we provide detailed information , including the reaction name , chemical reaction , gene association , and pathway , of all reactions critical for p. falciparum dna synthesis . experimental observations ( cohen and yielding , 1965 , polet and barr , 1968a ) and the present simulations ( fig . 3 ) indicate that chloroquine treatment inhibits replication of p. falciparum dna , which in turn suggests that pathways essential for dna synthesis are inhibited . 4 shows the relative change in the amount of dna synthesized by the parasite during the idc period in response to chloroquine treatment . table s2 lists the actual amounts of synthesized dna for both strains under each simulated condition . we observed a dose - dependent decrease in the amount of dna synthesized by the parasite during the idc ( fig . 4 , red markers ) . we used bic scores ( schwarz , 1978 ) and employed the algorithm described in section 2.7 to identify core genes that caused this net decrease of synthesized dna . preventing only the core genes from being down - regulated by chloroquine was sufficient to restore the amount of dna synthesized to control values ( fig . 4 , green markers ) ; for these simulations , the reaction expressions of the core genes were set to be equal for control and chloroquine - treated conditions . a list of the core genes identified for each strain and chloroquine dose ( table s3 ) suggests that thioredoxin reductase and ribonucleotide reductase substantially affect the amount of synthesized dna . for pfdd2 , only two genes ( thioredoxin reductase and ribonucleotide reductase ) caused the nearly 50% reduction in the net amount of synthesized dna ( table s3 and fig . 4 ) . these genes were not mentioned in the original transcriptome analysis of hu and colleagues , who used a fundamentally different approach to interpret their transcriptome data ( hu et al . , 2010 ) . these authors focused on genes that were either co - regulated or showed strongly differential expression ( 8 log2-fold change ) in response to a drug . in contrast , our methodology of coupling transcriptomic changes with a metabolic network model can predict physiological outcomes as they relate to parasite metabolism , regardless of the magnitude of transcriptional change for an individual gene . transcriptional changes are not proportional to changes in metabolic reaction fluxes ( moxley et al . , 2009 ) . our analysis suggests that low - amplitude alterations in these identified genes ( table s3 ) were sufficient to cause a significant reduction in parasite dna synthesis . several existing methods integrate whole - genome metabolic networks and transcriptome data ( colijn et al . , 2009 , song et al . , 2014 , recently , we developed a method that predicts metabolic flux profiles of p. falciparum during the idc ( fang et al . the transcriptome data of p. falciparum obtained under different stress conditions ( e.g. , drug exposure , genetic mutation , etc . ) contain information about its functional state and can be used to predict qualitative alterations , if any , in metabolic flux profiles during the idc . however , such data are often collected at a limited number of time points ; for instance , hu and colleagues ( hu et al . , 2010 ) obtained p. falciparum transcriptome data at only 8 selected time points during the idc . currently , there is no approach to integrate limited data , such as those reported previously ( hu et al . , 2010 ) , with a whole - genome network to predict metabolic flux profiles during the idc . this is mainly because such an approach requires gene expression data throughout the idc to predict the corresponding metabolic flux profiles . therefore , we developed an approach that uses gene expression data obtained under a stress condition to predict alterations of metabolic fluxes that correspond to that condition . we validated the developed method by integrating transcriptome data obtained under a given stress condition and predicting metabolomic data corresponding to that stress ( ke et al . , , we simulated the effect of chloroquine on the metabolic flux profiles of p. falciparum during the idc . chloroquine inhibits the formation of hemozoin and leads to heme accumulation , which is toxic to the parasite ( fitch , 1998 ) . however , the specific mechanism underlying this toxicity is not known ( ginsburg et al . , 1998 , some experiments suggest inhibition of dna replication as a potential mechanism of chloroquine action ( cohen and yielding , 1965 , kwakye - berko and meshnick , 1989 , polet and barr , 1968a ) . we developed a method that successfully predicted a reduction in the ability of the parasite to synthesize dna in response to chloroquine treatment ( see fig . we identified twelve distinct genes that were substantially affected in pf3d7 and pfdd2 after chloroquine treatment ; importantly , preventing their down - regulation in response to chloroquine treatment was sufficient for restoring the p. falciparum dna synthesis inhibited by chloroquine treatment ( see table 1 ) . these genes belonged to pathways involved in glycolysis , redox metabolism , and pyrimidine metabolism . a closer look at these genes reveals a potential mechanism of chloroquine action that unifies previous theories and observations involving heme ( fitch , 1998 ) , dna replication ( polet and barr , 1968a ) , and oxidative stress ( lehane et al . , 2012 , radfar et al . , 5 , suggests that administration of chloroquine has two major metabolic effects : i.it interferes with the function of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , which reduces the synthesis of phosphoenolpyruvate , whose carbon fixation activity is essential for anaplerotic tca metabolism and redox balance ( storm et al . , 2014).ii.its interference of thioredoxin reductase ( trxr ) reduces conversion of oxidized thioredoxin trx ( s2 ) to reduced thioredoxin trx ( sh2 ) , which is essential for donating electrons to reactions catalyzed by thioredoxin peroxidase ( tpx ) and ribonucleotide reductase ( rnr ) . tpx is essential for converting h2o2 ( reactive ) to h2o ( harmless ) ( pannala and dash , 2015 ) , and rnr for converting ribonucleoside 5-diphosphates species ( adp , udp , gdp and cdp ) , to deoxy - species ( dadp , dudp , dgdp and dcdp ) which are used for dna synthesis ( munro and silva , 2012 ) . it interferes with the function of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , which reduces the synthesis of phosphoenolpyruvate , whose carbon fixation activity is essential for anaplerotic tca metabolism and redox balance ( storm et al . , 2014 ) . its interference of thioredoxin reductase ( trxr ) reduces conversion of oxidized thioredoxin trx ( s2 ) to reduced thioredoxin trx ( sh2 ) , which is essential for donating electrons to reactions catalyzed by thioredoxin peroxidase ( tpx ) and ribonucleotide reductase ( rnr ) . tpx is essential for converting h2o2 ( reactive ) to h2o ( harmless ) ( pannala and dash , 2015 ) , and rnr for converting ribonucleoside 5-diphosphates species ( adp , udp , gdp and cdp ) , to deoxy - species ( dadp , dudp , dgdp and dcdp ) which are used for dna synthesis ( munro and silva , 2012 ) . for example , our analysis points to trxr and gapdh as candidate molecules that determine the efficacy of chloroquine . here , we focused on candidates susceptible to a low dose of chloroquine because several pathways will be inhibited at higher concentrations ( see table s3 ; chloroquine = 144 nm ) . in addition , proteins that transport chloroquine out of food vacuoles should decrease the effective concentration of chloroquine . targeting these pathways while administering chloroquine treatment should enhance the efficacy of chloroquine at a given effective concentration . the hypothesis that chloroquine kills the malarial parasite by dna intercalation is decades old ( meshnick , 1990 ) . although it was the prime candidate mechanism of chloroquine from the 1950s to the 1970s , it has now largely been abandoned for the past two decades . our model analyses do not identify a drug target or targets ; however , they suggest that the effect of chloroquine administration leads to inhibition of dna synthesis . according to our model , the parasite responds to chloroquine by reducing the expression of genes critical for dna synthesis . although chloroquine could specifically intercalate with the identified core gene set in accord with the dna intercalation theory of chloroquine ( meshnick , 1990 , o'brien et al . , 1966a heme is a more plausible molecular inhibitor , given that it accumulates in large amounts within the parasite 's food vacuole and is capable of causing dna damage ( ishikawa et al . , 2010 , rahman et al . , 1997 ) , protein oxidation ( kitatsuji et al . , 2016 ) , nadph oxidation ( bodaness , 1983 ) , and apoptosis ( chiabrando et al . , 2014 ) . however , there is no direct evidence suggesting that heme causes any of these damages in p. falciparum . chloroquine , which was once a highly effective drug , is now abandoned in many malaria endemic regions because of widespread resistance . knowledge of this mechanism allowed us to identify metabolic pathways that could be exploited to increase chloroquine efficacy . for example , our model analyses suggest that administration of h2o2 would potentiate parasite killing by chloroquine , consistent with in vitro experiments ( malhotra et al . , similarly , our analyses suggest the hypothesis that targeting parasite - specific pathways that synthesize phosphoenolpyruvate and reduce thioredoxin would potentiate parasite killing . targeting these pathways , while administering amodiaquine ( a chloroquine analogue ) , should result in higher efficacy than administering chloroquine , especially because amodiaquine accumulates more than chloroquine in the parasite s food vacuole and has a mechanism similar to that of chloroquine ( hawley et al . , 1996 ) . our aim was to identify and investigate enzymes that contribute to decreased dna replication in response to chloroquine treatment . using a transcriptomics - based approach , we can identify cellular responses that are specific to chloroquine by expression data taken from chloroquine - treated parasites . although other antimalarial drugs may activate similar stress pathways , the details of the response will be specific to the particular conditions . the approach makes no assumptions on the underlying molecular actions that cause or affect the response . the effect of chloroquine on dna replication is likely to be indirect , being mediated by heme accumulation , which evokes multiple toxic effects in the cell . we simulated the effect of chloroquine over the entire idc based on the available transcriptome data taken from an eight - hour window during the trophozoite stage . ideally , for any specific inhibitor , it is desirable to have hourly transcriptional readouts to predict the effect of a systemic perturbation throughout the entire idc . in the case presented here , the largest effect of chloroquine occurs during the trophozoite stage and the protocol for collecting experimental transcriptomic data was designed to characterize transcriptional changes during this stage . our use of these data to identify changes in metabolite fluxes throughout the idc relative to untreated controls rests on the assumption that altering the expression of genes not associated with any biological function specific to a particular stage will have minimal physiological effects at other stages . this is an assumption that is specific to p. falciparum , because malaria relies on the execution of a highly regimented transcriptional program during the idc , which initiates specific stage - dependent cellular functions . these assumptions were ultimately corroborated because the observed metabolite phenotype changes are mainly associated with the trophozoite stage and metabolic alterations during previous time points were non - existent or immaterial to dna synthesis . herein , we investigated the potential mechanism by which chloroquine inhibits dna synthesis in p. falciparum and kills the parasite . we first developed an approach ( that requires limited transcriptome data ) to simulate the effect of exogenous stress on p. falciparum metabolic fluxes during the idc . we then developed an algorithm employing bayesian information theory to identify potential pathways that inhibit dna synthesis in response to chloroquine treatment . our analysis suggests that 1 ) chloroquine inhibits dna synthesis via inhibition of redox metabolism , carbon fixation , and anaplerotic tca cycle metabolism ; 2 ) p. falciparum may maintain dna synthesis after chloroquine treatment by preserving a reduced pool of thioredoxin and by invoking alternate mechanisms for synthesizing phosphoenolpyruvate , and 3 ) a drug or combination of drugs that target redox metabolism , carbon fixation , and heme accumulation could be an effective way to target the parasite . the method developed here used limited transcriptome data to identify pathways that underlie the inhibition of dna synthesis of p. falciparum in response to chloroquine treatment . this suggests that the developed method could also be suitable to simulate and capture the effect of other drugs ( or stress conditions ) on complex metabolic and physiological responses of p. falciparum , based solely on transcriptome data .

chloroquine , long the default first - line treatment against malaria , is now abandoned in large parts of the world because of widespread drug - resistance in plasmodium falciparum . in spite of its importance as a cost - effective and efficient drug , a coherent understanding of the cellular mechanisms affected by chloroquine and how they influence the fitness and survival of the parasite remains elusive . here , we used a systems biology approach to integrate genome - scale transcriptomics to map out the effects of chloroquine , identify targeted metabolic pathways , and translate these findings into mechanistic insights . specifically , we first developed a method that integrates transcriptomic and metabolomic data , which we independently validated against a recently published set of such data for krebs - cycle mutants of p. falciparum . we then used the method to calculate the effect of chloroquine treatment on the metabolic flux profiles of p. falciparum during the intraerythrocytic developmental cycle . the model predicted dose - dependent inhibition of dna replication , in agreement with earlier experimental results for both drug - sensitive and drug - resistant p. falciparum strains . our simulations also corroborated experimental findings that suggest differences in chloroquine sensitivity between ring- and schizont - stage p. falciparum . our analysis also suggests that metabolic fluxes that govern reduced thioredoxin and phosphoenolpyruvate synthesis are significantly decreased and are pivotal to chloroquine - based inhibition of p. falciparum dna replication . the consequences of impaired phosphoenolpyruvate synthesis and redox metabolism are reduced carbon fixation and increased oxidative stress , respectively , both of which eventually facilitate killing of the parasite . our analysis suggests that a combination of chloroquine ( or an analogue ) and another drug , which inhibits carbon fixation and/or increases oxidative stress , should increase the clearance of p. falciparum from the host system .

Introduction Materials and methods Results Discussion Limitations of the current study Conclusion

governments across the developed world are concerned with enabling older people to maintain their active contribution to society , and thereby their quality of life ( qol).1 qol has become a commonly used end point in the evaluation of multisector public policy , including health , social , community and environmental policy actions . for policy outcomes to be measured with any validity , measures of qol need to have social , as well as policy , relevance , to be meaningful to people 's lives , and to be carefully conceptualised and constructed . lawton26 developed a popularly cited quadripartite concept of qol , proposing that the good life ( qol ) may be represented by behavioural and social competence ( health , cognition , time use , social behaviour ) , perceptions of qol ( subjective evaluation of each domain of life ) , psychological well - being ( mental health , cognitive judgements of life satisfaction , positive - negative emotions ) and the external , objective , physical environment ( housing , economic indicators ) . however , there is no consensus about its conceptual definition or measurement,7 and most investigators have based their concepts on expert opinions rather than the perspectives of lay people . this has the consequence that there are few empirical data on the extent to which the items included in measurement scales have any relevance to people . thus , it is increasingly important to develop a multidimensional model and measure of quality of life , for use in descriptive and evaluative multisector policy research , which reflects the views of the population concerned , with cross - sectional and longitudinal applicability . elicitation of people 's own views of qol in this process is particularly important because qol is a subjective concept . survey and qualitative research with people aged 65 + , living at home in britain , reported that the central planks of qol emphasised by respondents were psychological well - being and positive outlook , having health and functioning , social relationships , leisure activities , neighbourhood resources , adequate financial circumstances and independence.710 this research led to the development of the older people 's quality of life questionnaire ( opqol ) , which is unique in being derived from the views of a representative sample of older people , cross - checked against theoretical models for assessment comprehensiveness . the aim here is to compare the psychometric properties of the opqol , with the casp-19 and whoqol - old among people 65 + participating in three national surveys of older people living at home in britain . two of these three surveys were cross - sectional , and the third was longitudinal ( see supplementary appendix 1 ) : ethnibus survey of people aged 65 + responding to two waves of the national ethni surveys ( http://www.ethnicfocus.com ) in 2008 . this is a rolling face - to - face interview survey with adults aged 16 + , living at home , based on focused enumeration , stratified random sampling of postcodes in britain , and statistically robust sampling of people in common ethnic minority groups in britain ; the response rate was 70% ( n=400 ) . ons survey of people aged 65 + responding to two waves of the office for national statistics ( ons ) national omnibus survey ( http://www.statistics.gov.uk ) in 2008 . this is a rolling face - to - face interview survey with adults aged 16 + , living at home , based on a stratified random sample of postcodes across britain ; the response rate was 61% ( n=589 ) . qol follow - up survey in 20072008 , of people living at home in britain , aged 65 + at baseline , who had responded to four ons national omnibus interview surveys . these were based on stratified random samples of postcodes across britain during 1999/2000 ; response was 77% ( n=999 ) at baseline and 58% among survivors ( n=287 ) at 20072008 follow - up . the qol follow - up survey is included here as the longitudinal design provided the opportunity to test the causal model of the opqol , as well as a willing sample for test - retest reliability assessment . the opqol was administered in all three surveys . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . the casp-1911 and the whoqol - old12 13 were administered in the two face - to - face interview surveys only ; it would have been too cognitively burdensome to have included all three scales in the postal , self - administration mode . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . this would not necessarily be applicable to populations in other countries , because it reflects close connections between new commonwealth countries and ethnic minority groups in the uk.15 descriptive analyses included frequencies , tests , and spearman 's r correlations . tests of scale reliability were applied in order to assess the extent to which scale items measure the same construct , with freedom from random error ( internal consistency ) . this is the strength of the association between each scale item and the full scale , item - item and item - total correlations . test - retest reliability of the stability of the newly developed opqol was assessed by mailing a second copy of the questionnaire to a random subsample of 50 follow - up qol survey respondents , 4 weeks after return of the first questionnaire ( response rate : 76%/38 ) . criterion ( concurrent ) validity is the independent corroboration that the scale is measuring what it intends to measure . this can only be measured by proxy with subjective measures , as there is no gold standard . proxy variables used here included independent self - ratings of qol overall and of qol domains ( health , social relationships , independence / control over life / freedom , home and neighbourhood , psychological / emotional well - being , financial circumstances , social and leisure activities ) . construct ( convergent and discriminant ) validity requires corroboration that scales measure the underlying construct they purport to measure . this was tested by assessing spearman 's r correlations between the qol scales and similar variables ( for convergent validity that the scale should correlate with similar or hypothesised variables ) and dissimilar variables ( for discriminant validity that there should be low correlations between scales and variables not expected to be associated ) . multiple regression was used to assess validity further by examining the ability of theoretically relevant variables to predict total qol scores . a hierarchical approach was used , with independent variables entered in their theoretical order of importance . statistical significance was set at p<0.05 . the variables entered did not correlate by more than 0.732 ; tests for multicollinearity were satisfied . the opqol was administered in all three surveys . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . the casp-1911 and the whoqol - old12 13 were administered in the two face - to - face interview surveys only ; it would have been too cognitively burdensome to have included all three scales in the postal , self - administration mode . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . this would not necessarily be applicable to populations in other countries , because it reflects close connections between new commonwealth countries and ethnic minority groups in the uk.15 tests of scale reliability were applied in order to assess the extent to which scale items measure the same construct , with freedom from random error ( internal consistency ) . this is the strength of the association between each scale item and the full scale , item - item and item - total correlations . test - retest reliability of the stability of the newly developed opqol was assessed by mailing a second copy of the questionnaire to a random subsample of 50 follow - up qol survey respondents , 4 weeks after return of the first questionnaire ( response rate : 76%/38 ) . criterion ( concurrent ) validity is the independent corroboration that the scale is measuring what it intends to measure . this can only be measured by proxy with subjective measures , as there is no gold standard . proxy variables used here included independent self - ratings of qol overall and of qol domains ( health , social relationships , independence / control over life / freedom , home and neighbourhood , psychological / emotional well - being , financial circumstances , social and leisure activities ) . construct ( convergent and discriminant ) validity requires corroboration that scales measure the underlying construct they purport to measure . this was tested by assessing spearman 's r correlations between the qol scales and similar variables ( for convergent validity that the scale should correlate with similar or hypothesised variables ) and dissimilar variables ( for discriminant validity that there should be low correlations between scales and variables not expected to be associated ) . multiple regression was used to assess validity further by examining the ability of theoretically relevant variables to predict total qol scores . a hierarchical approach was used , with independent variables entered in their theoretical order of importance . the variables entered did not correlate by more than 0.732 ; tests for multicollinearity were satisfied . just over half of each sample comprised women ( 52%/207 ethnibus , 55%/324 ons , 54%/154 qol follow - up ) . whereas most ethnibus respondents were aged 65<75 ( 91%/363 ) , just over half of ons omnibus ( 55%/326 ) , and less than a fifth of qol follow - up respondents ( 17%/47 ) , were aged 65<75 . thirty - eight per cent ( 152 ) of the ethnibus sample were indian , 29% ( 117 ) were pakistani , 22% ( 86 ) were black caribbean and 11% ( 45 ) were chinese . most , 94% ( 555 ) of the ons omnibus sample were white british ; all qol follow - up respondents were white british . in reflection of their younger age , more of the ethnibus than other respondents were married or cohabiting ( 58%/230 , 49%/285 , 49%/138 respectively ) . fewer ethnibus than other respondents were home - owners ( 532%/208 , 73%/429 , 85%/239 respectively ) and fewer lived alone ( 5%/19 , 48%/286 , 49%/137 respectively ) ( all differences were statistically significant at least at p<0.01 . ) for detailed characteristics of the samples , see supplementary table 1 . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . the reliability criterion for item - total correlations ( the correlation of the item with the scale total with that item omitted ) is that the item should correlate with the total scale by at least 0.20 . with three exceptions , the 35 full opqol items met this criterion for all three samples ( the exceptions were in the ethnibus sample with items 10 , 12 and 32 ; but as cronbach 's was not improved by their removal , and they performed well in validity tests , they were retained ) . six of the 19 casp items failed to meet this criterion ( ethnibus : items 1 , 2 , 5 , 17 , 18 ; ons : item 6 ) . fourteen of the 24 whoqol - old items failed this criterion in the ethnibus sample only . as expected , all items correlated more highly with similar , than dissimilar , items in the scales . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . the 4 week test - retest correlations , assessed among qol follow - up survey respondents , ranged from moderate to high ( r 0.4030.782 ) . lower correlations were explained by reported life changes in the intervening month , demonstrating the difficulties of test - retest exercises in older populations . respondents ' comments at follow - up about life changes in the last 4 weeks illustrate this:about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free;my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard;my daughter and her young son have now left our home and acquired her own house . we miss them a lot;my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free ; my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard ; my daughter and her young son have now left our home and acquired her own house . we miss them a lot ; my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. the criterion validity of all three qol scales was indicated by their moderate to strong , significant correlations with global self - rated qol : the spearman 's r correlations for the opqol by self - rated qol overall in each sample were ethnibus 0.347 , ons 0.602 and qol follow - up 0.659 . for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . all correlations were significant at least at p<0.01 , with the exception of whoqol - old in the ethnibus sample which was p<0.05 . the validity of the opqol was further supported by significant correlations between its subscales and the independent qol domain ratings , in theoretically expected , similar directions7 ( eg , opqol health and functioning subscale correlated with self - rated health : spearman 's r ethnibus 0.122 ( p<0.05 ) , ons omnibus 0.679 ( p<0.01 ) and qol follow - up 0.713 ( p<0.01 ) . there were no significant correlations with dissimilar pairs ( eg , health and religion ) , again as expected . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old sensory abilities subscale correlated significantly , again as expected , with self - rated health in the ons ( r 0.322 , p<0.01 ) , but not the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . for comparability , the same independent variables were entered into each model . on the basis of the literature,7 optimum scores on each measure were hypothesised to be associated with optimum qol : self - rated active ageing , independent self - ratings of qol domains , social activities and help from social network members , self - rated health status and physical functioning ( adl ) , age , sex , marital status and housing tenure . the qol follow - up sample also provided an opportunity to test the causal model underpinning the opqol . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . just over half of each sample comprised women ( 52%/207 ethnibus , 55%/324 ons , 54%/154 qol follow - up ) . whereas most ethnibus respondents were aged 65<75 ( 91%/363 ) , just over half of ons omnibus ( 55%/326 ) , and less than a fifth of qol follow - up respondents ( 17%/47 ) , were aged 65<75 . thirty - eight per cent ( 152 ) of the ethnibus sample were indian , 29% ( 117 ) were pakistani , 22% ( 86 ) were black caribbean and 11% ( 45 ) were chinese . most , 94% ( 555 ) of the ons omnibus sample were white british ; all qol follow - up respondents were white british . in reflection of their younger age , more of the ethnibus than other respondents were married or cohabiting ( 58%/230 , 49%/285 , 49%/138 respectively ) . fewer ethnibus than other respondents were home - owners ( 532%/208 , 73%/429 , 85%/239 respectively ) and fewer lived alone ( 5%/19 , 48%/286 , 49%/137 respectively ) ( all differences were statistically significant at least at p<0.01 . ) for detailed characteristics of the samples , see supplementary table 1 . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . the reliability criterion for item - total correlations ( the correlation of the item with the scale total with that item omitted ) is that the item should correlate with the total scale by at least 0.20 . with three exceptions , the 35 full opqol items met this criterion for all three samples ( the exceptions were in the ethnibus sample with items 10 , 12 and 32 ; but as cronbach 's was not improved by their removal , and they performed well in validity tests , they were retained ) . six of the 19 casp items failed to meet this criterion ( ethnibus : items 1 , 2 , 5 , 17 , 18 ; ons : item 6 ) . fourteen of the 24 whoqol - old items failed this criterion in the ethnibus sample only . as expected , all items correlated more highly with similar , than dissimilar , items in the scales . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . the 4 week test - retest correlations , assessed among qol follow - up survey respondents , ranged from moderate to high ( r 0.4030.782 ) . lower correlations were explained by reported life changes in the intervening month , demonstrating the difficulties of test - retest exercises in older populations . respondents ' comments at follow - up about life changes in the last 4 weeks illustrate this:about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free;my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard;my daughter and her young son have now left our home and acquired her own house . we miss them a lot;my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free ; my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard ; my daughter and her young son have now left our home and acquired her own house . we miss them a lot ; my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. the criterion validity of all three qol scales was indicated by their moderate to strong , significant correlations with global self - rated qol : the spearman 's r correlations for the opqol by self - rated qol overall in each sample were ethnibus 0.347 , ons 0.602 and qol follow - up 0.659 . for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . all correlations were significant at least at p<0.01 , with the exception of whoqol - old in the ethnibus sample which was p<0.05 . the validity of the opqol was further supported by significant correlations between its subscales and the independent qol domain ratings , in theoretically expected , similar directions7 ( eg , opqol health and functioning subscale correlated with self - rated health : spearman 's r ethnibus 0.122 ( p<0.05 ) , ons omnibus 0.679 ( p<0.01 ) and qol follow - up 0.713 ( p<0.01 ) . there were no significant correlations with dissimilar pairs ( eg , health and religion ) , again as expected . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old sensory abilities subscale correlated significantly , again as expected , with self - rated health in the ons ( r 0.322 , p<0.01 ) , but not the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . for comparability , the same independent variables were entered into each model . on the basis of the literature,7 optimum scores on each measure were hypothesised to be associated with optimum qol : self - rated active ageing , independent self - ratings of qol domains , social activities and help from social network members , self - rated health status and physical functioning ( adl ) , age , sex , marital status and housing tenure . the qol follow - up sample also provided an opportunity to test the causal model underpinning the opqol . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . this study describes the psychometric performance of a qol questionnaire , developed from the perspectives of older people themselves : the opqol . it was tested in two cross - sectional , and one longitudinal , surveys of older people across britain . the longitudinal survey enabled the opqol to be tested in a dynamic , ageing population and an assessment of its underlying model , although its self - administration mode necessitated the assessment of the opqol only ( and not the casp-19 or whoqol ) in this older sample . the surveys used statistically robust sampling methods , and the response rates were fairly to very good . the characteristics of respondents to the ons omnibus and ethnibus surveys ( and the qol survey at baseline ) were comparable with population estimates from the last census . the qol follow - up sample , by its longitudinal design , reflected the healthy survivors . also , although the sampling approach of the ethnibus survey was statistically robust , it used focused enumeration . there is no other practical methodology for attempting to obtain representative samples of people in ethnic minority groups in national samples . this study reported that ethnibus respondents obtained poorer ( worse ) qol scores than the other sample respondents , with the opqol , casp-19 and whoqol - old . this is not unexpected given that people in ethnic minority groups are often more economically disadvantaged than the wider population.15 further research is needed to examine whether differences in qol reflect real variations , methodology , and cultural variations in expectations or in reporting . ethnic minority groups in britain live in a wide range of different communities , and their diversity may also have affected responses in some way . it should also be noted that the standard question for ethnic status used , largely reflected britain 's new commonwealth groups , and may not be appropriate for use in other countries . multiple regression models supported its validity and underlying constructs . despite the ethnibus sample 's consistently worse qol scores , compared with the other samples , the casp-19 and whoqol - old did not meet all criteria for internal consistency ( reliability ) in the ethnically diverse ethnibus sample . the casp-19 and whoqol - old also had relatively large numbers of items that failed to meet the reliability criterion for item - total scale correlations ; they frequently failed correlation tests for validity in the ethnibus sample . this may have been due to this sample 's ethnic diversity , or because the casp-19 and whoqol - old were not sufficiently sensitive . the opqol is currently being tested with older people living in italy ; initial results for cultural equivalence and understanding are positive ( personal communication , dr claudio bilotta , university of milan).what is already known on this subjectincreasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age.qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions.what this study addsthis study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness.the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . increasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age . qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions . this study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness . the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives .

backgroundmost measures of quality of life ( qol ) are based on expert opinions . this study describes a new measure of qol in older age , the older people 's qol questionnaire ( opqol ) , which is unique in being derived from the views of lay people , cross - checked against theoretical models for assessment of comprehensiveness . its performance was assessed cross - sectionally and longitudinally . it was compared with two existing qol measures in the cross - sectional studies in order to identify the optimal measure for use with older populations.methodsdata were taken from three surveys of older people living at home in britain in 20072008 : one population survey of people aged 65 + , one focused enumeration survey of ethnically diverse older people aged 65 + , one follow - up of a population survey of people aged 65 + at baseline in 1999/2000 . measures were qol ( using opqol , control , autonomy , satisfaction , pleasure - 19 items ( casp-19 ) , world health organization quality of life questionnaire - version for older people ( whoqol - old ) ) , health , social and socioeconomic circumstances . the casp-19 and whoqol - old were not administered to the longitudinal sample in order to reduce respondent burden.resultspsychometric tests were applied to each qol measure . the opqol , casp-19 and whoqol - old performed well with the cross - sectional samples ; however , only the opqol met criteria for internal consistency in the ethnibus samples.conclusionthe opqol is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . further research is needed to examine whether differences by ethnicity reflect real differences in qol , methodological issues , variations in expectations or cultural differences in reporting .

Background Methods Measures Statistical analysis Results Characteristics of samples Distributions of samples on QoL scales Reliability Validity Multivariable analyses OPQOL CASP-19 WHOQOL-OLD Conclusion

myiasis , the infestation of organs and tissues of human and animals with fly larvae , is a common phenomenon especially in people of tropical and subtropical areas . in spite of occasional reports of obligatory myiasis , the majority of reported cases are facultative myiasis and more than a dozen families of flies can cause this form ( gullan and durden 2009 ) . phoridae , commonly known as humpback or scuttle flies , is the most important family of dipteran that causes myiasis and various problems for human . in this family genus megaselia , perhaps the largest genera of living organisms , has a wide variety of life styles and polyphagus diet ( disney 2008 ) . among identified species of this genus , it has been unwittingly carried around the world by human ( disney 2008 ) and previously has been reported from alborz province in iran ( zamani 2009 ) . adults of scuttle flies which are very small ( 24 mm in length ) , can explore a large variety of environmental and ecological habitats . females of these flies are highly attracted to strong of foul odors and lay their eggs on different decomposing materials such as fruits , stole , meat , excrement and carrions ( disney 2008 ) . the scuttle flies which are important agent of human facultative myiasis , exhibit a greater diversity of larval habitat than other insects . these flies can explore a large variety of environmental and ecological niches ( disney 2008 ) . in spite of predation and parasitation of some arthropods by this species ( costa et al . 2007 ) , previous studies showed them as a cause of human myiasis ( francesconi lupi 2012 ) . moreover , they have already been a polyphagus species and may be harmful to health as they become vector of pathogens ( disney 2008 ) . 1978 , carpenter and chastain 1992 , hira et al . 2004 ) and intestinal ( kaneko et al . 1975 , sing et al . however , the urogenital form is rarely seen and all of reported cases are restricted to females . so the study of men clinical manifestation as well as morphology of third instars larvae of these flies seems to be important . the aim of this paper is to report a case of man urogenital myiasis involving and to discourse the importance of its diagnosis and management in clinical science . an 18 year - old man living in zanjan city , northwest of iran , presented to ayatollah mosavi hospital at zanjan university of medical sciences with history of difficulty in urination and maggot discharging . , he was medicated by venlafazine , alpirazolam and nortriptrin . physical examination and sonography of cortex of bladder and kidneys he received mebendazole ( 2 mg tablet ) orally and washed his urogenital area with solution of xylocain ( 2% ) and iodine ( 1% ) . the urine was yellow in color and laboratory examination showed microhematuria , proteinuria and leukocyturia . the larvae were forwarded to the department of medical entomology and vector control for further studies . sonography of cortex of bladder with , small echogenous floating particles microscopic examination showed that all larvae were alive , whitish in color and pale intestinal content in the third terminal part ( fig . one larva was preserved in 70 % ethylic alcohol and the other cultured in nutrient rich medium to complete its life cycle but it died after 1 day . the length and width of larvae ranged from 6.8 to 6.9 and 0.68 to 0.69 mm respectively ( fig . precision microscopic examination of larvae revealed the presence of short spinous ( peglike ) processes on the integument . the larval head was noncapsulate , being reduced to mouth hooks and supporting cephalopharyngeal skeleton which was developed and supported large areas of the head ( fig . the anterior spiracles were located on each latero - posterior edge of the prothorax each had about 810 rays . a pair of posterior spiracles protruded dorsally on the 12 segment each appears with large and slender plate . in addition to their anterior and posterior union the dorsal and lateral trunks were joined by transverse connectives ( fig . 4 ) . third instars larva of megaselia scalaris anterior section of third instars larva of megaselia scalaris posterior section of third instars larva of megaselia scalaris based on characters and previous descriptions of researchers ( sukantason et al . 2004 , disney 2008 ) the larvae were definitely identified as megaselia scalaris , facultative myiasis parasite belonging to the phoridae family . the voucher specimens were retained in the department of entomological systematics at university of tarbiat modarres . urogenital myiasis , the infestation of genitourinary organs by maggots , based on the anatomical location could be classified as external or internal myiasis . external urogenital myiasis is clinically , epidemiologically , and entomologically similar to wound myiasis but it affects women more commonly . internal urogenital myiasis is a rare event and occurs when the maggot reaches an internal genitourinary organ . all described cases are considered as accidental cases and uncommon myiasis agents are associated with m. scalaris , psychoda albipenis and muscoid flies ( francesconi and lupi 2012 ) . several previous researchers reported cases of wound ( disney 2008 ) , intestinal ( mazyard and rifaat 2005 ) and nasocominal myiasis ( hira et al . 2004 ) , due to m. scalaris . however , the published cases of genital myiasis are very few in number and these reports refer to patients of various ages . the first report of human urogenital myiasis due to this species was reported in 1978 by disney and kurahashi and then other cases were reported ( ramalingam et al . 1997 , delir et al . 1999 , perez - eid c , mouffok 1999 , rodriguez and , rashid 2001 , passos et al . 2002 ) . there is a paucity of such information from this country , probably because specimens tend to be discarded without study , due to lack of experience in identifying fly larvae and in the absence of reference center . we hope that our report will stimulate others to present similar clinical findings and to contribute to the pool of knowledge on the subject in iran . to the best of our knowledge , so we hope that in the coming future special aspects of this species will be studied in iran . the identification of larvae of megaselia sp posed difficulties here because none could be reared to adult for conformation based on adult morphology . the passing of live m. scalaris larvae in the urine indicated infestation of the urogenital system . the reported infestations ( priyanond et al . 1973 , biery et al . 1989 ) were normally , a secondary consequence of some obstructions to the normal flow of the urine , such as stone formation in the bladder . our microscopic examination showed that the larvae were probably mature ; at least second to third instars transition , and may have been seeking a place to pupate . it is worth mentioning that identification of the immature larvae of parasite causing myiasis posed difficulties particularly the parasite with little available knowledge . however , the available morphological characters of the larvae especially the cephalopharyngeal skeleton and the structure of spiracles can be used in differentiation of specimens . however the global warming and the globalization of commerce have established conditions suitable for expand their wider distribution . the flies infesting the patients probably originated from a natural population near home in the residential area . it s likely that female of megaselia deposited her eggs on the underwear clothing or directly on the urogenital area after being attracted by urine odor . the rational treatment of genitourinary myiasis is to remove the offending larvae . in many cases when the diagnosis is done several substances such as antiseptic and anesthetic solutions have been used in the form of lavage ( francesconi and lupi 2012 ) . it seems , in our case , oral administration of 2 mg mebendazole and washing the urogenital area with solution of xylocain ( 2% ) and iodine ( 1% ) , cured the myiasis . since the urogenitoury tract has inaccessible area , removed of maggots is difficult by conventional instruments , to overcome this problem we used urogenital lavage with xylocaine and iodine solution in our patient . xylocaine causes spastic paralysis in parasites straight muscles and this way the paralyzed larva was easily removed . medical personnel should take care of susceptible patients especially those with nocturnal enuresis , need to bear in mind the possibility of infestation with scuttle flies larvae , be able to make a prompt diagnosis of myiasis and implement relevant intervention to prevent tissue infestation . prevention of this condition is important and involves use of insect repellents and insecticides for control of fly population , adequate protective clothing , good skin and wound hygiene to keep flies from reaching the skin , covering open wounds , change dressing daily , hang clothes to dry in bright sunlight and/or iron them and improve hygiene and sanitation . because the size of gravid flies is very small besides , so much attention is paid to large muscoid flies that may cause myiasis , it is likely that the small phorids are unnoticed or ignored , which may lead to an increase in the occurrence of phorid myiasis . we need to emphasize that this myiasis rarely occurs in healthy individuals and neglected personal hygiene is the single most important factor in human infestation . nowadays the global warming and commerce globalization change the geographical map of scuttle flies and observation of the scarce case of urogenital myiasis in iran , represents their wide distribution . since m. scalaris can possess considerable larval habitat , its creation of various clinical forms of myiasis is imminent . therefore , we hope that our reported case will stimulate researches to present similar findings and we expect several aspects of this species will be studied in future . in addition medical personell should take special attention to susceptible cases and be able to diagnose myiasis as well as implicate relevant interventions to prevent new infections .

myiasis is the infestation of organs and tissues of human and animals with fly larvae . this article reports an 18 year - old man with urogenital myiasis , the passing of live megaselia scalaris larvae in the urine , from zanjan city , northwest of iran . we discourse the importance of diagnosis and management of urogenital myiasis in medicine .

Introduction Case presentation Discussion Conclusion

although malaria is preventable and curable , it still causes high morbidity and mortality ( 1 ) . according to the recent 2013 who report , globally an estimated of 3.4 billon people are at risk of malaria . in this report , who estimated 207 million malaria cases and 627,000 deaths occurred globally in 2012 ( 2 ) the majority of the global burden of human malaria is caused by plasmodium falciparum and p. vivax ( 3 ) . p. falciparum is the most deadly plasmodium species responsible for about 90% of malaria deaths , mainly in africa ( 4 ) and p. vivax is the most cause of malaria infection in the world ( 1 ) . p. vivax is accountable for 2540% of the annual bouts of malaria worldwide ( 4 ) . in iran , 2,714,648 individuals ( 4% of the total population ) mainly living in southern provinces namely sistan and baluchistan , kerman and hormozgan are at risk of malaria ( 5 ) . p. vivax is the most prevalent species reported among the malaria patients in iran annually ( 6 ) . however , a considerable decrease of malaria cases has been reported within the past few years in iran . since malaria elimination program has commence from a few years ago in the country ( 7 ) , for steady continuation of the program rapid and accurate diagnosis of malaria parasites play an important role in opportune case finding and treatment which result in on time control and elimination of the infection . conventional microscopic examination of giemsa stained thick and thin blood smears has been accepted as golden standard method for malaria diagnosis up to now . although malaria microscopy contains some advantages including cost , availability and relative sensitivity ( 810 ) , it bears some disadvantages such as time consuming and labor intensity ( 9 ) . the who has recently reiterated the urgent need for simple and cost - effective diagnostic tests for malaria to overcome the deficiencies of light microscopy and clinical diagnosis ( 10 , 11 ) . based on who advise rapid diagnostic tests can be replaced with microscopic method in remote and isolated areas particularly when trained and skilled personnel is not available ( 1214 ) . utilizing parasite lactate dehydrogenase ( pldh ) in rdts has shown better sensitivity for diagnosing low level of parasitemia in comparison with other malaria proteins . moreover , the amount of pldh indicates to metabolically presence of p. vivax due to short stability of pldh in the body ( 13 ) . pldh plays role of a coenzyme due to involving the oxidation of lactate to pyruvate with nicotinamide adenine dinucleotide ( nad ) ( 15 ) . inhibition of the malarial ldh enzyme prevents the production of atp and results to death of the plasmodium parasites ( 13 ) ; it becomes an attractive drug target candidate ( 16 ) . the genetic diversity of pldh might influence its drug target candidacy and the sensitivity of rdt kits . as far as we know , until now the genetic variation of pldh gene in p. vivax and p. falciparum infections were not reported in iran . this study aimed to detect the polymorphism of pldh gene from iranian strains of p. vivax and p. falciparum . obviously , understanding such polymorphism is important for designing or improving rdt kits . it can also give information about the molecular details of p. falciparum ldh ( pfldh ) and p. vivax ldh ( pvldh ) genes for designing a new drug . totally 43 whole blood samples were collected from p. vivax and p. falciparum infected patients in sistan and baluchestan province located in southeast of iran from 2012 to 2013 . sistan and baluchestan province is bordered with afghanistan and pakistan to the east and oman sea in south . thirty - three samples for p. vivax and 10 samples for p. falciparum were confirmed positive by light microscopic examination of giemsa stained thick and thin blood smears . one ml of blood was collected into tubes containing edta anticoagulant , placed immediately at 20 c for further analysis . dna was extracted from 200 l of whole blood samples of 33 p. vivax and 10 p. falciparum malaria infected patients using , accu - prep kit , genomic dna extraction kit ( bioneer , seoul , korea ) based on the manufacturer instructions . nucleotide sequences corresponding to pvldh and pfldh genes were amplified using the following sets of primers using conventional pcr . pvldh gene amplification was conducted using : forward : 5-atgacgccgaaacccaaaat-3 and reverse : 5-acctttaaatgagcgccttcat-3 , on the other hand pfldh gene was also amplified by f : 5-agatggcaccaaaagcaaaaat-3 and r : 5-acctttaagctaatgccttcat-3. pvldh primers were designed based on p. vivax sal-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) strains from genbank whereas pfldh primers designed based on the reference sequence p. falciparum 3d7 ( xm_001349953.1 ) strain in genbank . dna was extracted from whole blood of a healthy person living in non - endemic area as a negative control for using in amplification process . pcr reaction was performed in 25l reaction volumes containing 1lof each forward and reverse primers ( 10 pmol ) , 10 l of ready to use master mix ( ampliqon , denmark ) contains ( tris - hcl ph 8.5 , 1.5 mm mgcl2 , dntps and taqdna polymerase ) , 3 l of genomic dna samples and 10 l distilled water . pcr cycle parameters for pvldh gene amplification were as follows : 5minutes initial denaturation at 95 c followed by 30 cycles with 30 s at 95 c , 30 at 56 c , 1 at 72 c and final extension at 72 c for 5 min . all the pcr parameters were the same for pfldh gene amplification except the annealing temperature was 58 c . the fragment sizes of pcr products were determined using 1 kb dna ladder marker ( solis biodyne , estonia ) . twenty - two sequences including 15 p. vivax and 7 p. falciparum were analyzed to investigate polymorphism in pvldh and pfldh genes respectively . these genes were sequenced by applied biosystems 3730/3730xl dna analyzers , ( bioneer , seoul , korea ) using sanger method . nucleotide sequences of pvldh and pfldh were aligned and compared using clustal w2 software ( embl - ebi , http://www.ebi.ac.uk/tools/msa/clustalw2/ ) . pvldh gene sequences were compared with genbank sequences of p. vivax belem ( dq060151.1 ) and p. vivax sai-1 ( xm_001615570.1 ) . on the other hand , pfldh gene sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) . moreover , amino acid sequences related to each samples of p. vivax and p. faliparum were derived using expasy translate tool ( http://web.expasy.org/tran-slate/ ) . pvldh amino acid sequences were compared with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) whereas pfldh amino acid sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains registered in genbank . finally phylogenic tree was prepared to illustrate the distance among sequences of isolates using average distance ( ad ) method in clustal w2 jalview software ( http://www.eb-i.ac.uk/ ) . thelactate dehydrogenase gene from iranian plasmodium strains were submitted with the accession numbers of ( km226649-km226654 and km226656-km226664 ) for p. vivax , and ( km226665-km226671 ) for p. falciparum in genbank ( blast ) . dna was extracted from 200 l of whole blood samples of 33 p. vivax and 10 p. falciparum malaria infected patients using , accu - prep kit , genomic dna extraction kit ( bioneer , seoul , korea ) based on the manufacturer instructions . nucleotide sequences corresponding to pvldh and pfldh genes were amplified using the following sets of primers using conventional pcr . pvldh gene amplification was conducted using : forward : 5-atgacgccgaaacccaaaat-3 and reverse : 5-acctttaaatgagcgccttcat-3 , on the other hand pfldh gene was also amplified by f : 5-agatggcaccaaaagcaaaaat-3 and r : 5-acctttaagctaatgccttcat-3. pvldh primers were designed based on p. vivax sal-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) strains from genbank whereas pfldh primers designed based on the reference sequence p. falciparum 3d7 ( xm_001349953.1 ) strain in genbank . dna was extracted from whole blood of a healthy person living in non - endemic area as a negative control for using in amplification process . pcr reaction was performed in 25l reaction volumes containing 1lof each forward and reverse primers ( 10 pmol ) , 10 l of ready to use master mix ( ampliqon , denmark ) contains ( tris - hcl ph 8.5 , 1.5 mm mgcl2 , dntps and taqdna polymerase ) , 3 l of genomic dna samples and 10 l distilled water . pcr cycle parameters for pvldh gene amplification were as follows : 5minutes initial denaturation at 95 c followed by 30 cycles with 30 s at 95 c , 30 at 56 c , 1 at 72 c and final extension at 72 c for 5 min . all the pcr parameters were the same for pfldh gene amplification except the annealing temperature was 58 c . the fragment sizes of pcr products were determined using 1 kb dna ladder marker ( solis biodyne , estonia ) . twenty - two sequences including 15 p. vivax and 7 p. falciparum were analyzed to investigate polymorphism in pvldh and pfldh genes respectively . these genes were sequenced by applied biosystems 3730/3730xl dna analyzers , ( bioneer , seoul , korea ) using sanger method . nucleotide sequences of pvldh and pfldh were aligned and compared using clustal w2 software ( embl - ebi , http://www.ebi.ac.uk/tools/msa/clustalw2/ ) . pvldh gene sequences were compared with genbank sequences of p. vivax belem ( dq060151.1 ) and p. vivax sai-1 ( xm_001615570.1 ) . on the other hand , pfldh gene sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) . moreover , amino acid sequences related to each samples of p. vivax and p. faliparum were derived using expasy translate tool ( http://web.expasy.org/tran-slate/ ) . pvldh amino acid sequences were compared with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) whereas pfldh amino acid sequences were compared with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains registered in genbank . finally phylogenic tree was prepared to illustrate the distance among sequences of isolates using average distance ( ad ) method in clustal w2 jalview software ( http://www.eb-i.ac.uk/ ) . thelactate dehydrogenase gene from iranian plasmodium strains were submitted with the accession numbers of ( km226649-km226654 and km226656-km226664 ) for p. vivax , and ( km226665-km226671 ) for p. falciparum in genbank ( blast ) . a 955 bp band was observed in gel electrophoresis of pcr products of pfldh and pvldh amplified genes ( fig . 1 and fig . the amplified pvldh gene was yielded approximately 955 base pairs , coding for 316 amino acids . fifteen of the amplified genes were sequenced to analyze the genetic variation of pvldh gene using clustal w2 software . after comparing the sequences with the chromatogram with p. vivax sal-1 reference sequence , two single nucleotide substitution were detected at 666 , 899 positions from g to c and c to t respectively ( fig . thirteen of the 15 isolates displayed 100% nucleotide sequence homology with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) ( table 1 & fig . was brought an amino acid change from ( t , neutral polar amino acid to me , non - polar amino acid ) whereas the nucleotide substitution at 666 positions from g to c did not result any change in amino acid ( fig . dna was extracted from 10 p. falciparum confirmed whole blood samples and pfldh gene was amplified using specific primers . dna sequences of pfldh gene displayed three nucleotide substitutions at 36 , 814 and 891positions from a to g , g to a and g to a respectively ( fig . five of the 7 isolates had 100% nucleotide homology with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains submitted in genbank ( table 3 ) . : accession number , if : iran - baluchistan falciparum only one of the nucleotide changes at 814 positions from g to a was brought an amino acid change from aspartic acid ( d , acidic polar amino acid to n , neutral polar amino acid ) ( fig . 7 ) . the rest six isolates showed 100% amino acid homology withpfmzr-1 and pf3d7 strains from genbank ( table 4 ) . : accession number , if : iran - baluchistan falciparum the nucleotide homology between pvldh and pfldh in iranian isolates of p. vivax and p. falciparum was 75.876% . all p. vivax ldh nucleotide sequences had 75.79% homology with six of p. falciparum isolates . the amino acids sequence homology between pvldh and pfldh iranian isolates were 90.4% exception of one isolate which had 90.76% homology . generally , the amino acids sequence homology between pvldh and pfldh iranian isolates were more than 90% . the amplified pvldh gene was yielded approximately 955 base pairs , coding for 316 amino acids . fifteen of the amplified genes were sequenced to analyze the genetic variation of pvldh gene using clustal w2 software . after comparing the sequences with the chromatogram with p. vivax sal-1 reference sequence , two single nucleotide substitution were detected at 666 , 899 positions from g to c and c to t respectively ( fig . thirteen of the 15 isolates displayed 100% nucleotide sequence homology with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) ( table 1 & fig . was brought an amino acid change from ( t , neutral polar amino acid to me , non - polar amino acid ) whereas the nucleotide substitution at 666 positions from g to c did not result any change in amino acid ( fig . 5 ) . dna was extracted from 10 p. falciparum confirmed whole blood samples and pfldh gene was amplified using specific primers . dna sequences of pfldh gene displayed three nucleotide substitutions at 36 , 814 and 891positions from a to g , g to a and g to a respectively ( fig . five of the 7 isolates had 100% nucleotide homology with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains submitted in genbank ( table 3 ) . : accession number , if : iran - baluchistan falciparum only one of the nucleotide changes at 814 positions from g to a was brought an amino acid change from aspartic acid ( d , acidic polar amino acid to n , neutral polar amino acid ) ( fig . 7 ) . the rest six isolates showed 100% amino acid homology withpfmzr-1 and pf3d7 strains from genbank ( table 4 ) . the nucleotide homology between pvldh and pfldh in iranian isolates of p. vivax and p. falciparum was 75.876% . all p. vivax ldh nucleotide sequences had 75.79% homology with six of p. falciparum isolates . the amino acids sequence homology between pvldh and pfldh iranian isolates were 90.4% exception of one isolate which had 90.76% homology . generally , the amino acids sequence homology between pvldh and pfldh iranian isolates were more than 90% . pldh antigen is assumed to be a specific marker for the presence of viable plasmodium in blood , and is used for screening in malaria - endemic countries ( 17 ) . inhibition of the malarial ldh enzyme prevents the producing atp and causes death of the plasmodium parasites,(13 ) so it becomes an attractive drug target candidate ( 16 ) . therefore , the protein obtained from this gene can be used in any diagnostic test ( 18 ) . diversity in the pldh gene may influence specificity and sensitivity of rdts in any malaria endemic area . investigation of polymorphism in p. vivax and p. falciparum lactate dehydrogenase gene can lead to produce more specific and sensitive rdts kit . the nucleotide homology among 15 pvldh sequences of p. vivax was 100% with the exception of two isolates displayed 99.9% homology ( table 1 , 2 & fig . 100% pvldh nucleotide sequence homology was reported among chinese p. vivax , sal-1 and belem ( 19 ) . another study done in china reported 99.89% nucleotide identity of chinese isolates with belem strain ( 20 ) . this point out iranian pvldh nucleotide sequences had more homology with belem strain than chinese isolates . reported pvldh genes from chinese p. vivax anhui isolates had more than 99% sequence homology compared with strains in gene bank ( 21 ) . this outcome strongly agreed with findings from our study , which also showed more than 99% homology with all compared p. vivax strains registered in genbank . in the present study , pvldh gene sequences showed two nucleotide substitutions with one resulted an amino acid change from t , neutral polar amino acid to i , non - polar amino acids . antigen variability is unlikely to explain variability in implementation of rdts detecting pldh in p. falciparum , p. vivax cases ( 22 ) . in contrast to our finding , shin et al . in korea reported one snp which did not bring any change in amino acid ( 23 ) jianget al . in china also reported a single nucleotide difference at the position 666 between pvldh gene and p. vivax belem ( dq060151)(24 ) . pvldh genes from iranian isolates of p. vivax were displayed more nucleotide changes than korean and chinese isolates . in earlier chinese studies from jianghuai region and anhui isolates of p. vivax , there were no nucleotide changes among isolates ( 21 , 25 ) . compared to these reports the nucleotide changes among pvldh from iranian isolates of p. vivax was higher than both jianghuai region and anhui isolates of p. vivax . talman et al . reported four different type of dna sequence of p. vivax from 10 isolates ; the mutations were synonymous ( 22 ) . in our study , less number of nucleotide changes was seen and the mutations were not synonymous . fourteen isolates had the same amino acid sequences with p. vivax sai-1 ( xm_001615570.1 ) and p. vivax belem ( dq060151.1 ) . this finding was agreed with a study conducted in china , which reported 100% pvldh gene homology among chinese isolates , p. vivax sal - i and belem ( 19 ) . studies done in korea and china from korean and hainan isolates respectively , also reported 100% amino acid homology with p. vivax belem ( dq060151.1 ) ( 20 , 23 ) . this indicated rdts produced from korean and chinese isolates can be used in iran . on the other hand , pfldh homology among iranian strains of p. falciparum was 100% with the exception of two isolates . in contrast to our finding , talman et al . reported no variability among all sequences p. falciparum ( n = 49 ) in worldwide isolates of plasmodium spp ( 22 ) . five of the seven isolates had 100% nucleotide identity with p. falciparum 3d7 ( xm_001349953.1 ) and p. falciparum mzr-1 ( jn547219.1 ) strains registered in genbank . iranian pfldh genes and reference sequence ( pf3d7 ) had high homology about 99.9%100% ( table 1 , 2 & fig . 8) . this indicated pfldh gene is relatively conserved and can be a good target for antimalarial drug and producing rdt . in our study , these amino acid sequences also had 100% homology with p. falciparum mzr-1 and p. falciparum 3d7 strains from gen - bank . our finding was supported by the study conducted in indonesia , which reported 100% amino acid sequences between indonesian pfldh and pf 3d7 reference sequence ( 26 ) . in this study however , only the substitution at 891 positions from g to a was brought an amino acid change from aspartic acid to asparagine ( d , acidic polar amino acid to n , neutral polar amino acid ) . in madagascar , two snps at 73 and 814 positions among the 137 dna sequences of p. falciparum isolates were displayed . the nucleotide change in 10 isolates at 814 position resulted in an amino acid change ( d , acidic polar amino acid to n , neutral polar amino acid ) . in addition , another amino acid change ( at codon 25 : q , neutral polar amino acid to a , basic polar amino acid ) was seen due to the snp at 73 position ( 18 ) . the position of nucleotide change ( 814bp ) and the resulted amino acid change ( aspartic acid ( d ) to asparagine ( n ) in one of the isolates in our study was the same with the madagascar study . the nucleotide change at 814 positions in our study might be a single nucleotide polymorphism given the madagascar study snp report at the same position . iranian pfldh demonstrated less number of amino acid changes in comparison with the report that released from madagascar study . the nucleotide sequences homology between iranian isolates of pvldh and pfldh were 75.7976% . in china , jiang et al . akbulut et al . also reported 74.8% homology between pvldh and pfldh ( 27 ) . compared to jiang et al . and akubulut et al . report , the homology of iranian pvldh and pfldh was high . in our study , the amino acid sequences homology among iranian isolates of pvldh and pfldh was 90.4% with the exception of one isolate . reported 89.5% and 90.2% amino acid sequence homology between pvldh and pfldh respectively ( 23 , 28 ) . this indicated the amino acid homology between pvldh and pfldh genes from iranian isolates of p. vivax and p. falciparum were higher than previously reports . generally , in our study the amino acid homology between pvldh and pfldh was more than 90% . pldh gene from iranian p. falciparum and p.vivax isolates displayed 98.8100% homology with 13 nucleotide substitutions . this relatively stability indicated pvldh and pfldh genes can be a good antimalaria target and used for producing rdt kits . this indicated some techniques like drug discovery , vaccine development and other activities , which were applied on p. falciparum , could also be tried for p. vivax . the homology among pldh of p. vivax and p. falciparum should be further investigated with large enough sample size . in general , before using pldh for producing rdt kits the genetic variation of this gene should be investigated since its polymorphism varies with geographical locations .

background : parasite lactate dehydrogenase ( pldh ) is extensively employed as malaria rapid diagnostic tests ( rdts ) . moreover , it is a well - known drug target candidate . however , the genetic diversity of this gene might influence performance of rdt kits and its drug target candidacy . this study aimed to determine polymorphism of pldh gene from iranian isolates of p. vivax and p. falciparum.methods:genomic dna was extracted from whole blood of microscopically confirmed p. vivax and p. falciparum infected patients . pldh gene of p. falciparum and p. vivax was amplified using conventional pcr from 43 symptomatic malaria patients from sistan and baluchistan province , southeast iran from 2012 to 2013.results:sequence analysis of 15 p. vivax ldh showed fourteen had 100% identity with p. vivax sal-1 and belem strains . two nucleotide substitutions were detected with only one resulted in amino acid change . analysis of p. falciparum ldh sequences showed six of the seven sequences had 100% homology with p. falciparum 3d7 and mzr-1 . moreover , pfldh displayed three nucleotide changes that resulted in changing only one amino acid . pvldh and pfldh showed 75%76% nucleotide and 90.4%90.76% amino acid homology.conclusion:pldh gene from iranian p. falciparum and p. vivax isolates displayed 98.8100% homology with 13 nucleotide substitutions . this indicated this gene was relatively conserved . additional studies can be done weather this genetic variation can influence the performance of pldh based rdts or not .

Introduction Materials and Methods Genomic DNA extraction PCR amplification DNA sequencing Results None None None Discussion Conclusion

the fundamental problems with the personality disorders ( pd ) diagnostic system in the previous version of dsm , such as all - or - nothing diagnostic categories , considerable heterogeneity within categories , extensive overlap or comorbidity among categories , indistinct boundaries with normal personality , and incomplete coverage of personality psychopathology , led to the revision of the dsm approach ( 1 , 2 ) . since 2000 , after the revision of dsm - iv , pd researchers largely agree that personality pathology should be represented dimensionally rather than categorically ( 3 ) . thus , many alternative dimensional models of personality have been considered ( 4 - 7 ) , and ongoing research has been performed to delineate the conceptual and empirical structure of personality traits in the pathological range ( 6 , 8) . finally , a multidimensional system was proposed for representing and diagnosis of personality disorder features in dsm-5 . the new approach for the assessment of personality pathology identifies core impairments in levels of personality functioning , pathological personality traits , and prominent personality pathology types ( 9 ) . however , there is wide consensus that severity assessment is essential for any dimensional system of personality psychopathology ( 10 ) . moreover , the dsm-5 personality disorders work group has proposed impairments in personality functioning as the central element of pd ( 11 ) . thus , in the dimensional model of personality disorder any pd ( schizotypal , antisocial , borderline , narcissistic , obsessive - compulsive and avoidant ) is associated with fundamental disturbances of self and interpersonal relations , problems with identity , self - direction , empathy and intimacy . the review of literature showed that levels of personality functioning or self - other approach for diagnosis are informative in determining both the type and the severity of personality pathology ( 12 - 14 ) . the severity of impairment in personality functioning has also been shown to be important in planning treatment and predicting its outcome . despite the good advantages and empirical background of levels of personality functioning , practical studies in clinical settings with specific pds and cross - cultural studies in this field also , researchers need to determine which levels of personality functioning better predict and explain severity than others . the purpose of the current study was firstly , to examine the relationship between dsm-5 levels of personality functioning on an iranian sample with antisocial and borderline personality disorders , and second , to explore which levels of personality functioning in patients with antisocial and borderline personality disorders can better predict severity . of the 300 participants , 252 individuals with antisocial ( n = 122 ) and borderline personality disorders ( n = 130 ) were selected from september to november 2013 . with regard to = 0.05 and based on partial r = 0.095 , the sample size for antisocial personality disorder ( aspd ) was calculated to be 119 . for borderline personality disorder ( bpd ) , and were 0.05 and 80% , respectively , and four variables were tested by multiple regression analysis . based on partial r = 0.1 , the adequate sample size for bpd was 113 . participants were selected from prisoners ( 48.8% ) , outpatients ( 16.5% ) and inpatients ( 7.5% ) . they were recruited from tehran prisons , and clinical psychology and psychiatry centers of razi psychiatric hospital and taleghani general hospital , tehran , iran . inclusion criteria were diagnosis of antisocial and borderline personality disorders ( for personality disorders ) , being at least 18 years old , having at least secondary education , and the exclusion criteria were presence of a psychotic disorder , presence of severe mood disorder , presence of mental retardation , and presence of physical condition that impairs the person 's mental state . participants were 90.5% males and 9.5% females , aged 18 to 60 years ( sd = 1.37 ) , with guidance school degree education level or higher . disorders of axis i ; 106 patients ( 42.06% ) had no impairment , 78 patients ( 30.97% ) had a history of substance - related disorders , 35 patients ( 13.88% ) had mood disorder , 15 patients ( 5.96% ) had anxiety disorders , 8 patients ( 3.17% ) had had a history of psychotic spectrum disorder and 10 patients ( 3.96% ) had other disorders . data gathering measurements included psychological reports , scid - ii - pq , scid - ii , and dsm-5 levels of personality functioning checklist . the scid and its versions are considered to be the most comprehensive of the structured diagnostic interviews , which are currently available . in fact , in 1987 they were new and wide range of instruments , by spitzer , gibbon , williams and built in compliance with the criteria of the dsm - iv ( 16 ) . this instrument has been established as the gold standard for reliable assessment of psychiatric disorders . inter - rater reliability for scid - i was above 70 for mood , anxiety , schizophrenic disorders and alcohol abuse ; it was somewhat lower for a few other disorders ( 17 ) , while for scid - ii it was reported between 0.48 and 0.98 for the categorical diagnoses ( cohen s ) and 0.90 to 0.98 for the dimensional judgments ( intra - class correlation coefficient ) ( 18 ) . cronbach s was found between 0.71 and 0.94 for the scid - ii personality disorder scales ( 18 ) . due to high accuracy of the diagnostic criteria and extraordinary compliance with dsm - iv criteria , translated to and adapted with different languages . in iran scid - ii and scid - ii - pq the duration of the scid - i is 30 to 90 minutes , the duration of the scid - ii is 30 to 60 minutes . severity was determined in this study by the scores obtained from scid - ii . levels of personality functioning refer to the core capacities of personality - related self and interpersonal functioning and determining the severity of any impairment in these areas ( 12 ) . the dsm-5 levels of personality functioning include identity , self - direction , empathy and intimacy . the levels of personality functioning scale use each of these elements to differentiate five levels of impairment on a continuum of severity ranging from no impairment , i.e. healthy functioning ( level = 0 ) , to extreme impairment ( level = 4 ) ( 11 ) . the more impairment in these areas , the more likely that person has pd . in preliminary analysis of a sample of 424 psychiatric patients , morey et al . ( 14 ) found that a score of greater than three ( out of five ) on a short five - item scale , had a sensitivity of 79% and a specificity of 54% for a semi - structured interview diagnosis of pd . ( 20 ) have translated dsm-5 levels of personality functioning scale to farsi , and developed a semi - structured interview . inter - rater reliability for semi - structured interview of dsm-5 levels of personality functioning items was above 0.80 . the dsm-5 levels of personality functioning scale domains correlation with dsm - iv was between 0.30 and 0.69 . the duration of the semi - structured interview for dsm-5 levels of personality functioning is 15 to 25 minutes . in the implementation process , the researchers included three post - graduates of clinical psychology . to avoid probable bias outcome from these people , they were not informed of the exact goal of the research in detail and they were told that the research goal was to study personality disorders . they were entirely uninformed of the concerned disorder types . to control the probable bias , the research associates began to collect data periodically per step while they were blinded to the outcome of the previous or next step . the colleagues were trained to use these instruments . after training under the supervision of the researcher , some people were actually interviewed , and the interviewers bug was fixed . as mentioned above , there were two groups of patients ( patients with personality disorders and normal subjects ) . prior to the research onset , the participants were fully informed and provided a written consent . to avoid fatigue and to increase motivation in subjects , study for each subject was conducted during two days . in the days following the completion of the demographic questionnaire furthermore , cases , which had both symptoms of antisocial and borderline personality disorders , were examined by the structured clinical interview for personality disorders ( scid - ii ) . after the definitive diagnosis of antisocial personality disorder and borderline , they were invited to attend the next stage of the interview process based on dsm-5 levels of personality functioning . ethics approval was obtained from the university of social welfare and rehabilitation sciences research ethics committee , and the registered ethical code was 92/801/3110/2/a . we computed bivariate correlations between the dsm-5 levels of personality functioning and aspd , and bpd symptoms . to examine which levels of personality functioning in patients with antisocial and borderline personality disorders can better predicted severity , stepwise regression analysis was used , in which scid - ii results ( aspd and bpd severity ) were the dependent variables and levels of personality functioning were the predictors . of the 300 participants , 252 individuals with antisocial ( n = 122 ) and borderline personality disorders ( n = 130 ) were selected from september to november 2013 . with regard to = 0.05 and based on partial r = 0.095 , the sample size for antisocial personality disorder ( aspd ) was calculated to be 119 . for borderline personality disorder ( bpd ) , and were 0.05 and 80% , respectively , and four variables were tested by multiple regression analysis . based on partial r = 0.1 , the adequate sample size for bpd was 113 . participants were selected from prisoners ( 48.8% ) , outpatients ( 16.5% ) and inpatients ( 7.5% ) . they were recruited from tehran prisons , and clinical psychology and psychiatry centers of razi psychiatric hospital and taleghani general hospital , tehran , iran . inclusion criteria were diagnosis of antisocial and borderline personality disorders ( for personality disorders ) , being at least 18 years old , having at least secondary education , and the exclusion criteria were presence of a psychotic disorder , presence of severe mood disorder , presence of mental retardation , and presence of physical condition that impairs the person 's mental state . participants were 90.5% males and 9.5% females , aged 18 to 60 years ( sd = 1.37 ) , with guidance school degree education level or higher . disorders of axis i ; 106 patients ( 42.06% ) had no impairment , 78 patients ( 30.97% ) had a history of substance - related disorders , 35 patients ( 13.88% ) had mood disorder , 15 patients ( 5.96% ) had anxiety disorders , 8 patients ( 3.17% ) had had a history of psychotic spectrum disorder and 10 patients ( 3.96% ) had other disorders . data gathering measurements included psychological reports , scid - ii - pq , scid - ii , and dsm-5 levels of personality functioning checklist . the scid and its versions are considered to be the most comprehensive of the structured diagnostic interviews , which are currently available . in fact , in 1987 they were new and wide range of instruments , by spitzer , gibbon , williams and built in compliance with the criteria of the dsm - iv ( 16 ) . this instrument has been established as the gold standard for reliable assessment of psychiatric disorders . inter - rater reliability for scid - i was above 70 for mood , anxiety , schizophrenic disorders and alcohol abuse ; it was somewhat lower for a few other disorders ( 17 ) , while for scid - ii it was reported between 0.48 and 0.98 for the categorical diagnoses ( cohen s ) and 0.90 to 0.98 for the dimensional judgments ( intra - class correlation coefficient ) ( 18 ) . cronbach s was found between 0.71 and 0.94 for the scid - ii personality disorder scales ( 18 ) . due to high accuracy of the diagnostic criteria and extraordinary compliance with dsm - iv criteria , translated to and adapted with different languages . in iran scid - ii and scid - ii - pq the duration of the scid - i is 30 to 90 minutes , the duration of the scid - ii is 30 to 60 minutes . severity was determined in this study by the scores obtained from scid - ii . levels of personality functioning refer to the core capacities of personality - related self and interpersonal functioning and determining the severity of any impairment in these areas ( 12 ) . the dsm-5 levels of personality functioning include identity , self - direction , empathy and intimacy . the levels of personality functioning scale use each of these elements to differentiate five levels of impairment on a continuum of severity ranging from no impairment , i.e. healthy functioning ( level = 0 ) , to extreme impairment ( level = 4 ) ( 11 ) . the more impairment in these areas , the more likely that person has pd . in preliminary analysis of a sample of 424 psychiatric patients , morey et al . ( 14 ) found that a score of greater than three ( out of five ) on a short five - item scale , had a sensitivity of 79% and a specificity of 54% for a semi - structured interview diagnosis of pd . ( 20 ) have translated dsm-5 levels of personality functioning scale to farsi , and developed a semi - structured interview . inter - rater reliability for semi - structured interview of dsm-5 levels of personality functioning items was above 0.80 . the dsm-5 levels of personality functioning scale domains correlation with dsm - iv was between 0.30 and 0.69 . the duration of the semi - structured interview for dsm-5 levels of personality functioning is 15 to 25 minutes . the scid and its versions are considered to be the most comprehensive of the structured diagnostic interviews , which are currently available . in fact , in 1987 they were new and wide range of instruments , by spitzer , gibbon , williams and built in compliance with the criteria of the dsm - iv ( 16 ) . this instrument has been established as the gold standard for reliable assessment of psychiatric disorders . inter - rater reliability for scid - i was above 70 for mood , anxiety , schizophrenic disorders and alcohol abuse ; it was somewhat lower for a few other disorders ( 17 ) , while for scid - ii it was reported between 0.48 and 0.98 for the categorical diagnoses ( cohen s ) and 0.90 to 0.98 for the dimensional judgments ( intra - class correlation coefficient ) ( 18 ) . cronbach s was found between 0.71 and 0.94 for the scid - ii personality disorder scales ( 18 ) . due to high accuracy of the diagnostic criteria and extraordinary compliance with dsm - iv criteria , translated to and adapted with different languages . in iran scid - ii and scid - ii - pq the duration of the scid - i is 30 to 90 minutes , the duration of the scid - ii is 30 to 60 minutes . levels of personality functioning refer to the core capacities of personality - related self and interpersonal functioning and determining the severity of any impairment in these areas ( 12 ) . the dsm-5 levels of personality functioning include identity , self - direction , empathy and intimacy . the levels of personality functioning scale use each of these elements to differentiate five levels of impairment on a continuum of severity ranging from no impairment , i.e. healthy functioning ( level = 0 ) , to extreme impairment ( level = 4 ) ( 11 ) . the more impairment in these areas , the more likely that person has pd . in preliminary analysis of a sample of 424 psychiatric patients , morey et al . ( 14 ) found that a score of greater than three ( out of five ) on a short five - item scale , had a sensitivity of 79% and a specificity of 54% for a semi - structured interview diagnosis of pd . ( 20 ) have translated dsm-5 levels of personality functioning scale to farsi , and developed a semi - structured interview . inter - rater reliability for semi - structured interview of dsm-5 levels of personality functioning items was above 0.80 . the dsm-5 levels of personality functioning scale domains correlation with dsm - iv was between 0.30 and 0.69 . the duration of the semi - structured interview for dsm-5 levels of personality functioning is 15 to 25 minutes . in the implementation process , the researchers included three post - graduates of clinical psychology . to avoid probable bias outcome from these people , they were not informed of the exact goal of the research in detail and they were told that the research goal was to study personality disorders . they were entirely uninformed of the concerned disorder types . to control the probable bias , the research associates began to collect data periodically per step while they were blinded to the outcome of the previous or next step . the colleagues were trained to use these instruments . after training under the supervision of the researcher , some people were actually interviewed , and the interviewers bug was fixed . as mentioned above , there were two groups of patients ( patients with personality disorders and normal subjects ) . prior to the research onset , the participants were fully informed and provided a written consent . to avoid fatigue and to increase motivation in subjects , study for each subject was conducted during two days . in the days following the completion of the demographic questionnaire furthermore , cases , which had both symptoms of antisocial and borderline personality disorders , were examined by the structured clinical interview for personality disorders ( scid - ii ) . after the definitive diagnosis of antisocial personality disorder and borderline , they were invited to attend the next stage of the interview process based on dsm-5 levels of personality functioning . ethics approval was obtained from the university of social welfare and rehabilitation sciences research ethics committee , and the registered ethical code was 92/801/3110/2/a . we computed bivariate correlations between the dsm-5 levels of personality functioning and aspd , and bpd symptoms . to examine which levels of personality functioning in patients with antisocial and borderline personality disorders can better predicted severity , stepwise regression analysis was used , in which scid - ii results ( aspd and bpd severity ) were the dependent variables and levels of personality functioning were the predictors . data are presented as mean standard deviation ( sd ) or no ( % ) . the mean and standard deviation scores of the levels of personality functioning in antisocial and borderline personality disorder groups are showed in table 2 . bivariate correlation between dsm-5 levels of personality functioning ( identity , self - directedness , intimacy , empathy ) and antisocial and borderline personality symptoms are presented in table 3 . as seen in table 3 , positive and significant correlations were found between antisocial personality symptoms and dsm-5 levels of personality functioning . the total levels are highly correlated with aspd symptoms ( p value < 0.001 ) . in borderline personality disorder , except identity and empathy , other variables had significant correlations with bpd symptoms ( p value < 0.001 and < 0.05 ) . to examine the relationship between dsm-5 levels of personality functioning and aspd and bpd severity , a stepwise regression analysis was conducted , where aspd and bpd severity were the dependent variables and levels of personality functioning ( identity , self - directedness , intimacy , empathy ) were the independent variables . the results of regression analysis for levels of personality functioning and aspd severity are presented in table 4 . these results indicate that approximately 21.7% , 31.4% , and 33% of the variance of the aspd severity could be accounted by identity , intimacy and self - directedness , respectively . the data showed that identity , intimacy , and self - directedness significantly predicted antisocial personality disorder severity . the results of regression analysis for levels of personality functioning and bpd severity are displayed in table 5 . the results showed that intimacy and empathy were good predictors of borderline personality disorder severity . this means that approximately 16.5% and 25.1% of the variance of the bpd severity could be accounted for by these variables . the first aim of this study was to examine the dsm-5 levels of personality functioning on an iranian sample with antisocial and borderline personality disorders . the study findings revealed that all levels of personality functioning had significant positive correlations with aspd . this means that dsm-5 levels of personality functioning had a good relationship with antisocial personality disorder symptoms of an iranian sample . also , the findings indicate that except identity and intimacy , other variables ( self - directedness and empathy ) had a significant correlation with borderline personality symptoms . it seems that the study population could be one of the reasons for these findings . previous studies indicated that severity is the most important single predictor of concurrent and prospective dysfunction in assessing personality psychopathology ( 11 , 21 , 22 ) . therefore , the second aim of this study was to explore which levels of personality functioning in patients with antisocial and borderline personality disorders can better predict severity . the stepwise regression analysis results showed that identity , intimacy , and self - directedness are positively and significantly correlated with antisocial personality disorder severity , indicating that those with higher scores on these variables tend to have higher symptoms of aspd . the analysis showed that intimacy and empathy had a significant association with borderline personality disorder severity . it means that interpersonal level of personality functioning ( intimacy and empathy ) could explain the severity on patients with bpd in this study . in addition , the results showed that the dsm-5 levels of personality functioning are an essential part of antisocial and borderline personality disorders . also , the results revealed that levels of personality functioning were the same in other cultures and the iranian sample . the present study is one of the first in which researchers examined the dsm-5 levels of personality functioning relationship with severity , and is the first study on iranian patients with personality disorders based on the dsm-5 approach . overall , our findings showed that the levels of personality functioning are a significant predictor of antisocial and borderline personality disorders severity . nevertheless , there are a number of important limitations in this work and future research is required to overcome such limitations . firstly , the results are based on a relatively small number of cases and so caution should be taken in interpreting the data . secondly , data was gathered by a semi - structured interview designed to assess a dimensional model of personality disorders , and future work should focus on other relevant instruments . the third limitation of the current study was the nature of the sample , which was drawn from antisocial and borderline personality disorders . thus , future research should examine the role of axis i disorders in personality disorders severity . fifth , most participants in this study were males . therefore , other research is needed to investigate dsm-5 levels of personality functioning and severity in females . seventh and finally , our work focused on the assessment of dsm-5 levels of personality functioning and severity in adults .

background : fundamental problems with personality disorders ( pd ) diagnostic system in the previous version of dsm , led to the revision of dsm . therefore , a multidimensional system has been proposed for diagnosis of personality disorder features in dsm-5 . in the dimensional approach of dsm-5 , personality disorders diagnosis is based on levels of personality functioning ( criteria a ) and personality trait domains ( criteria b).objectives : the purpose of this study was firstly , to examine the dsm-5 levels of personality functioning in antisocial and borderline personality disorders , and second , to explore which levels of personality functioning in patients with antisocial and borderline personality disorders can better predicted severity than others.patients and methods : this study had a cross sectional design . the participants consisted of 252 individuals with antisocial ( n = 122 ) and borderline personality disorders ( n = 130 ) . they were recruited from tehran prisoners , and clinical psychology and psychiatry centers of razi and taleghani hospitals , tehran , iran . the sample was selected based on judgmental sampling . the scid - ii - pq , scid - ii and dsm-5 levels of personality functioning were used to diagnose and assess personality disorders . the data were analyzed by correlation and multiple regression analysis . all statistical analyses were performed using the spss 16 software.results:firstly , it was found that dsm-5 levels of personality functioning have a strong correlation with antisocial and borderline personality symptoms , specially intimacy and self - directedness ( p < 0.001 ) . secondly , the findings showed that identity , intimacy and self - directedness significantly predicted antisocial personality disorder severity ( p < 0.0001 ) . the results showed that intimacy and empathy were good predictors of borderline personality disorder severity , as well ( p < 0.0001).conclusions : overall , our findings showed that levels of personality functioning are a significant predictor of personality disorders severity . the results partially confirm existing studies .

1. Background 2. Objectives 3. Patients and Methods 3.1. Study Population and Sampling 3.2. Materials 3.2.1. SCID 3.2.2. The DSM-5 levels of personality functioning scale 3.3. Procedure 3.4. Statistical Analysis 4. Results 5. Discussion

homeostasis of the brain is maintained owing to its rigidly controlled communication with the peripheral tissues . entry of metabolites from the periphery to the brain is controlled by the blood brain barrier ( bbb ) . the major structural constituents of the bbb are the cerebral microvascular endothelial cells , and their barrier function relies on so- called tight - junctions ( tjs ) , consisting of transmembrane components : junctional adhesion molecule ( jam)-1 , occludin , and the claudins and intracellular proteins : zo-1 , zo-2 , and zo-3 , which link transmembrane proteins to the actin filaments of cytoskeleton and in this way improve stability and functioning of the tj . adherent junctions which are located in the basal region below the tjs , also contribute to the barrier function . cadherins stabilize adhesion between neighboring endothelial cells , while intracellularly , catenins link cadherins to the cytoskeleton ( fig . 1 ) . physically , the tjs limit free paracellular diffusion of low molecular weight compounds and make the transcellular transport of larger molecules dependent on specific transport systems , which can be grouped accordingly to the class of molecules transported ( hawkins and davis 2005 ; carvey et al . these transport systems are located in endothelial cells , and are modulated both intrinsically and by other cells of the neurovascular unit : astrocytes and pericytes ( simard and nedergaard 2004 ) . fine - tuning of the transport involves its polarization by differential location of the transport systems in the luminal versus abluminal membranes , which holds in particular for the different amino acid transport systems ( hawkins et al . two ultimate and complementary goals are reached : ( i ) control of the inflow and outflow of metabolic precursors and products , ( ii ) prevention of entry to the brain of undesired compounds.fig . 1composition of the tight junction and adherence junction which collectively restrict the paracellular passage of solutes across the bbb composition of the tight junction and adherence junction which collectively restrict the paracellular passage of solutes across the bbb the sections below describes the evolution of views on the role of bbb changes in the pathogenesis of diseases associated with increased exposure of the brain to blood - derived ammonia . studies on bbb penetration by different compounds in he models : a historical account section gives a historical perspective on the experimental studies on ammonia- and he - induced changes in bbb penetration of different compounds , without emphasis on the underlying mechanisms . transcellular passage of different molecules across the endothelium : roles of active transport section of the review will elaborate on the relatively well explored subject of modulation of transcellular passage , which represents active transport of medium- to large - molecules , and channel- or transporter - mediated ion fluxes across the capillary endothelial cell membranes . bbb leakage induced by ammonia and inflammatory molecules : new vistas on the underlying mechanisms section is devoted to the new findings regarding the mechanisms underlying alterations in the paracellular transport which is defined as bbb leakage , the role of which in ammonia neurotoxicity has so far been underestimated . pioneering studies pertinent to the effect of ammonia on bbb permeability were performed on animals with portacaval anastomosis ( pca)a model which mimics the condition of portal - systemic shunting in patients with liver cirrhosis . ( 1975 ) showed that bbb in pca rats is leaky to horseradish peroxidase ( hrp ) . this observation has been confirmed by sumner ( 1982 ) in a similar experimental setting , and by others using different bbb permeability markers and/or he models : by zaki ( 1983 ) also in pca rats who measured amino acid influx using the oldendorf perfusion technique ( oldendorf 1971 ) , and by horowitz et al . ( 1983 ) in galactosamine - induced animal model of acute liver failure ( alf ) , where permeability changes to aminoisobutyric acid were measured . however , other contemporary animal studies often performed in similar he models and using similar markers , revealed no brain vascular permeability changes . examples include the absence of changes of sucrose and methyl - aminoisobutyric acid permeation in galactosamine induced he ( lo et al . 1987 ) , and to mannitol or ions in the pca model ( sarna et al . 1977 ; alexander et al . transcellular passage of different molecules across the endothelium : roles of active transport and bbb leakage induced by ammonia and inflammatory molecules : new vistas on the underlying mechanisms sections , controversies about the bbb status as assessed with different compounds have lasted until the present time , with bbb changes being either confirmed ( wang et al . incoherent results were also obtained with regard to the passage of ammonia through the bbb , as monitored with n - labeled ammonia ( pet technique ) . lockwood et al . ( 1991 ) showed that ammonia enters the brain more easily in advanced he patients than in healthy controls . ( 2010 ) did not see any differences in bbb permeability for ammonia between patients with and without liver failure . ( 2007 ) observed increased ammonia accumulation in cirrhotic patients , but in their hands the increase was solely attributable to increased blood ammonia content . understanding of the effects of hyperammonemia on ammonia passage will require separate analysis of the two different forms of ammonia . at physiological ph overwhelming proportion of ammonia occurs as a cation ( warren 1962 ) , so it enters the brain mainly by a transcellular route , using an array of potassium channels and transporters or by substituting other cations with similar hydrated radius ( ott and larsen 2004 ) . one article indicated the presence of a specific nh4 carrier the rhesus associated glycoprotein rhcg in the brain capillaries ( huang and liu 2001 ) but its location ( luminal vs. abluminal side ) and functionality remains to be confirmed . however , recently the pericellular penetration by gaseous ammonia is being taken under consideration as a significant alternative ( ott and larsen 2004 ) . it is not known which of the two routes would be affected under excessive ammonia load . on the top of these controversies , increased vesicular transport across endothelial cells and swelling of astrocytic end - feet has been observed in different he models ( pilbeam et al . 1983 ) , with tjs remaining intact ( kato et al . it would thus appear that altered transcellular passage maybe a frequent phenomenon , albeit bbb changes in he are often too subtle to be detected with markers of gross bbb leakage . pca in rats is associated with amino acid imbalance in csf and brain due to enhanced blood to brain transport of tryptophan and other members of large neutral amino acid group ( lnaa ) ( james et al . in addition , increased concentration of aromatic amino acids ( aaa ) was found in brains of rats with pca , while the level of branched - chain amino acids was decreased ( smith et al . the above observations prompted a hypothesis that these alterations may contribute to impaired neurotransmission in he by producing ( i ) excessive amounts of neurotransmitters from which they derive , and/or ( ii ) false instead of authentic neurotransmitters , which are similar in structure but are either not active at the postsynaptic membrane , or their activity differs from their true counterparts ( curzon et al . 1975 ) . hypothesis appears attractive , because aaa are also precursors of these false modulators : tyrosine for octopamine ; phenylalanine for phenylethanolamine . ( 1982 ) showed elevated brain octopamine and phenylethanolamine levels in the brains of pca rats , and hilgier et al . however , the contribution of these false neurotransmitters to the neurotransmission imbalance associated with he has insofar not been examined in more detail . a plausible explanation for the ammonia - induced increase of blood brain aaa transport activity was proposed by james and colleagues ( 1979 ) who hypothesized that during hyperammonemia , increased brain glutamine ( gln ) production is followed by increased gln efflux from the brain , resulting in increased inward transport of these amino acids . this inference has been proven directly in studies in which increased tryptophan ( try ) uptake in exchange with gln via the l - transport system was recorded in cerebral capillary microvessels isolated from pca rats ( cangiano et al . vice versa , release of newly loaded gln from the capillaries was promoted by the try and leucine ( leu ) , and the effect was more pronounced when the capillaries were isolated from taa rats or following their incubation with ammonia than in control preparations ( hilgier et al . ( 1985 ) showed that treatment of pca rats with an inhibitor of gln synthesis , methionine sulfoximine ( mso ) , reduced the increased accumulation of the aaa in the brain in a manner correlated with increased ammonia accumulation . ( 1993 ) showed that administration of mso to pca rats normalized amino acid imbalance ascribed to excessive gln production . hyperammonemia was shown to be directly responsible for pca - induced alterations in the metabolism and transport of amino acids ( jessy et al . 1990 ) , including elevated brain try content and rise in the brain level of a serotonin metabolite , 5-hydroxyindoleacetic acid . these effects appeared to be due to ammonia - induced functional impairment of lnaa transport at the bbb . in rats in which ha was executed by urease administration , the impairment was found closely correlated with the rise in brain gln content ( bachmann and colombo 1983 ) , while in the cortical capillaries the increased try - gln exchange could be related to the raised -glutamyl - transpeptidase ( ggt ) activity ( stastn et al . 1988 ) . because ggt participates in lnaa transport and its activity was found to be increased in brain capillaries from hyperammonemic rats , a hypothesis has been put forward that ggt is involved in triggering the outward transport of the excess of gln from brain ( gorgievski - hrisoho et al . 1986 ) . in this way , enhanced activation of ggt could contribute to raised try and other lnaa levels as observed in rats with taa - induced alf ( hilgier et al . the above speculations were confirmed in a follow up study from the author s laboratory showing that ggt affects the l system - mediated amino acid exchange ( hilgier et al . the bbb transport of the cationic amino acids arginine ( arg ) and ornithine ( orn ) was investigated in different he models , and contradictory results have been obtained . zaki et al . ( 1984 ) showed a 30% increase in the brain uptake of arg in the galactosamine model of hepatic failure ; however , the effect was not specific to this amino acid and possibly secondary to bbb leakage also revealed by high molecular weight markers . by contrast , arg uptake from blood to brain was found decreased in chronic he ( pca ) rats ( zanchin et al . 1979 ) and in rats with thioacetamide ( taa)-induced he ( albrecht et al . 1996 ) . with regard to orn , increased brain uptake index of this amino acid coincident with its increased content in the blood was found in the taa model of he ( albrecht and hilgier 1986 ; albrecht et al . increased bbb transport of orn in the taa model has been considered as auto - protective response and in the same line has been speculated to facilitate intracerebral therapeutic action of the ammonia - trapping drug , l - ornithine - l - aspartate ( lola ) ( albrecht et al . . however , the benefits of orn may not apply to he in a chronic setting , where blood to brain transport of orn appears to remain unchanged ( zanchin et al . the mechanism underlying alterations of bbb transport of arg and orn has been hypothesized to involve changes in the basic amino acid transporter y activity and competition between these two amino acids for the transport site ( albrecht et al . 1996 ) , but experimental evidence in support of this hypothesis has not been provided as yet . the effects of he on arg transport are also likely to be mediated by gln , which accumulates intracerebrally in consequence of increased ammonia influx ( cooper and plum 1987 ) , overloading different cellular and subcellular compartments of the cns ( albrecht 2010 ) . it has been shown that gln added exogenously reduces no generation in the brain by inhibiting arg transport via the arg / gln exchanger , ylat2 , and that this effect is potentiated when ammonia is infused directly to the brain ( hilgier et al . if the above mechanism operates not only in the cns cells but also in the cerebral capillary endothelial cells forming the bbb , enhanced gln accumulation would modulate arg transport in these cells . the final outcome of this interaction would depend on whether gln accumulates intra- or extra - cellularly . a hypothesis that such an interaction may occur is supported by the observation that , gln infusion in the absence of hyperammonemia impairs cerebrovascular co2 reactivity , most likely by reducing arg availability and no synthesis , because co - infusion of arg counteracts the effect caused by glutamine ( okada et al . consistent with the role of arg / gln exchange at the bbb , our preliminary data indicate that ammonia increases the expression of the ylat2 transporter in a cerebral capillary endothelial cell line ( manuscript in preparation ) , as it does in the brain in the course of ha in situ ( zieliska et al . further studies on the mechanisms and pathophysiological implications of the changes in arg or orn influx to the brain are warranted in view of the proven or suspected contributions of the amino acids to the pathogenesis of he . arg is a precursor of no , a compound whose increased accumulation is engaged in the inflammatory response of the brain to ammonia ( jalan et al . 2011 ) , and in ammonia - induced brain swelling ( hussinger and grg 2010 ) , while decreased no synthesis has been implicated in impairment of cognition associated with prolonged hyperammonemia ( felipo 2006 ) . moreover , ha increases arg uptake to the different cell types within the cns ( rao et al . 1998 ) , and he in the taa model stimulates arg conversion to the neurotransmitter amino acids glu and gaba as measured in the whole brain ( albrecht and hilgier 1986 ) and in synaptosomes derived from these rats , which is likely to alter the balance between the inhibitory and excitatory neurotransmission ( albrecht et al . evaluation of the contribution of changes in arg transport across the bbb to the availability of this amino acid in the brain can not be accomplished without accounting for the variability in blood arg content in the different hyperammonemic models . the plasma arg level was shown to be decreased in pca rats ( zanchin et al . 1979 ) , but was elevated in rats subjected to prolonged hyperammonemia ( ishihara et al . 1998 ) , and fluctuated from increase to decrease during the development of taa - induced he ( albrecht and hilgier 1986 ) . orn plays a role in ammonia detoxification and gives rise to polyamines which exert hepato- and neuroprotection ( sikorska et al . treatment with lola , where orn contributes to urea formation , reduces blood ammonia level and in consequence improves the general condition of he patients ( kircheis et al . , increased brain uptake of orn as found in the taa model of he would further promote protection ( albrecht et al . orn also contributes in some degree to the biosynthesis of the neurotransmitter amino acids glu and gaba ( shank and campbell 1983 ) . similar to arg , conversion of its product orn to glu / gaba is stimulated during he ( albrecht and hilgier 1986 ; albrecht et al . however , implications for this increased conversion for neurotransmission imbalance associated with he are not known . taurine ( tau ) is a sulfur amino acid largely implicated in osmoregulatory and neuroprotective responses of the brain in various diseases , including hyperammonemia and he ( bosman et al . volume regulatory properties of tau are thought to be of particular importance in the case of brain edema , a major consequence of hyperammonemia , which results from impaired water homeostasis followed by swelling of astrocytes ( blei 2005 ) . he but not ha was associated with elevated blood content and increased brain uptake from blood to brain of tau , which collectively contributed to the increase of tau level in cerebral cortex ( hilgier et al . similar observation that liver failure induces elevation of tau in the blood were also made by other authors ( hamberger and nystrm 1984 ; zimmermann et al . because increased passage of tau was not due to massive breakdown of bbb ( as manifested by the absence of penetration of l - aspartate , which is not transported by intact capillary endothelial cells ) , it was believed to reflect activation of a tau transport system ( hilgier et al . of note in this context , treatment of an endothelial cell line with ammonia led to up - regulation and increased function of tau transporter ( blanger et al . hyperammonemia by affecting bbb transport of different substances and molecules can also lead to disturbances in cerebral energy homeostasis . hepatic encephalopathy evoked by pca was demonstrated to be associated with decreased brain glucose use and energy metabolism ( dejoseph and hawkins 1991 ) , and a similar effect was noted in rats with taa - induced he ( hilgier et al . brain uptake index of glucose was reduced after pca in rats ( sarna et al . 1979 ; crinquette et al . 1982 ) and this decrease was almost entirely due to the decrease in plasma glucose concentrations ( mans et al . glut-1 , the principal glucose transporter at the bbb responsible for supplying cns cells with blood - borne glucose was demonstrated to be induced by alf ( blanger et al . 2006 ) . since inhibition of glucose oxidative metabolism and subsequent activation of cerebral glycolysis are a hallmark of brain energy metabolism in he animals ( zwingmann et al . 2003 ; rao and norenberg 2001 ) , increased expression of glut-1 maybe considered as a compensatory response aimed at supporting higher glycolysis and maintaining brain atp levels . creatine ( cr ) a key substrate of the creatine / phosphocreatine / creatine kinase pathway is involved in regeneration of atp and in this way it also contributes to brain energy metabolism . moreover , cr was shown to affect gaba - ergic neurotransmission by acting as partial agonist on post - synaptic gaba(a ) receptors ( cupello et al . 2008 ) and to be crucial in dendritic and axonal elongation ( braissant et al . 2002 ) . exposure to ammonia was shown to generate a deficiency in cr in cns cells and to lead to neuronal cell loss , while co - treatment with cr was neuroprotective under ammonia exposure , but only in the presence of astrocytes ( braissant 2002 ) . ammonia treatment was demonstrated to increase cr uptake in cultured microcapillary brain endothelial cells ( blanger et al . pca in rats is associated with amino acid imbalance in csf and brain due to enhanced blood to brain transport of tryptophan and other members of large neutral amino acid group ( lnaa ) ( james et al . in addition , increased concentration of aromatic amino acids ( aaa ) was found in brains of rats with pca , while the level of branched - chain amino acids was decreased ( smith et al . the above observations prompted a hypothesis that these alterations may contribute to impaired neurotransmission in he by producing ( i ) excessive amounts of neurotransmitters from which they derive , and/or ( ii ) false instead of authentic neurotransmitters , which are similar in structure but are either not active at the postsynaptic membrane , or their activity differs from their true counterparts ( curzon et al . 1975 ) . hypothesis appears attractive , because aaa are also precursors of these false modulators : tyrosine for octopamine ; phenylalanine for phenylethanolamine . ( 1982 ) showed elevated brain octopamine and phenylethanolamine levels in the brains of pca rats , and hilgier et al . however , the contribution of these false neurotransmitters to the neurotransmission imbalance associated with he has insofar not been examined in more detail . a plausible explanation for the ammonia - induced increase of blood brain aaa transport activity was proposed by james and colleagues ( 1979 ) who hypothesized that during hyperammonemia , increased brain glutamine ( gln ) production is followed by increased gln efflux from the brain , resulting in increased inward transport of these amino acids . this inference has been proven directly in studies in which increased tryptophan ( try ) uptake in exchange with gln via the l - transport system was recorded in cerebral capillary microvessels isolated from pca rats ( cangiano et al . vice versa , release of newly loaded gln from the capillaries was promoted by the try and leucine ( leu ) , and the effect was more pronounced when the capillaries were isolated from taa rats or following their incubation with ammonia than in control preparations ( hilgier et al . ( 1985 ) showed that treatment of pca rats with an inhibitor of gln synthesis , methionine sulfoximine ( mso ) , reduced the increased accumulation of the aaa in the brain in a manner correlated with increased ammonia accumulation . ( 1993 ) showed that administration of mso to pca rats normalized amino acid imbalance ascribed to excessive gln production . hyperammonemia was shown to be directly responsible for pca - induced alterations in the metabolism and transport of amino acids ( jessy et al . 1990 ) , including elevated brain try content and rise in the brain level of a serotonin metabolite , 5-hydroxyindoleacetic acid . these effects appeared to be due to ammonia - induced functional impairment of lnaa transport at the bbb . in rats in which ha was executed by urease administration , the impairment was found closely correlated with the rise in brain gln content ( bachmann and colombo 1983 ) , while in the cortical capillaries the increased try - gln exchange could be related to the raised -glutamyl - transpeptidase ( ggt ) activity ( stastn et al . 1988 ) . because ggt participates in lnaa transport and its activity was found to be increased in brain capillaries from hyperammonemic rats , a hypothesis has been put forward that ggt is involved in triggering the outward transport of the excess of gln from brain ( gorgievski - hrisoho et al . 1986 ) . in this way , enhanced activation of ggt could contribute to raised try and other lnaa levels as observed in rats with taa - induced alf ( hilgier et al . the above speculations were confirmed in a follow up study from the author s laboratory showing that ggt affects the l system - mediated amino acid exchange ( hilgier et al . the bbb transport of the cationic amino acids arginine ( arg ) and ornithine ( orn ) was investigated in different he models , and contradictory results have been obtained . zaki et al . ( 1984 ) showed a 30% increase in the brain uptake of arg in the galactosamine model of hepatic failure ; however , the effect was not specific to this amino acid and possibly secondary to bbb leakage also revealed by high molecular weight markers . by contrast , arg uptake from blood to brain was found decreased in chronic he ( pca ) rats ( zanchin et al . 1979 ) and in rats with thioacetamide ( taa)-induced he ( albrecht et al . 1996 ) . with regard to orn , increased brain uptake index of this amino acid coincident with its increased content in the blood was found in the taa model of he ( albrecht and hilgier 1986 ; albrecht et al . increased bbb transport of orn in the taa model has been considered as auto - protective response and in the same line has been speculated to facilitate intracerebral therapeutic action of the ammonia - trapping drug , l - ornithine - l - aspartate ( lola ) ( albrecht et al . . however , the benefits of orn may not apply to he in a chronic setting , where blood to brain transport of orn appears to remain unchanged ( zanchin et al . the mechanism underlying alterations of bbb transport of arg and orn has been hypothesized to involve changes in the basic amino acid transporter y activity and competition between these two amino acids for the transport site ( albrecht et al . 1996 ) , but experimental evidence in support of this hypothesis has not been provided as yet . the effects of he on arg transport are also likely to be mediated by gln , which accumulates intracerebrally in consequence of increased ammonia influx ( cooper and plum 1987 ) , overloading different cellular and subcellular compartments of the cns ( albrecht 2010 ) . it has been shown that gln added exogenously reduces no generation in the brain by inhibiting arg transport via the arg / gln exchanger , ylat2 , and that this effect is potentiated when ammonia is infused directly to the brain ( hilgier et al . if the above mechanism operates not only in the cns cells but also in the cerebral capillary endothelial cells forming the bbb , enhanced gln accumulation would modulate arg transport in these cells . the final outcome of this interaction would depend on whether gln accumulates intra- or extra - cellularly . a hypothesis that such an interaction may occur is supported by the observation that , gln infusion in the absence of hyperammonemia impairs cerebrovascular co2 reactivity , most likely by reducing arg availability and no synthesis , because co - infusion of arg counteracts the effect caused by glutamine ( okada et al . consistent with the role of arg / gln exchange at the bbb , our preliminary data indicate that ammonia increases the expression of the ylat2 transporter in a cerebral capillary endothelial cell line ( manuscript in preparation ) , as it does in the brain in the course of ha in situ ( zieliska et al . further studies on the mechanisms and pathophysiological implications of the changes in arg or orn influx to the brain are warranted in view of the proven or suspected contributions of the amino acids to the pathogenesis of he . arg is a precursor of no , a compound whose increased accumulation is engaged in the inflammatory response of the brain to ammonia ( jalan et al . 2011 ) , and in ammonia - induced brain swelling ( hussinger and grg 2010 ) , while decreased no synthesis has been implicated in impairment of cognition associated with prolonged hyperammonemia ( felipo 2006 ) . moreover , ha increases arg uptake to the different cell types within the cns ( rao et al . 1998 ) , and he in the taa model stimulates arg conversion to the neurotransmitter amino acids glu and gaba as measured in the whole brain ( albrecht and hilgier 1986 ) and in synaptosomes derived from these rats , which is likely to alter the balance between the inhibitory and excitatory neurotransmission ( albrecht et al . evaluation of the contribution of changes in arg transport across the bbb to the availability of this amino acid in the brain can not be accomplished without accounting for the variability in blood arg content in the different hyperammonemic models . the plasma arg level was shown to be decreased in pca rats ( zanchin et al . 1979 ) , but was elevated in rats subjected to prolonged hyperammonemia ( ishihara et al . 1998 ) , and fluctuated from increase to decrease during the development of taa - induced he ( albrecht and hilgier 1986 ) . orn plays a role in ammonia detoxification and gives rise to polyamines which exert hepato- and neuroprotection ( sikorska et al . treatment with lola , where orn contributes to urea formation , reduces blood ammonia level and in consequence improves the general condition of he patients ( kircheis et al . , increased brain uptake of orn as found in the taa model of he would further promote protection ( albrecht et al . orn also contributes in some degree to the biosynthesis of the neurotransmitter amino acids glu and gaba ( shank and campbell 1983 ) . similar to arg , conversion of its product orn to glu / gaba is stimulated during he ( albrecht and hilgier 1986 ; albrecht et al . however , implications for this increased conversion for neurotransmission imbalance associated with he are not known . taurine ( tau ) is a sulfur amino acid largely implicated in osmoregulatory and neuroprotective responses of the brain in various diseases , including hyperammonemia and he ( bosman et al . volume regulatory properties of tau are thought to be of particular importance in the case of brain edema , a major consequence of hyperammonemia , which results from impaired water homeostasis followed by swelling of astrocytes ( blei 2005 ) . he but not ha was associated with elevated blood content and increased brain uptake from blood to brain of tau , which collectively contributed to the increase of tau level in cerebral cortex ( hilgier et al . similar observation that liver failure induces elevation of tau in the blood were also made by other authors ( hamberger and nystrm 1984 ; zimmermann et al . because increased passage of tau was not due to massive breakdown of bbb ( as manifested by the absence of penetration of l - aspartate , which is not transported by intact capillary endothelial cells ) , it was believed to reflect activation of a tau transport system ( hilgier et al . of note in this context , treatment of an endothelial cell line with ammonia led to up - regulation and increased function of tau transporter ( blanger et al . hyperammonemia by affecting bbb transport of different substances and molecules can also lead to disturbances in cerebral energy homeostasis . hepatic encephalopathy evoked by pca was demonstrated to be associated with decreased brain glucose use and energy metabolism ( dejoseph and hawkins 1991 ) , and a similar effect was noted in rats with taa - induced he ( hilgier et al . 1991 ) . brain uptake index of glucose was reduced after pca in rats ( sarna et al . 1979 ; crinquette et al . 1982 ) and this decrease was almost entirely due to the decrease in plasma glucose concentrations ( mans et al . glut-1 , the principal glucose transporter at the bbb responsible for supplying cns cells with blood - borne glucose was demonstrated to be induced by alf ( blanger et al . 2003 ; rao and norenberg 2001 ) , increased expression of glut-1 maybe considered as a compensatory response aimed at supporting higher glycolysis and maintaining brain atp levels . creatine ( cr ) a key substrate of the creatine / phosphocreatine / creatine kinase pathway is involved in regeneration of atp and in this way it also contributes to brain energy metabolism . moreover , cr was shown to affect gaba - ergic neurotransmission by acting as partial agonist on post - synaptic gaba(a ) receptors ( cupello et al . 2008 ) and to be crucial in dendritic and axonal elongation ( braissant et al . exposure to ammonia was shown to generate a deficiency in cr in cns cells and to lead to neuronal cell loss , while co - treatment with cr was neuroprotective under ammonia exposure , but only in the presence of astrocytes ( braissant 2002 ) . ammonia treatment was demonstrated to increase cr uptake in cultured microcapillary brain endothelial cells ( blanger et al . recent studies confirmed the view that hyperammonemia produces subtle changes in bbb integrity and partly unraveled the underlying mechanism . brain extravasation and edema in azoxymethane - induced alf were found to be secondary to tight junction ( tj ) protein degradation mediated by activation of matrix metalloproteinase-9 ( mmp-9 ) ( nguyen et al . specifically , it has been shown that tj proteins occludin and claudin-5 are significantly degraded in the brains of mice with galactosamine - induced alf , and this effect was reversed by treatment with inhibitor of mmp-9 , gm6001 ( chen et al . 2009 ) . a recent study delineated the most likely sequence of events linking activation of mmp-9 to occludin degradation in alf mice ; the intermediate steps include transactivation of epidermal growth factor receptor ( egfr ) and p38 mapk / nfb ( mitogen - activated protein kinase / nuclear factor - kappa b ) ( chen et al . 2011 ) . ( 2011 ) observed that progression of intracranial pressure in the course of alf is strictly correlated with the increase in bbb permeability and mmp-9 content . basing on this study the authors proposed a sequence of events of alf - induced brain damage , in which increase in bbb permeability is an initial step leading to vasogenic edema followed by ammonia excitotoxicity and cytotoxic edema . inflammatory molecules , including cytokines ( il-1 and/or il-6 ) and tumor necrosis factor - alpha ( tnf- ) are increased in plasma during acute and chronic liver failure in patients ( tilg et al . 2007 ) , and in animals with experimentally - induced he ( jiang et al . 2009 ) . circulating levels of tnf- correlate positively with the severity of he ( odeh et al . 2005 ) , moreover , its involvement in the development of intracranial pressure in patients with alf was demonstrated ( jalan et al . plasma il-6 level was also found well correlated with the severity of he and morbidity of the patients ( sheron et al . because massive breakdown of bbb is not observed during he , it is believed that the effects of inflammatory cytokines are transduced to the cns by vaso - active agents such as nitric oxide or prostanoids , which are synthesized by bbb - forming endothelium ( licinio and wong 1997 ) . brain barrier permeability in alf animals in the galactosamine ( lv 2010 ) and apap model ( wang et al . 2011 ) and in human alf patients ( lv 2010 ) , by disrupting tjs and inducing loss of the tj - associated protein occludin ( lv 2010 ) . data presented in this review provide considerable evidence that ammonia alters the passage of different molecules across the bbb , both by the transcellular route representing active or facilitated transport , and paracellularly , which occurs due to changes in the integrity of bbb constituents and thus reflects bbb leakage . as discussed above , increased bbb permeability adds a vasogenic component to the cytotoxic brain edema associated with he ( cauli et al . the effects of ammonia on the carrier - mediated transport of different molecules by the cerebral endothelial cells have been studied in considerable detail and the outlines of the changes in amino acid or energy metabolite transport are relatively well described . by contrast , the transcellular transport has long been given little consideration , mainly because in most he models , the ammonia - or he - induced changes have been too subtle and spatially restricted to be visualized by standard light- and electron microscopic techniques . the advent of more sensitive techniques has made it possible to identify the changes in tj proteins and their environment in a microscale , and provided tools to bridge the observations to the molecular mechanisms underlying the bbb leakage . further studies in this direction should allow to distinguish between the bbb changes in he which are induced directly by ammonia and those related to inflammatory toxins , mostly cytokines . one aspect deserving consideration in the future studies is the potential role of free radicals of oxygen and nitrogen , which have been found to be generated in excess by ammonia in different models and cell types of the cns and are responsible for the oxidative / nitrosative stress ( ons ) ( bemeur et al . 2010 ; hussinger 2010 ; skowroska et al . preliminary results from our laboratory disclosed that ons markers accumulate in an ammonia - treated brain microvascular endothelial cell line and increase permeability of these cells to a high molecular weight marker ( skowroska et al . this line of investigation appears attractive in view of the fact that ons causes bbb dysfunction in brain pathologies of varying etiology and severity ( lehner et al . many of the intracellular derangements known to be induced by ammonia in the cells within the cns or in peripheral tissues are likely to hold for the bbb - forming cerebral vascular endothelial cells , and may converge with events triggered in the different cells by ons . of note activation of the p38 mapk / nfb pathway which underlies mmp-9-induced tj protein damage ( chen et al . 2011 ) , is also involved in ammonia - induced oxidative damage of astrocytes ( jayakumar et al . other targets may include , for instance , altered nrf2-mediated synthesis of heme oxygenase i , an effect common to the response to various blood brain barrier damaging conditions ( lehner et al . 2011 ) and to the ammonia - induced ons in astrocytes ( warskulat et al . clearly , the above described mechanisms do not exhaust the list of possibilities that are worth further investigation . data presented in this review provide considerable evidence that ammonia alters the passage of different molecules across the bbb , both by the transcellular route representing active or facilitated transport , and paracellularly , which occurs due to changes in the integrity of bbb constituents and thus reflects bbb leakage . as discussed above , increased bbb permeability adds a vasogenic component to the cytotoxic brain edema associated with he ( cauli et al . 2011 ) . the effects of ammonia on the carrier - mediated transport of different molecules by the cerebral endothelial cells have been studied in considerable detail and the outlines of the changes in amino acid or energy metabolite transport are relatively well described . by contrast , the transcellular transport has long been given little consideration , mainly because in most he models , the ammonia - or he - induced changes have been too subtle and spatially restricted to be visualized by standard light- and electron microscopic techniques . the advent of more sensitive techniques has made it possible to identify the changes in tj proteins and their environment in a microscale , and provided tools to bridge the observations to the molecular mechanisms underlying the bbb leakage . further studies in this direction should allow to distinguish between the bbb changes in he which are induced directly by ammonia and those related to inflammatory toxins , mostly cytokines . one aspect deserving consideration in the future studies is the potential role of free radicals of oxygen and nitrogen , which have been found to be generated in excess by ammonia in different models and cell types of the cns and are responsible for the oxidative / nitrosative stress ( ons ) ( bemeur et al . preliminary results from our laboratory disclosed that ons markers accumulate in an ammonia - treated brain microvascular endothelial cell line and increase permeability of these cells to a high molecular weight marker ( skowroska et al . this line of investigation appears attractive in view of the fact that ons causes bbb dysfunction in brain pathologies of varying etiology and severity ( lehner et al . many of the intracellular derangements known to be induced by ammonia in the cells within the cns or in peripheral tissues are likely to hold for the bbb - forming cerebral vascular endothelial cells , and may converge with events triggered in the different cells by ons . of note activation of the p38 mapk / nfb pathway which underlies mmp-9-induced tj protein damage ( chen et al . 2011 ) , is also involved in ammonia - induced oxidative damage of astrocytes ( jayakumar et al . other targets may include , for instance , altered nrf2-mediated synthesis of heme oxygenase i , an effect common to the response to various blood brain barrier damaging conditions ( lehner et al . 2011 ) and to the ammonia - induced ons in astrocytes ( warskulat et al . clearly , the above described mechanisms do not exhaust the list of possibilities that are worth further investigation .

ammonia is a neurotoxin involved in the pathogenesis of neurological conditions associated with hyperammonemia , including hepatic encephalopathy , a condition associated with acute(alf ) or chronic liver failure . this article reviews evidence that apart from directly affecting the metabolism and function of the central nervous system cells , ammonia influences the passage of different molecules across the blood brain barrier ( bbb ) . a brief description is provided of the tight junctions , which couple adjacent cerebral capillary endothelial cells to each other to form the barrier . ammonia modulates the transcellular passage of low - to medium - size molecules , by affecting their carriers located at the bbb . ammonia induces interrelated aberrations of the transport of the large neutral amino acids and aromatic amino acids ( aaa ) , whose influx is augmented by exchange with glutamine produced in the course of ammonia detoxification , and maybe also modulated by the extracellularly acting gamma - glutamyl moiety transferring enzyme , gamma - glutamyl - transpeptidase . impaired aaa transport affects neurotransmission by altering intracerebral synthesis of catecholamines ( serotonin and dopamine ) , and producing false neurotransmitters ( octopamine and phenylethylamine ) . ammonia also modulates bbb transport of the cationic amino acids : the nitric oxide precursor , arginine , and ornithine , which is an ammonia trap , and affects the transport of energy metabolites glucose and creatine . moreover , ammonia acting either directly or in synergy with liver injury - derived inflammatory cytokines also evokes subtle increases of the transcellular passage of molecules of different size ( bbb leakage ) , which appears to be responsible for the vasogenic component of cerebral edema associated with alf .

Introductory Comments Studies on BBB Penetration by Different Compounds in HE Models: A Historical Account Transcellular Passage of Different Molecules Across the Endothelium: Roles of Active Transport Amino Acids Energy Substrates BBB Leakage Induced by Ammonia and Inflammatory Molecules: New Vistas on the Underlying Mechanisms Conclusions and Perspectives

focal and diffuse mesangial proliferative glomerulonephritis reveal the most common histologic lesions observed on renal biopsies from patients with iga nephropathy ( igan ) . however , histologic variability is also observed in this disease , which is the same as with lupus nephritis . in some cases , endocapillary hypercellularity is accompanied by segmental duplication of the glomerular basement membrane , and there is rarely diffuse endocapillary proliferation with lobular accentuation and multiple double contours , resembling type i membranoproliferative glomerulonephritis ( mpgn ) . tanaka and waga have reported a biopsied patient with acute igan following the formation of keloid scars due to a burn injury . however , further reports regarding the correlation between igan and burn injury are rare . a 46-year - old male patient with scrotal and pedal edema was admitted to our department in august 2012 . laboratory examination showed proteinuria ( 4 + ) and more than 775 red blood cells in the urine without casts . this patient had been treated for his 2nd- to 3rd - degree burn injuries in a local burn center . he had no inhalation injury or sepsis during the whole treatment process . at the admission time , he had a body temperature of 36.6c , a pulse rate of 75 beats / min , a respiratory rate of 16/min , and a blood pressure of 118/80 mm hg . physical examination revealed cutaneous convalescence after full - thickness burns of both hands , the coronal plane of the body and both legs . the skin showed fuscous , slight exudation , bleeding cracks , mild scaling and itch during the treatment process ( fig . the laboratory test results of the urine and blood for this patient are listed in table 1 . he was diagnosed as having nephrotic syndrome on the basis of a diminished serum albumin level of 23 g / l and an increased proteinuria excretion of 10.0 g/24 h. during the treatment course , microbiological examination of wound secretion was performed twice a week and no infection was observed . a percutaneous renal biopsy was performed on september 11 , 2012 ( day 57 after burn injury ) to explore the cause of proteinuria . the examination under light microscopy revealed a diffuse and moderate proliferation of mesangial cells and endothelial cells without observable crescents in 20 glomeruli . arterioles presented as normal status . neither tubular atrophy nor interstitial fibrosis was found , as shown in figure 2a c . immunofluorescence revealed iga ( 3 + ) , trace igm , c3 ( 3 + ) , igg ( ) , c4 ( ) and c1q ( ) in a coarse granular pattern with mesangial and capillary distribution ( fig . 2d , e ) , and inconspicuous staining for igm along the glomerular capillary walls in a granular pattern . in addition , this patient showed negative staining of hbsag and hbcag . the mesangial hypercellularity and increased mesangial matrix were present between the glomerular basement membrane and glomerular endothelium . the thickened capillary wall was composed of basement membrane - like materials , interposed mesangial cells , and electron - dense immune - type deposits ( fig . based on the proliferative properties of glomerular lesions , the patient was started on therapy with oral prednisone at a dose of 1 mg / kg / day . after treatment , the excretion of urinary protein revealed a significant decrease from 10.0 to 5.20 g/24 h in 2 weeks . the patient had been followed up to date and showed normal renal function and mild proteinuria . the glomerular filtration rate is increased after burn injuries , which is accompanied by vascular dysfunction and increased cardiac output . renal disease and acute kidney injury remains prevalent and is associated with the increased mortality of patients with severe burn injuries . this case shows the pattern of diffuse proliferative type i mpgn - like symptoms with mesangial interposition and double contours of the glomerular basement membrane present on periodic acid - schiff and silver stains ( fig . the following reasons are considered as a close correlation between burn injury and the onset of igan . first , this patient did not have any urinary abnormalities or chronic kidney diseases before and at the early stage of burn injury . second , nephrotic syndrome was observed at the 2nd week after the injury , and the patient did not have a history of infection such as inflammation of the respiratory tract although concurrent nephrotic syndrome in convalescence was observed . in addition , the patient suffered from skin problems with slight exudation , bleeding cracks , mild scaling and itch during the treatment process ( fig . although a causal relationship between igan and burn injury has not been established , an association of cutaneous lesions after burn injury with igan has been reported in a few patients [ 2 , 4 ] . tanaka and waga mentioned a biopsied patient with acute igan following the formation of keloid scars due to burn injury . the patient revealed a rapid recovery after complete removal of the scars without any medication . wang et al . have reported that acute igan is closely related to high - voltage electrical burn injury . in this study , all those case reports revealed a close relationship between igan and burn injury . because of trauma , infection , tissue necrosis , allogeneic plasma transfusion , increased concentrations of circulating immune complexes were observed . this situation is similar to the immune dysfunction of igan . both have inherent genetic predisposition and external infections , which may induce igan . if the pathogenesis of igan is not correlated with the concurrent burn injury , what is the cause of the nephrotic sydrome ? we hypothesize that an autoimmune dysregulation due to a sequestered antigen induced by skin infection and pruritus after burn injury may have an important role in the pathogenesis of igan . the persistent skin lesions with dry , itch and crusting properties can trigger iga deposition and promote the depositions in the mesangium and subendothelium of the patient . after serious burns , the immune system is disordered , the immune complex is increased in the circulation , and macrophages are excessively activated . numerous studies have provided evidence that burns upregulate inflammatory cytokines such as tnf- and il-1 . those cytokines can interfere with the hemodynamics of intraglomerular microcirculation , the coagulation - fibrinolysis system and the infiltration of inflammatory cells after burns . nephritis is a rare complication of burn injury , and once this happens , it will affect the recovery and prognosis of the patient . regular urine analysis will contribute to the early detection and diagnosis of glomerular diseases while treating burn injury . it is difficult to measure the blood pressure of burned patients , but blood pressure should be monitored as early as possible . because of wound exudation , infection and other reasons , the patients with large burned areas often have some degree of hypoalbuminemia . once persistent hypoalbuminemia exist , the burned patients need further examination to diagnose whether they have chronic glomerulonephritis or nephrotic syndrome . if the burned patient has nephrotic syndrome , enough glucocorticoid should be administered in order to alleviate proteinuria effectively and for the recovery of burn wounds . the casual relationship between the onset of igan and burn injury remains speculative and needs to be explored further .

iga nephropathy ( igan ) is the most common primary glomerulonephritis worldwide , accounting for approximately 3040% of patients undergoing renal biopsy in asia . the characteristic and diagnostic lesion of igan is the deposition of glomerular iga . the morphological lesions observed by light microscopy are extremely variable . a causal relationship between igan and burn injury has not been established , and the correlation between them is not clear if they appear at the same time . we have explored the cause of severe proteinuria of a chinese patient with burns of 2nd or 3rd degree after a gas leakage accident 2 weeks ago . the diffuse proliferative glomerulonephritis of this patient revealed type i membranoproliferative glomerulonephritis - like symptoms . moreover , this patient showed a sensitive response to prednisone . this case report demonstrates the intrinsic relationship between kidney disease and burn injury , which will facilitate a feasible treatment strategy for proteinuria after burn injury .

Introduction Case Report Discussion Disclosure Statement

chronic total occlusion of coronary arteries ( cto ) is observed in 35%50% of patients with significant coronary artery disease ( cad ) undergoing diagnostic angiography . percutaneous coronary intervention ( pci ) for cto has become a widely accepted treatment strategy and accounts for up to 20% of all pcis [ 36 ] . successful pci of cto has been associated with improved left ventricular ( lv ) systolic function , reduced anginal symptoms , increased exercise capacity , decreased need for bypass surgery , and , most importantly , an increase in survival . qt dispersion ( qtd ) , first introduced by campbell et al . , is the maximum inter - lead variation between the longest and shortest qt intervals recorded in the standard 12-lead electrocardiogram ( ecg ) . ventricular contraction and recovery are recorded as qt interval in ecg ; hence , qtd represents the regional heterogeneity of ventricular repolarization duration in the 3-dimensional structure of ventricular myocardium [ 1012 ] . it has been previously demonstrated by many studies that the defect in myocardial microvascular perfusion was presented as an increase in qtd [ 1315 ] and the qtd was decreased following successful revascularization . heart rate variability ( hrv ) has been shown to be a reliable non - invasive technique for the quantitative analysis of the activity of the components of the autonomic nervous system . analysis of hrv consists of a series of measurements of successive rr interval variations of sinus origin , which provide information about autonomic tone . increased sympathetic tone may increase the incidence and severity of arrhythmias in the setting of ischemia . the significance of autonomic factors for arrhythmogenesis following early reperfusion of ctos has not yet been well established . the myocardial defect and/or ongoing myocardial ischemia in cto patients may lead to impairment in ventricular repolarization . the change in autonomic tone in these patients may also be related to ventricular arrhythmias . the aim of this study was to assess short - term changes in qtd and hrv parameters after successful pci of cto patients . in this prospective , observational study , conducted from april 2011 to february 2013 , we enrolled 139 successfully revascularized patients with cto . all patients underwent physical examination , chest x - ray , ecg , and transthoracic echocardiographic evaluation . cto was defined as the lumen compromise resulting in either thrombolysis in myocardial infarction ( timi ) flow grade 0 or 1 , with a likely duration of > 3 months . all patients included had a native vessel occlusion estimated to be of at least 3-month duration on the basis of a history of sudden chest pain , a previous myocardial infarction ( mi ) in the same target vessel territory , or the time between diagnosis made on coronary angiography and pci . we excluded patients with mi within the previous 6 months , second or third - degree atrioventricular conduction disturbances , atrial fibrillation or flutter , frequent ( > 10/min ) ventricular extrasystoles , sinus node disease , lv hypertrophy , permanent st changes on ecg , permanent cardiac pacemaker , abnormal serum electrolyte levels , congenital long - qt syndrome , an ecg with more than 6 missing leads , and patients taking drugs that modify the qt interval . after pci , all patients were prescribed lifelong aspirin ; in addition , clopidogrel was prescribed for at least 12 months in all participating sites . procedural success was defined as successful recanalization and dilation of at least 1 cto per patient with or without stent implantation , residual stenosis of < 50% , and timi flow > 2 . the study protocol was approved by the institutional ethics committee , and all patients provided written informed consent . the qt intervals and qtd were measured manually from the standard ecgs available before and after pci . standard 12-lead ecg was recorded at a 25 mm / s paper speed and a gain of 10 mm / mv ( montara instrument eu 250 electrocardiograph , milwaukee , wi , usa ) . all ecgs were scanned at a 600 dpi resolution and computer - based analysis was performed by 2 independent cardiologists who were blind to the timing of the ecgs and patients data . to measure qtd , qt interval was defined as the interval between beginnings of qrs complex to the point the t wave returned to isoelectric line and for each lead the mean of qt interval in 4 consecutive beats was measured . when the u wave was present , the end of the t wave was defined as the nadir of the curve between the t and the u waves . dispersion of the qt interval was measured as the difference between the maximum and minimum mean qt intervals recorded in any of the 12 leads of the standard ecg . for each patient , qtd was measured twice ; once 612 hours before , and once 6 hours after pci . both the qt interval and qtd were rate - corrected with a modification of bazett s formula ( corrected qt interval qt / square root of the rr interval ) ( qtcd ) . after completing the computer - based measurements , qtd was tested to identify intra - observer variability in 25 randomly selected patients using the bland - altman method . the 95% limit of agreement for qtd was acceptable ( 7.4 and 7.1 ms , respectively ) ( figure 1a ) . the 95% limit of agreement for qtd was 9.2 and 5.8 ms , respectively ( figure 1b ) . hrv was analyzed using data from holter recordings ( synetec version 1.10 , ela medical , montrouge , france ) , which were started on hospital admission . r data from holter recordings were assessed using power spectral analysis before and within 24 hours after pci . we calculated time - domain hrv indices of standard deviations of the normal - to - normal qrs intervals ( sdnn ) , square roots of the mean squared differences of successive n n intervals ( rmssd ) , and percentage of consecutive rr differences > 50 ms ( pnn50 ) . for frequency domain hrv indices , we used the fourier transform method for the spectral measurements , and the heart rate spectrum between 0.003 and 0.40 hz was defined as total energy ( ms ) . this energy was divided into 2 components : low frequency ( lf : 0.040.15 hz ) and high frequency ( hf : 0.160.40 hz ) . continuous variables are reported as mean standard deviations ( sd ) and categorical variables are expressed as percentages . comparison of categorical and continuous variables between the 2 groups was performed using the test and unpaired t - test , respectively . the qt intervals and qtd were measured manually from the standard ecgs available before and after pci . standard 12-lead ecg was recorded at a 25 mm / s paper speed and a gain of 10 mm / mv ( montara instrument eu 250 electrocardiograph , milwaukee , wi , usa ) . all ecgs were scanned at a 600 dpi resolution and computer - based analysis was performed by 2 independent cardiologists who were blind to the timing of the ecgs and patients data . to measure qtd , qt interval was defined as the interval between beginnings of qrs complex to the point the t wave returned to isoelectric line and for each lead the mean of qt interval in 4 consecutive beats was measured . when the u wave was present , the end of the t wave was defined as the nadir of the curve between the t and the u waves . dispersion of the qt interval was measured as the difference between the maximum and minimum mean qt intervals recorded in any of the 12 leads of the standard ecg . for each patient , qtd was measured twice ; once 612 hours before , and once 6 hours after pci . both the qt interval and qtd were rate - corrected with a modification of bazett s formula ( corrected qt interval qt / square root of the rr interval ) ( qtcd ) . after completing the computer - based measurements , qtd was tested to identify intra - observer variability in 25 randomly selected patients using the bland - altman method . the 95% limit of agreement for qtd was acceptable ( 7.4 and 7.1 ms , respectively ) ( figure 1a ) . the 95% limit of agreement for qtd was 9.2 and 5.8 ms , respectively ( figure 1b ) . hrv was analyzed using data from holter recordings ( synetec version 1.10 , ela medical , montrouge , france ) , which were started on hospital admission . r data from holter recordings were assessed using power spectral analysis before and within 24 hours after pci . we calculated time - domain hrv indices of standard deviations of the normal - to - normal qrs intervals ( sdnn ) , square roots of the mean squared differences of successive n n intervals ( rmssd ) , and percentage of consecutive rr differences > 50 ms ( pnn50 ) . for frequency domain hrv indices , we used the fourier transform method for the spectral measurements , and the heart rate spectrum between 0.003 and 0.40 hz was defined as total energy ( ms ) . this energy was divided into 2 components : low frequency ( lf : 0.040.15 hz ) and high frequency ( hf : 0.160.40 hz ) . continuous variables are reported as mean standard deviations ( sd ) and categorical variables are expressed as percentages . comparison of categorical and continuous variables between the 2 groups was performed using the test and unpaired t - test , respectively . the mean patient age was 58.39.6 years , 118 ( % 84 ) were male and 59 ( % 42 ) had diabetes mellitus . the cohort included 47 patients ( 34% ) with prior mi and 14 patients ( 10% ) with a history of congestive heart failure . at the baseline , most of the patients were on aspirin ( n=139 , 100% ) , angiotensin - converting enzyme ( ace ) inhibitors ( n=98 , 70% ) , lipid - lowering medications ( n=113 , 81% ) , and -blockers ( n=124 , 89% ) however , use of nitrate ( n=29 , 20% ) , calcium channel blockers ( n=23 , 16% ) , and angiotensin receptor blockers ( n=34 , 24% ) was limited . both qtd and qtcd showed significant improvement following successful revascularization of cto ( 55.8314.79 vs. 38.8711.69 ; p<0.001 and 61.0216.28 vs. 42.9213.41 ; p<0.001 , respectively ) . regarding hrv parameters , the revascularization of the left coronary artery ( lad ) ( n=38 ) resulted in a decrease in hrv indices , including sddn , rmssd , and pnn50 , but none of them reached statistical significance . similar findings were observed after revascularization of the circumflex branch of the left coronary artery ( cx ) ( n=28 ) and right coronary artery ( rca ) ( n=73 ) lesions ( table 3 ) . we investigated changes in qtcd and hrv parameters in cto patients undergoing successful percutaneous revascularization . the major findings of this study are : ( 1 ) successful revascularization may improve qtcd in patients with cto , ( 2 ) the revascularization in cto lesions does not seem to have a significant impact on hrv , and ( 3 ) the impact on hrv does not change with the intervention to the lad , cx , or rca . myocardial necrosis and reversible myocardial ischemia both impact qtd . a direct relationship between the prolongation of the qt interval and myocardial ischemia has been reported by roukema et al . , who observed increased qtd in patients with exercise - induced myocardial ischemia . in experimental animal studies and in human studies it has been shown that the qt interval shortened in acutely hypoperfused areas , whereas in infarcted myocardium there was a prolonged repolarization time associated with qt prolongation on the ecg . the heterogeneity of the ventricular excitability was presumed to increase the propensity for arrhythmic manifestations and arrhythmic death , especially in patients with previous mi or history of cad . reported that increased qtd is related to susceptibility to reentry ventricular tachyarrhythmias , independent of degree of lv dysfunction or clinical characteristics of the patient , suggesting that the simple , noninvasive measurement of this interval from a standard 12-lead ecg significantly contributes to identifying patients at risk for life - threatening arrhythmias after a previous mi . the strong heart study of assessment of qt interval and qtd for prediction of all - cause cardiovascular ( cv ) mortality showed that qtcd was a strong predictor of all - cause mortality and a weaker predictor of cv mortality , and that qtd is a significant predictor of cv mortality . in the present study we showed that successful revascularization of ctos resulted in significant decrease in qtd and qtcd . our results are consistent with data reported by yunus et al . , who assessed qtd in patients with ischemia due to 1-vessel cad without prior mi and who underwent successful pci . however , our patient population included patients with only ctos and demonstrated the improvement in qtd in this challenging group . we suggest that the electrophysiological mechanism of action is based on a decrease in ischemia - induced prolongation of conduction and in dispersion of conduction times . the improvement in qtd with pci in may suggest a role for revascularization in achieving more homogenous repolarization and , perhaps , greater clinical stability in cto patients . hrv analysis is a safe and convenient method for the evaluation of the function of the autonomic nervous system activity in accordance with guidelines for standardization . significant decrease in autonomic tone has been shown to be an independent predictor of mortality in patients with cad . it has been shown that hrv indices decreased following coronary revascularizations . in our study , although there was a trend in decrease in some hrv parameters following pci of cto , none of the parameters reached statistical significance . similar results were also reported by szwoch et al . , but they repeated the hrv analysis after 3 months and observed improvement in most of the parameters . we speculate that the decrease in hrv parameters in early periods following recanalization of ctos could be transient and might be associated with acute endothelial injury during the procedure or microembolization to the vascular bed that was previously protected from ischemia by collateral support . consistent with the results of a previous study , localization of totally occluded coronary arteries in this study did not have significant impact on hrv results after the revascularization procedure . because the sinus node blood supply primarily comes from this artery , a significant change in hrv following successful pci to rca might be expected . the reason may not only be the incomplete disclosed mechanisms of hrv , but also a common impact of each artery on the sinus node artery or other mechanisms that might be responsible from hrv changes in addition to changes in blood supply . the main limitations of our study were lack of long - term follow - up and heterogeneous patient population with concomitant diseases . nearly half of our patients ( 42% ) had diabetes . diabetes itself may be associated with depressed hrv due to diabetic autonomic neuropathy and may result in reduced hrv , inducing hemodynamic changes that involve adrenergic activation and vagal tone reduction . however , our patient group may not be representative of the overall patient population with cad . we analyzed hrv parameters before and after pci , but we did not measure respiratory rate . in addition , because hrv was affected by various factors such as age of the patient and the use of ace inhibitors and -blockers , these factors might have affected our results [ 3436 ] . in conclusion , successful pci of cto patients may improve ventricular repolarization abnormalities , and thus may reduce the incidence of ventricular arrhythmias . early assessment of hrv parameters does not seem to be changed following recanalization of cto lesions .

backgroundqt dispersion ( qtd ) , which is a measure of inhomogeneity of myocardial repolarization , increases following impaired myocardial perfusion . its prolongation may provide a suitable substrate for life - threatening ventricular arrhythmias . we investigated the changes in qtd and heart rate variability ( hrv ) parameters after successful coronary artery revascularization in a patient with chronic total occlusions ( cto).material / methodsthis study included 139 successfully revascularized cto patients ( 118 men , 21 women , mean age 58.39.6 years ) . qtd was measured from a 12-lead electrocardiogram and was defined as the difference between maximum and minimum qt interval . hrv analyses of all subjects were obtained . frequency domain ( lf : hf ) and time domain ( sdnn , pnn50 , and rmssd ) parameters were analyzed . qt intervals were also corrected for heart rate using bazett s formula , and the corrected qt interval dispersion ( qtcd ) was then calculated . all measurements were made before and after percutaneous coronary intervention ( pci).resultsboth qtd and qtcd showed significant improvement following successful revascularization of cto ( 55.8314.79 to 38.8711.69 ; p<0.001 and 61.0216.28 to 42.9213.41 ; p<0.001 ) . the revascularization of lad ( n=38 ) , cx ( n=28 ) and rca ( n=73 ) resulted in decrease in hrv indices , including sddn , rmssd , and pnn50 , but none of the variables reached statistical significance.conclusionssuccessful revascularization of cto may result in improvement in regional heterogeneity of myocardial repolarization , evidenced as decreased qtcd after the pci . the revascularization in cto lesions does not seem to have a significant impact on hrv .

Background Material and Methods Electrocardiographic and QT dispersion analysis Heart rate variability Statistical analysis Results Discussion Conclusions

ocular cicatricial pemphigoid ( ocp ) is an uncommon , chronic autoimmune disease that affects mucous membranes , particularly the conjunctiva.1 the disease typically results in chronic conjunctivitis and causes conjunctival and corneal scarring , which can result in limbal stem cell deficiency and blindness.2 adequate control of ocular inflammation usually requires systemic as well as topical immunosuppressants . commonly used systemic medications include prednisone , methotrexate , mycophenolate mofetil ( mmf ) , and cyclophosphamide.3 the porphyrias are metabolic disorders caused by defective enzymes within the heme synthetic pathway.4 these defective enzymes cause an accumulation of intermediates from the heme synthetic pathway that results in various clinical manifestations.4,5 porphyria cutanea tarda is the most common of the porphyrias and results from a deficiency in uroporphyrin decarboxylase , which is the fifth enzyme in the heme synthetic pathway.6 this deficiency results in the accumulation of porphyrins in the liver and plasma . on exposure to light with a wavelength near 400 nm , the porphyrins enter an excited state that can lead to the damage of proteins , lipids , and basement membranes.6 this process results in blisters , fibrosis , and scarring of the skin in areas of the body exposed to sunlight.6 park et al7 have reported a case of a 31-year - old with cicatricial conjunctivitis who was biopsy negative for ocp and was later diagnosed with porphyria cutanea tarda ; in this case the patient s clinical symptoms significantly improved after initiating phlebotomy treatments . the present authors report a similar case , in which pathology and direct immunofluorescence confirmed a diagnosis of ocp and where the patient s clinical condition also improved significantly upon the diagnosis and treatment of porphyria cutanea tarda . a 64-year - old caucasian male complaining of redness and tearing for 3 years in both eyes was referred for evaluation of cicatricial conjunctivitis . he had been treated with tobramycin and dexamethasone ophthalmic ointment in both eyes as needed and doxycycline 100 mg by mouth daily with no improvement in his symptoms . on slit lamp examination the patient had subconjunctival fibrosis , symblepharon , forniceal foreshortening , and trichiasis in both eyes ( figure 1 ) . examination of the corneas revealed multiple punctate epithelial erosions . a schirmer s test was performed without anesthesia , showing 22 mm of wetting in the right eye and 13 mm in the left after 5 minutes . direct immunofluorescence studies of the conjunctival biopsy specimen revealed immunoglobulin g4 deposits in the basement membrane zone of the junctional area ( figure 2 ) , consistent with ocp . given the findings of subconjunctival fibrosis and symblepharon formation in both eyes , the patient was diagnosed with bilateral stage iii pemphigoid . the patient was started on methotrexate 15 mg by mouth weekly and prednisone 20 mg by mouth daily . the patient demonstrated gradual improvement in the conjunctival inflammation after starting the methotrexate and prednisone . the patient was subsequently tapered off the prednisone , while the methotrexate 15 mg by mouth weekly was continued . after 4 months of treatment with the methotrexate , the patient s conjunctival inflammation began to worsen and his regimen was subsequently changed from methotrexate to mmf 1000 mg by mouth twice daily . approximately 3 months after initiating mmf treatment , the patient was diagnosed with porphyria cutanea tarda . the patient s conjunctival inflammation appeared stable following initiation of the phlebotomy treatments and the mmf was subsequently discontinued . approximately 6 weeks following discontinuation of the mmf , the patient returned with mildly increased conjunctival injection and trichiasis in both eyes . epilation was performed and the patient was started on 1% prednisolone acetate ( one drop in both eyes twice daily ) . the patient s conjunctival inflammation stabilized and he was tapered down to one drop of 1% prednisolone acetate in both eyes once daily . since initiation of the phlebotomy treatments , the patient s conjunctival inflammation and subconjunctival fibrosis has remained quiescent for 4 months without requiring mmf ( figure 3 ) . ocp is believed to be an autoimmune disease of genetic predisposition , and it is likely that a second - hit environmental trigger is required to initiate onset of the disease.1 it has been thought this could include chemical exposure or microbial environmental triggers . the present case suggests that the patient s porphyria could be a causal factor associated with the ocp and could even have been the environmental trigger that stimulated the disease to occur . the porphyrins are present in plasma and therefore they would be present in the ocular surface vasculature.5 the ocular surface is constantly exposed to light . exposure to ultraviolet light would lead the porphyrins to enter into an excited state , resulting in inflammation and damage to the ocular surface . this process could be the trigger to either initiate or exacerbate ocp . in the present case , the patient s conjunctival inflammation was observed to significantly improve following initiation of treatment for his porphyria . a similar response was observed in the aforementioned case reported by park et al,7 although that particular patient was biopsy negative for ocp and was positive for hepatitis c virus infection ; in addition , the patient remained on methotrexate . the patient in the present case was unique in that he was biopsy positive for ocp and his clinical improvement was significant enough after starting phlebotomy treatments that his mmf was discontinued . the authors consider that this case , as well as the case reported by park et al,7 sheds new light on the search for the etiology of ocp and the subsequent treatment options for patients with this disease .

a 64-year - old caucasian male complaining of redness and tearing for 3 years in both eyes was referred for evaluation of cicatricial conjunctivitis . ocular cicatricial pemphigoid was suspected and this diagnosis was confirmed through biopsy . the patient s condition showed moderate improvement following treatment with methotrexate and mycophenolate mofetil . the patient was later diagnosed with porphyria cutanea tarda and phlebotomy treatments were subsequently initiated . the patient s ocular symptoms improved further after he began receiving these phlebotomy treatments , and conventional treatment was discontinued . the authors hypothesize that circulating porphyrins activated by ultraviolet light could be the cause of the ocular cicatricial pemphigoid in this patient .

Introduction Case report Discussion Conclusion

the incidence of endometrial cancer has remained stable , but the number of deaths annually from this disease has doubled since 1987 . despite the fact that many gynecologists believe endometrial cancer is harmless , when compared stage by stage , 5-year survival is identical to that of cervical cancer , which is considered virulent . thus , we are obligated to reassess the screening , diagnostic , staging , and therapeutic aspects and most importantly the debate on lymphadenectomy during hysterectomy . currently , an extremely low prevalence is inferred for lymph node involvement in stages ia , ib , and grade 1 neoplasm , which comprises 40% to 60% of newly diagnosed patients . furthermore , it is postulated that in early disease stages and when regional nodes are clinically unaffected , nodes should only be sampled for prognostic significance and not removed radically in the vain hope of curing the patient . indeed , evidence from laboratory studies has shown that many lymphatic and lymphatico - venous shunts that bypass regional lymph nodes exist and allow an early stage lymphatic and hematogenous dissemination of malignant cells . on the other hand , removal of lymph nodes if they are grossly positive , lessening the tumor burden , should decrease the amount of suppressive tumor antigens present in the host and reduce the amount of adjunctive therapy required to treat residual disease . indeed , ineffective nodes are no longer useful to the host but now contribute to increased tumor - induced immunologic suppression . for tumors infiltrating the inner third of the myometrium , the actual risk of node metastases is substantial for grade 3 histologic type tumors , and for the middle third invasion , the risk of node metastases is substantial for grade 2 and 3 lesions . for tumors infiltrating the outer third , thus , the risk is ignorable for superficial invasion and substantial for deep myometrial invasion for all grades ( 20% to 45% ) . the negligible risk of node metastases ( 0% to 4% ) is also valid for inner one - third myometrial involvement for grades 1 and 2 , for middle one - third involvement with grade 1 tumors , and for all aforementioned conditions with no vascular space involvement and no spread to the cervix or adnexa , or both , with histologic type adenocarcinoma and adenoacanthoma . this group comprises 75% of patients . nevertheless , the common belief among gynecologists of just to take out the uterus as it is small and well differentiated is not valid as 10% to 15% of the grade 1 lesions have substantial myometrial invasion exposing the patient to substantial risk for node metastases and thereby necessitating intraoperative assessment of myometrial invasion in those instances as well . thus , the need for lymph node assessment during surgery is the most challenging issue among gynecologists . several methods of lymph node dissection have been proposed for different situations although these differ from one surgeon to the next . the adequacy of clinical assessment of lymph nodes and sampling lymphadenectomy has been a subject of controversy for years . meticulous inspection and palpation bidigitally are refined exercises for the gynecologic oncologist , but slightly more than one half of the suspicious pelvic and paraaortic nodes reveal metastases while only 5% of the unsuspicious nodes bear malignant cells . thus , one should bear in mind the possibility of undetected metastases of regional nodes by traditional methods during surgery . in addition , some tumor cells can only be detected by immunohistochemical analysis and thereby pass unrecorded . thus , the real incidence of tumor involvement might have been underestimated due to the technique used for pathologic assessment and limited node dissection . do we need to do complete dissection for positive detection , because negative staging does not necessarily identify a subset that does not need further therapy is another debate . on the other hand , the concern about what would be the false negativity rate to be accepted by the surgeon and the patient ? is another important issue . hence , being the most prevalent gynecological cancer , endometrial cancer does not require a radical hysterectomy but lymph node evaluation in its early stage . as gynecologic oncology surgeons have gained more experience and skill in different techniques and procedures , and more interest in minimally invasive surgery , the laparoscopic approach has found applications in the treatment of endometrial cancer . with the application of video and improvement in the armamentarium of video , laparoscopic surgery changed from being performed in an operating room by one man frustrated because of the lack of the ability of other operating room staff to follow and participate in this surgical procedure , to being performed in an amphitheater , allowing not only the assistant and other operating room staff to see the actual procedure and follow the sequences but these advances resulted in accelerated improvement in operative laparoscopy and proved to be an important aid to education because of the advantage of using monitors during the operation and taping the surgery for repetitive education . laparoscopic - assisted surgical staging ( lass ) of early stage endometrial cancer , although remaining a controversial subject , is an attractive alternative to the traditional laparotomic approach . nevertheless , one should bear in mind that the first purpose of the staging is to provide a common language and uniformity for comparison instead of aiding therapy and management issues . staging must be the first aim despite there still existing some conflicting ideas regarding the system of staging . laparoscopy has a similar success rate when compared with laparotomic staging , and it can be accepted as less invasive . studies have shown that the lymph node yield from laparoscopy is equivalent to that of laparotomy . feasibility of laparoscopic staging in incompletely staged endometrial cancer patients is another subject of interest . although laparoscopic pelvic and paraaortic lymphadenectomy is accepted as feasible , randomized studies are still awaited to standardize its indications and applications . the patient population consisted of 52 early stage endometrial cancer patients who presented to akdeniz university obstetrics and gynecology department between 1998 and 2002 . all underwent staging surgery comprising total hysterectomy , bilateral salpingo - oophorectomy , and pelvic lymph node sampling by the same 2 surgeons ( cgz , ts ) , using the same technique and instruments . the patients were randomly assigned to the surgical approach and informed before surgery about the type of surgery they were to undergo . one group comprised 26 patients managed by traditional laparotomic surgery , while the other comprised 26 patients who underwent laparoscopic surgery . the patients who were clinically thought to have advanced disease were not included in the study . the main outcomes studied were operative time , blood transfusion , intraoperative and postoperative complications , duration of hospital stay , number of lymph nodes obtained and lymph node positivity . statistical analysis was performed by the mann whitney u test , and p values < 0.05 were considered statistically significant . the laparotomy group had an average age of 54.9 ( range , 36 to77 ) and an average gravidity of 3.8 ( range , 0 to 9 ) . the laparoscopy group had an average age of 56.6 ( range , 40 to 72 ) and an average gravidity of 3.6 ( range , 0 to 8) . body mass indexes were not significantly different between laparotomy and laparoscopy groups , being 26.2 vs 24.4 respectively . the majority of the patients had stage i disease ( 67.3% ) , and endometrioid type of endometrial cancer was more common ( tables 1 , 2 , and 3 ) . the number of lymph nodes was not significantly different between the groups ( p>0.05 ) . the laparoscopic group had an average number of 18.2 ( range , 9 to 31 ) , and the laparotomic group 21.1 ( range , 9 to 38 ) . two ( 7.7% ) patients in the laparoscopy group and 4 ( 15.4% ) in the laparotomy group had pelvic lymph node metastasis . eleven patients ( 42.3% ) in the laparoscopy group and 10 ( 38.5% ) in the laparotomy group were later scheduled for adjuvant radiation therapy . study groups compared according to stage histologic grades of the study groups histologic types of endometrial cancer in the study groups the laparoscopic group had significantly shorter hospitalization than did the laparotomy group ( 4.1 vs 8.2 days , z 1.96 , p<0.05 ) . operative time of laparoscopy being close to that of laparotomy was encouraging ( 155 vs 144 minutes , p>0.05 ) . in terms of postoperative and intraoperative complications , the laparoscopic approach had none , while wound complications occurred in 5 patients in the laparotomy group , of which one was evisceration and needed reoperation for closure . eight units of red blood cell suspension were transfused to the laparotomy group patients and 6 units to the laparoscopy group patients . nowadays , the laparoscope allows us to carry out almost any procedure that can be done by laparotomy in gynecologic practice . laparoscopically assisted vaginal hysterectomies and laparoscopic total hysterectomies for benign conditions are being applied widely even in patients with large uteri . however , in the management of gynecologic cancers , the laparoscope had a lesser role , until pelvic and paraaortic lymph node dissections became feasible in the 1990s . following this achievement , the laparoscope has gained importance among gynecologic oncologists especially in patients with early endometrial and cervical cancers . every surgeon was doubtful about the outcome of this type of surgery . because of such doubt , many institutions established pilot studies and some prospective small - scale trials . at any rate , some were successful with speeded up publication encouraging surgeons to switch to this type of surgery . laparoscopically assisted vaginal hysterectomy with lymph node evaluation is now an alternative treatment for endometrial cancer in properly selected patients . the goal is to minimize the morbidity of treatment instead of satisfying surgeons ' new enthusiasm to use the technique . laparoscopically assisted surgical staging for endometrial cancer in experienced hands can be performed with equal success and safety with minimal morbidity . it has the advantages of less pain , early resumption of normal activities , and overall improved quality of life . many authors are in agreement that it results in significantly less blood loss and shorter hospitalization . scribner et al compared laparoscopy and laparotomy in a similar number of patients as in our study . they concluded that although the early hospital discharge is an advantage , longer surgical time and higher anesthetic costs of the laparoscopic approach offset this gain , and total costs appear not to differ statistically . in contrast to scribner 's study , gemignani et al , found the overall charges in the laparoscopy group significantly lower than that of laparotomy group , considering fewer postoperative complications seen with the laparoscopic approach , a factor that lowers overall hospital charges . in these 2 studies , the laparoscopy group had a significantly longer operating room time ( 237 vs 157 minutes in scribner 's study and 214 vs 144 minutes in gemignani 's study ) ; however , in our study , the duration of the operation for the 2 groups was similar ( 155 vs 144 minutes ) . another randomized study comparing the laparoscopicvaginal approach with the conventional abdominal approach for treatment of patients with endometrial cancer was performed including 70 patients with endometrial cancer figo stage i - iii . thirty - seven patients were treated in the laparoscopic group versus 33 patients in the laparotomy group . lymph node dissection was performed in 25 patients by laparoscopy and in 24 patients by laparotomy . blood loss and yield of pelvic and paraaortic lymph nodes , duration of surgery , and incidence of postoperative complications were similar for both groups . while comparing laparotomy and laparoscopy , the number of residual nodes following lymphadenectomy has also been studied because lymph nodes left in situ might have had microscopic metastases . in a review by lecuru and taurelle , laparoscopic pelvic lymphadenectomy was declared able to retrieve 90% to 95% of the nodes , which was similar to that of laparotomy . laparoscopic paraaortic lymphadenectomy can be substituted for its open counterpart regarding lymph node yield and accuracy in recovery of positive nodes as well . port - site metastases are one of the most often addressed issues in patients undergoing a laparoscopic procedure for any kind of gynecologic cancer . dragging cancerous tissue through a small incision and exfoliation from the surface and implantation into the healing wounds are the main reasons for an increased likelihood of metastases or recurrences at that sites . however , in gynecologic cancers port - site recurrences are always associated with either disseminated intraperitoneal disease or cyst rupture . the question of contamination and increasing tumor growth because of co2 laparoscopy and the effect of pneumoperitoneum on survival remains obscure . however , surgeons should take some preventive measures , such as avoiding cyst rupture , gentle handling of the cancerous tissue , avoiding the rupture of the lymph node capsule , and including the port sites in the radiation field in patients undergoing postoperative irradiation . in this study morbidities related only to laparoscopic surgery are of some concern , and injuries are generally related to trocar installation . these morbidities are mostly bowel , vascular , and bladder injuries . increasing numbers of advanced laparoscopic applications like expanded lymph node dissections and more radicalness in treating gynecologic cancers have caused surgeons to face other complications as well . injury to vein tributaries during laparoscopic lymphadenectomy may cause major hemorrhage from the adjacent veins . it might not be easy sometimes to overcome such bleeding even during laparotomy because of the depth of the location of bleeding , preventing easy access to the area . a laparoscope with 5-to 7-fold magnification could sometimes be superior in identifying such deep bleeding locations ; however , difficulty in suturing or unavailability of vascular clamps may prevent the appropriate approach . in our study group , almost 90% of the patients in the laparoscopy group had stage i or ii disease following surgical staging , whereas only 70% of the patients in the laparotomy group had early stage disease . however , the breakout of stage iii subsets showed us that such patients are not identifiable before surgery , and therefore this could be incidental because we never felt that we 're glad that we opened this patient ; it would be dreadful if we put the scope for the first time during surgery . we conclude that the laparoscope seems to be very useful in a select group as it reduces postoperative morbidity offering quick recovery with the same success and efficacy . however , experience is of the utmost importance , and there is no way to start scoping without proper training . feeling ready does not mean that one will be good in the operating room , and thus being supervised should not be overlooked before integrating such a procedure into clinical practice . shorter hospital stay and less postoperative morbidity are advantages of laparoscopic surgery . duration of laparoscopic surgery does not seem to be different from that of laparotomy .

objective : the aim of this study was to evaluate the feasibility of laparoscopy in the management of early stage endometrial cancer.methods:fifty-two patients with endometrial cancer who underwent surgical staging consisting of total hysterectomy , bilateral salpingo - oophorectomy with pelvic lymph node dissection , and cytology between 1998 to 2002 were included in the study . laparotomy and laparoscopy were randomly offered to patients upon admittance.results:of 52 patients , 26 underwent laparotomy and the remaining 26 underwent laparoscopic staging surgery . no significant difference existed between the demographic characteristics of the 2 groups . the mean number of harvested lymph nodes was 18.2 in the laparoscopic group and 21.1 in the laparotomic group ( p>0.05 ) . pelvic lymph node metastases were detected in 7.7% of the patients in the laparoscopy group and 15.4% in the laparotomy group , and the difference was not significant . adjuvant radiotherapy was applied later to 42.3% of the laparoscopy group and 38.5% of the laparotomy group . operative morbidity was higher in the laparotomy group mainly because of postoperative wound infection , and the patients in the laparotomy group had a longer hospital stay.conclusion:laparoscopic surgery is a method that can be applied as well as laparotomy in the management of endometrial cancer . lymph node number and detection of lymph node metastasis did not differ significantly in laparotomic and laparoscopic approaches . wound infections were more frequent in laparotomies .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

a mild catalytic asymmetric direct fluoro - arylation of styrenes has been developed . the palladium - catalyzed three - component coupling of selectfluor , a styrene and a boronic acid , provides chiral monofluorinated compounds in good yield and in high enantiomeric excess . a mechanism proceeding through a pd(iv)-fluoride intermediate is proposed for the transformation and synthesis of an sp3 c f bond .

Supplementary Material

genetic analysis of phenotypes and diseases has traditionally followed two approaches : family - based linkage analysis and population - based association studies . while in linkage analysis it is the co - segregation of alleles in families that is measured , population - based studies use non - random associations between phenotypes and alleles in populations to identify causative genes . linkage analysis has proven to be immensely successful as a means of identifying genes for a number of single gene diseases with simple mendelian inheritance ( eg see omim database ) . complex diseases are multifactorial , polygenic and often characterised by late age of onset , incomplete penetrance , locus heterogeneity and environmental exposures and , despite significant efforts , have not been amenable to family - based mapping . linkage disequilibrium ( ld ) is an important aspect of genetic association studies and is generated in a population through mutation , selection , drift , non - random mating and admixture . allelic associations due to ld are significant and are correlated with physical distance within small genomic regions but decay over time due to recombination [ 2 - 4 ] . ld - based association studies have been successful in both fine scale mapping and initial disease gene mapping in homogeneous populations that have undergone recent bottlenecks ( eg hirschsprung disease in mennonites , bardet- beidle syndrome in bedouins ) . allelic associations can result either from direct functional effects of the alleles tested or indirectly through non - random associations between the allele measured and nearby functional alleles . since functional alleles in most genes are still unknown and are indeed an object of the research , ld is an important feature of how genes can be screened for alleles that alter disease risk . thus , there has been substantial focus on the extent of ld across the genome and the definition of statistical methods for disease gene mapping using ld [ 9 - 11 ] . in large cosmopolitan populations , however , ld may be difficult to detect when the mutation is old , since the amount of remaining ld may be small . additionally , false - positive associations due to population stratification are important confounders in ld - based association studies . the process of admixture itself creates ld between all loci , linked and unlinked , that have different allele frequencies in the parental populations . the magnitude of admixture linkage disequilibrium ( ald ) in an admixed population depends on the allele frequency differential between the parental populations , the level of admixture , the admixture dynamics , the time since admixture and the recombination rate between the loci . while ald between unlinked markers decays rapidly ( within two to four generations ) the exponential decrease in ald with genetic distance facilitates the differentiation of ald that is high between markers that are close together and genetically linked , from ald generated at unlinked loci . thus , if the parental populations differ in a trait or disease due to different frequencies of risk alleles , it should be possible to identify the loci containing these alleles using admixture mapping ( am ) [ 12 - 14 ] . the european colonial period that started in the late 1400s brought together in the new world populations that had been geographically isolated , namely , europeans , west africans and native americans . given the recent and common origin of all human populations , this admixture had only a small average effect on the gene pools of these new populations . in other words , for most genomic regions , the pre - colonial ( or parental ) populations had similar allele frequencies and , at these , admixture was of little consequence . at some other loci , however , there had been some change in allele frequency in the time since the separation of parental populations and it is at these loci where admixture has had an important effect . since populations like african americans , african caribbeans and mexican americans were formed in the recent past , allelic associations in these populations that were created by admixture extend over large distances . admixed populations represent a useful resource for mapping complex - disease genes by using this long - range ald , which requires fewer markers to screen the genome than other populations or approaches . understanding the genetic consequences of admixture is important because it can be both a confounding factor and a source of statistical power in gene identification studies . two models of admixture dynamics have been described to represent the extremes of the process by which an admixed population is formed : the continuous gene flow ( cgf ) model and the hybrid isolation ( hi ) model . in the hi model , admixture occurs immediately in a single generation without further contribution from either parental population , hence , ald is generated in a single generation and gradually decays in successive generations through independent assortment and recombination between loci . few false - positive results are thus expected in an association study under the hi model . alternatively , the cgf model represents a situation where admixture occurs at a steady rate in each generation , with contributions from one ( or all ) of the parental populations into the admixed population . ald under the cgf model increases in each generation , since new admixture is constantly occurring . a point will be reached , however ( when the admixture proportion = 0.5 ) , where continued admixture will actually decrease the ald , since added gene flow will result in the conversion of the admixed population into the introgressing parental population . figure 1 shows the amount of ald expected under these two models for linked and unlinked loci . for both models , association between markers simulation studies have shown that populations that have a demographic history more consistent with the cgf model of admixture retain ald over larger chromosomal regions and show significant associations between unlinked marker loci . while associations between unlinked markers could potentially lead to false - positives , conditioning upon parental admixture allows the distinction between associations arising due to true linkage and those due to cgf stratification to be made , thereby providing greater power for detecting ald over larger chromosomal distances . the amount of admixture linkage disequilibrium ( ald ) expected under the continuous gene flow ( cgf ) and hybrid isolation ( hi ) models of admixture for unlinked loci and loci linked at 5 cm . the results shown are for two loci with = 0.54 and 0.49 , and with 50 per cent admixture in the first generation for the hi model and 1.9 per cent admixture for 36 generations under the cgf model ( equivalent to 50 per cent total ) . ald under the hi model decreases for both linked and unlinked loci , whereas ald under the cgf model for both linked and unlinked loci increases initially and then decreases ( adapted from pfaff et al . , 2001 ) there are several ways in which admixture can be an important resource in the elucidation of genetic factors that contribute to the risk of common disease . common diseases often have environmental components to their risk , and the clinical phenotype results from currently unknown interactions between environmental factors and underlying genotypes . decomposing the sources of variation it is possible to distinguish between the genetic and environmental explanations for ethnic differences in disease risk ( and investigating the mode of inheritance ) , by studying the relationship of disease risk to individual admixture [ 14,17 - 19 ] . for example , recent studies have demonstrated a strong relationship between proportional west african ancestry and the risk of systemic lupus erythematosus in admixed populations in trinidad . several common diseases ( eg hypertension , diabetes , obesity , prostate cancer and osteoporosis ) have differences in risk among population groups ( see table 1 ) . in situations where these differences have a genetic basis , genes underlying these differences can be identified by testing for locus ancestry by conditioning on parental admixture . as detailed by shriver et al . , this approach has a greater statistical power than family linkage studies for mapping polygenic traits . estimates of biogeographical ancestry ( bga ) , the proportional ancestry levels of an individual , can be used in conjunction with measured environmental effects for investigating the roles of environmental and inherited risks underlying complex traits [ 18 - 20 ] . it is important to recognise that associations between individual admixture and disease risk might reflect correlations between bga and socio - cultural variables and exposures . for example , hypothetically , if bga and years of education were to be correlated , hypertension might be correlated with bga , even though the causal risk factor was years of education or vice versa . diseases with possible genetic components based on ethnic differences in disease rates and hence amenable to admixture mapping admixture - based methods rely on using suitable markers and estimates of allele frequencies from appropriately identified parental populations . since ald is fairly new and extends over larger distances , fewer markers are required for am studies . markers informative for ancestry have been used in several contexts and have been referred to as ' ideal , ' ' private ' and ' unique ' . informativeness of such markers can be measured as the allele frequency differential ( ) , which is the absolute value of the difference of a particular allele between populations . microsatellites and insertion / deletion polymorphisms with > 0.3 were recently called ' ethnic - difference markers ' ( edms ) suitable for mapping by admixture linkage disequilibrium ( mald ) . additionally , markers with high and very high log likelihood allelic ratio ( llar ) between populations have been designated ' population specific alleles ' ( psas ) . this report followed from earlier work where markers with large allele frequency difference were identified to be appropriate for admixture studies , and most ( > 95 per cent ) of the arbitrarily identified biallelic markers had < 50 per cent . thus , the authors proposed that ideal psas should have > 50 per cent and also indicated that for multiallelic loci , a composite could be estimated as one half the summation of the absolute value of allelic frequency differences for all alleles at that locus . it has also been shown that markers with lower values , of approximately 30 per cent , can provide up to 80 per cent power for detecting associations at distances of 5 cm with a large enough sample size ( n = 1,000 ) . pfaff et al . , suggested referring to markers suitable for admixture studies as ' ancestry informative markers ' ( aims ) , given that the central feature of these markers is the ancestry information content ( f ) . the present authors agree that the term aim more accurately describes these markers and does so using language that is less likely to be misunderstood and misinterpreted . marker information content ' f ' denotes the locus - specific fst and is a value representative of the differentiation between two populations at a single locus . simulation studies for estimating the information content of markers with varying levels of f have shown that for 1,000 markers with average information content for ancestry at 40 per cent between two ancestral subpopulations , approximately 80 per cent of the information about ancestry can be extracted from an initial genome screen . after initial identification of regions showing admixture , more markers can be typed in these regions to increase extraction of information to nearly 100 per cent . it is well established , however , that only 5 - 15 per cent of the total genetic variation results from differences among human populations [ 30 - 32 ] . moreover , most alleles are shared between populations , and alleles common in one population are also common in other populations . thus , most genetic markers are unaffected by admixture and it is imperative to choose markers that show high levels of d ( and f ) between the parental populations . recent studies by several groups have focused on identifying panels of markers suitable for admixture studies . one notable study screened 744 microsatellite markers for composite d values and llar in four different populations and identified a genome spanning set of 315 markers ( average spacing 10 cm , 0.3 ) for mapping in african americans and 214 markers ( average spacing of 16 cm , 0.25 ) for mapping in hispanics . a dna pooling method was used to identify 151 aims ( microsatellites and short insertion / deletion polymorphisms ) , with > 0.3 for mapping in mexican american populations to distinguish between european - american and native - american contributions . ninety - seven aims were identified for mapping in african - american populations that show limited variation within africa . additional resources are available for obtaining marker frequency , and genotype and haplotype information , from the snp consortium ( tsc ; http://snp.cshl.org ) , the national center for biotechnology information 's ' dbsnp ' website ( http://www.ncbi.nlm.nih.gov/snp ) , the marshfield database ( http://research.marshfieldclinic.org/genetics/default.htm ) and the ongoing hapmap project . since the amount of ald created is proportional to the level of admixture in a population , it is important briefly to review studies on admixture levels across populations . those populations that are likely to be useful for admixture studies include african americans , mexican americans , cubans and puerto ricans in the usa , african caribbeans , various latin american populations , various groups in central and south america and the caribbean islands , anglo indians in india and ' coloured ' populations of south africa . various statistical approaches have been used to estimate admixture proportions in these populations and have been reviewed in detail elsewhere . these include a least squares method , a weighted least squares method and likelihood methods . a recent review of admixture studies and admixture proportions of various latin american populations is provided by sans . african americans are a well - studied group with substantial european and west african contributions and a smaller native american contribution . a survey of current literature indicates that european admixture ranges from 3.5 per cent in the gullah sea islanders of south carolina , to 28 per cent in new orleans . admixture estimates in african - american populations can be highly variable across the usa , which is likely to reflect local variation in the demographic histories and social norms . us hispanics form a complex socio - political conglomerate including puerto ricans , cubans , spanish americans , mexican americans . the proportional contributions from parental europeans are estimated to be the largest , followed by a substantial native american ancestry and varying amounts of west african ancestry . in a sample of mexican americans from arizona , the admixture estimates obtained using a weighted least squares method showed 29 4 per cent native american , 68 5 per cent european and 3 2 per cent west african contribution . a recent study reports the following estimates for a hispanic population from the san luis valley , colorado : 62.7 2.1 per cent european , 34.1 1.9 per cent native american , 3.2 1.5 per cent west african . in puerto ricans from new york city , the estimates obtained were 53.3 2.8 per cent european , 29.1 2.3 per cent west african , 17.6 2.4 per cent native american . in a separate mexican - american population sample from california , european ancestry was estimated to be 60 per cent and native american contribution was estimated at 40 per cent . from these results , it is evident that , when studying any new admixed population sample , it is important to accurately determine the proportional contributions and not to rely on previously obtained estimates from a similar population . additionally , it is instructive to have information on the levels of stratification related to admixture that are present in the population under consideration . traits and diseases more prevalent in one population than in others are amenable to admixture analysis and some examples are listed in table 1 . most of the diseases shown in this table have a complex aetiology affected by multiple genes and environmental factors . earlier studies focused on admixed populations as units of analysis in exploring relationships between ancestry and phenotypes . these authors showed that non - insulin - dependent ( type 2 ) diabetes mellitus prevalence is correlated with admixture proportions among a selection of populations with varying levels of native american ancestry . data like these provide compelling evidence for frequency differences in risk modifying alleles , but such data have not been collected for many diseases . another related approach is to test for individual admixture - phenotype correlations within an admixed population . correlations between ancestry and phenotypes have been detected and reported by various authors [ 14,17 - 19,44,45,47 ] . a prerequisite for testing ancestry / phenotype correlations is the presence of stratification related to admixture , which will be evident in variation in individual ancestry levels . figure 2 shows the distribution of bga estimates from three examples of hispanic population samples , puerto ricans from new york , mexicans from tlapa , mexico and hispanics from the san luis valley , colorado . substantial variation is observed in all three samples . with the san luis valley group , more variability is observed on the european - native american axis , while the new york group is more variable on the european - west african axis . following the argument of chakraborty and weiss , admixture proportions should be correlated with diseases / traits that differ in populations due to underlying genetic differences . in each of these population samples , strong positive correlation was observed between individual ancestry and skin pigmentation measured as melanin index ' m ' or lightness index ' l ' ( figures 3a , 3b and 3c ) . a significant negative correlation was also observed between the proportion of west african ancestry and bone mineral density ( bmd ) in the puerto rican sample . proportion west african ancestry and skin pigmentation ( measured as melanin index ) in individuals is also correlated in african americans from washington dc and african caribbeans from the uk , but not in european americans from state college , pennsylvania ( figure 4 ) . recently , correlations have been observed between proportion west african ancestry and lower insulin sensitivity , higher fasting insulin and acute insulin response to glucose in a combined sample of african - american and european - american children . in a separate sample of african - american females , west african admixture is associated with body mass index , fat mass , fat - free mass and bmd . it is important to keep in mind that ancestry - pheno - type correlations are dependent on both the existence of functional alleles at different frequencies in parental populations , and significant stratification related to admixture . although most admixed populations tested to date are structured , there is variation in the amount of stratification present , and this structure should be tested for explicitly when investigating a new population . each vertex represents a parental population , which for this plot are europeans , west africans and native americans . the three populations shown are hispanics from the san luis valley ( blank circles ) , puerto ricans from new york city ( grey diamonds ) and mexicans from tlapa , mexico ( grey triangles ) ( adapted from bonilla , 2003 ) the relationship between proportional ancestry and skin pigmentation in three hispanic populations . for all populations , proportional ancestry was estimated using the maximum likelihood ( ml ) method ( adapted from bonilla , 2003 ) . ( a ) percent native american ancestry versus lightness index ( l ) in hispanics from the san luis valley , colorado ( ancestry estimated using 22 aims ) . ( b ) percent native american ancestry versus melanin index in mexicans from tlapa , mexico ( ancestry estimates using 29 aims ) . ( c ) percent african ancestry versus melanin index ( m ) in puerto ricans from new york city ( ancestry estimated using 35 aims ) the relationship between percent african ancestry and skin pigmentation in three populations . percent african ancestry ( obtained using 34 aims and calculated by the maximum likelihood ( ml ) method ) and the melanin index ( m ) are shown for three populations , european americans from state college , pennsylvania ( diamonds ) , african americans from washington , dc and state college , pennsylvania ( squares ) and african caribbeans from britain ( triangles ) . 2003 ) theoretical and experimental studies have explored the parameters that characterise and affect admixture studies . the acronym mald was proposed to designate the mapping method proposed originally by chakraborty and weiss , which exploited the long range allelic associations created through ald . parameters critical for mald include the genetic distance between markers and disease locus ( ) ; number of generations since admixture ( t ) ; proportion of admixture ( m ) from one parental population ; the allele frequency differential ( ) between parental populations ; and sample size ( n ) . simulation studies suggest that sample sizes of 200 - 300 patients , typed for 200 - 300 evenly spaced markers , each having allele frequency differentials > 0.3 , have a > 95 per cent chance of locating the causative gene , when there has been no new admixture from the parental population in the last four generations and no other sources of population structure or sample heterogeneity . other approaches proposed for using admixture include a method based on the transmission disequilibrium test ( tdt ) that assesses excess transmission of alleles derived from high - risk ancestors to affected offspring of parents who are heterozygous at the marker locus , containing one allele from each of two ancestral populations . a second tdt - based likelihood approach was developed that compared the transmission of haplotypes with non - transmission in affected offspring in an admixed population following a multipoint method . it obtained a likelihood statistic to determine the significance of various models under different scenarios . one fundamental limitation of mald as initially described and in its early extensions , is the effects of stratification on causing false - positive association . marker data at all loci are combined to estimate ancestry of alleles at each locus . when allelic ancestry at marker loci is known , this approach is analogous to a linkage analysis , hence the term am is more appropriate than mald for describing this method and to distinguish it from ld approaches . the underlying variation in ancestry of chromosomes of mixed descent is modelled to extract all of the information about linkage that is generated by admixture . for example , where a locus is assumed to account for variation in skin pigmentation between two parental groups , eg west africans and europeans , individuals can be classified according to whether they have 0 , 1 or 2 alleles of west african descent at this locus . by comparing these three groups for mean pigmentation level , holding all other factors constant , variation in pigmentation can be observed depending upon the number of alleles of west african ancestry in an individual . controlling for parental admixture eliminates association of the trait with ancestry at unlinked loci . by removing the background effects of ancestry , it is possible to observe the locus - specific effects on a trait / disease . allelic ancestry at a locus is inferred from the marker by using the conditional probability of each allelic state given the ancestry - specific allele frequencies . a complex hierarchical model with many nuisance parameters this is implemented using the admixmap program ( at http://www.lshtm.ac.uk/eph.eu/geneticepidemiologygroup/htm ) , which follows a bayesian approach with markov chain simulation , and incorporates the admixture of each individual 's parents and the random variation of ancestry on chromosomes inherited from each of the parents in the model . variation in individual admixture introduces population stratification , which in turn can inflate the number of significant associations that are observed and is a potential confounder in association studies [ 29,57 - 59 ] . various statistical approaches have been developed to detect and control for stratification within a population sample [ 14,15,17,42,60 - 62 ] . for example , the dt / d0 test examines the relationship between the observed ld and the predicted ald between unlinked marker pairs for detecting structure within the sample . using individual ancestry as a conditioning variable in analysis of variance tests the bayesian approaches implemented by mckeigue et al . and pritchard et al . offer an advantage over classical maximum likelihood based methods by allowing for missing genotype and ancestry data and modelling admixture hierarchically . methods have been developed to control for parental admixture and to account for uncertain bga estimation . admixture - based methods rely on using suitable markers and estimates of allele frequencies from appropriately identified parental populations . since ald is fairly new and extends over larger distances , fewer markers are required for am studies . markers informative for ancestry have been used in several contexts and have been referred to as ' ideal , ' ' private ' and ' unique ' . informativeness of such markers can be measured as the allele frequency differential ( ) , which is the absolute value of the difference of a particular allele between populations . microsatellites and insertion / deletion polymorphisms with > 0.3 were recently called ' ethnic - difference markers ' ( edms ) suitable for mapping by admixture linkage disequilibrium ( mald ) . additionally , markers with high and very high log likelihood allelic ratio ( llar ) between populations have been designated ' population specific alleles ' ( psas ) . this report followed from earlier work where markers with large allele frequency difference were identified to be appropriate for admixture studies , and most ( > 95 per cent ) of the arbitrarily identified biallelic markers had < 50 per cent . thus , the authors proposed that ideal psas should have > 50 per cent and also indicated that for multiallelic loci , a composite could be estimated as one half the summation of the absolute value of allelic frequency differences for all alleles at that locus . it has also been shown that markers with lower values , of approximately 30 per cent , can provide up to 80 per cent power for detecting associations at distances of 5 cm with a large enough sample size ( n = 1,000 ) . pfaff et al . , suggested referring to markers suitable for admixture studies as ' ancestry informative markers ' ( aims ) , given that the central feature of these markers is the ancestry information content ( f ) . the present authors agree that the term aim more accurately describes these markers and does so using language that is less likely to be misunderstood and misinterpreted . marker information content ' f ' denotes the locus - specific fst and is a value representative of the differentiation between two populations at a single locus . simulation studies for estimating the information content of markers with varying levels of f have shown that for 1,000 markers with average information content for ancestry at 40 per cent between two ancestral subpopulations , approximately 80 per cent of the information about ancestry can be extracted from an initial genome screen . after initial identification of regions showing admixture , more markers can be typed in these regions to increase extraction of information to nearly 100 per cent . it is well established , however , that only 5 - 15 per cent of the total genetic variation results from differences among human populations [ 30 - 32 ] . moreover , most alleles are shared between populations , and alleles common in one population are also common in other populations . thus , most genetic markers are unaffected by admixture and it is imperative to choose markers that show high levels of d ( and f ) between the parental populations . recent studies by several groups have focused on identifying panels of markers suitable for admixture studies . one notable study screened 744 microsatellite markers for composite d values and llar in four different populations and identified a genome spanning set of 315 markers ( average spacing 10 cm , 0.3 ) for mapping in african americans and 214 markers ( average spacing of 16 cm , 0.25 ) for mapping in hispanics . a dna pooling method was used to identify 151 aims ( microsatellites and short insertion / deletion polymorphisms ) , with > 0.3 for mapping in mexican american populations to distinguish between european - american and native - american contributions . ninety - seven aims were identified for mapping in african - american populations that show limited variation within africa . additional resources are available for obtaining marker frequency , and genotype and haplotype information , from the snp consortium ( tsc ; http://snp.cshl.org ) , the national center for biotechnology information 's ' dbsnp ' website ( http://www.ncbi.nlm.nih.gov/snp ) , the marshfield database ( http://research.marshfieldclinic.org/genetics/default.htm ) and the ongoing hapmap project . since the amount of ald created is proportional to the level of admixture in a population , it is important briefly to review studies on admixture levels across populations . those populations that are likely to be useful for admixture studies include african americans , mexican americans , cubans and puerto ricans in the usa , african caribbeans , various latin american populations , various groups in central and south america and the caribbean islands , anglo indians in india and ' coloured ' populations of south africa . various statistical approaches have been used to estimate admixture proportions in these populations and have been reviewed in detail elsewhere . these include a least squares method , a weighted least squares method and likelihood methods . a recent review of admixture studies and admixture proportions of various latin american populations is provided by sans . african americans are a well - studied group with substantial european and west african contributions and a smaller native american contribution . a survey of current literature indicates that european admixture ranges from 3.5 per cent in the gullah sea islanders of south carolina , to 28 per cent in new orleans . admixture estimates in african - american populations can be highly variable across the usa , which is likely to reflect local variation in the demographic histories and social norms . us hispanics form a complex socio - political conglomerate including puerto ricans , cubans , spanish americans , mexican americans . the proportional contributions from parental europeans are estimated to be the largest , followed by a substantial native american ancestry and varying amounts of west african ancestry . in a sample of mexican americans from arizona , the admixture estimates obtained using a weighted least squares method showed 29 4 per cent native american , 68 5 per cent european and 3 2 per cent west african contribution . a recent study reports the following estimates for a hispanic population from the san luis valley , colorado : 62.7 2.1 per cent european , 34.1 1.9 per cent native american , 3.2 1.5 per cent west african . in puerto ricans from new york city , the estimates obtained were 53.3 2.8 per cent european , 29.1 2.3 per cent west african , 17.6 2.4 per cent native american . in a separate mexican - american population sample from california , european ancestry was estimated to be 60 per cent and native american contribution was estimated at 40 per cent . as with african - american populations , there is substantial variation across populations . from these results , it is evident that , when studying any new admixed population sample , it is important to accurately determine the proportional contributions and not to rely on previously obtained estimates from a similar population . additionally , it is instructive to have information on the levels of stratification related to admixture that are present in the population under consideration . traits and diseases more prevalent in one population than in others are amenable to admixture analysis and some examples are listed in table 1 . most of the diseases shown in this table have a complex aetiology affected by multiple genes and environmental factors . earlier studies focused on admixed populations as units of analysis in exploring relationships between ancestry and phenotypes . these authors showed that non - insulin - dependent ( type 2 ) diabetes mellitus prevalence is correlated with admixture proportions among a selection of populations with varying levels of native american ancestry . data like these provide compelling evidence for frequency differences in risk modifying alleles , but such data have not been collected for many diseases . another related approach is to test for individual admixture - phenotype correlations within an admixed population . correlations between ancestry and phenotypes have been detected and reported by various authors [ 14,17 - 19,44,45,47 ] . a prerequisite for testing ancestry / phenotype correlations is the presence of stratification related to admixture , which will be evident in variation in individual ancestry levels . figure 2 shows the distribution of bga estimates from three examples of hispanic population samples , puerto ricans from new york , mexicans from tlapa , mexico and hispanics from the san luis valley , colorado . substantial variation is observed in all three samples . with the san luis valley group , more variability is observed on the european - native american axis , while the new york group is more variable on the european - west african axis . following the argument of chakraborty and weiss , admixture proportions should be correlated with diseases / traits that differ in populations due to underlying genetic differences . in each of these population samples , strong positive correlation was observed between individual ancestry and skin pigmentation measured as melanin index ' m ' or lightness index ' l ' ( figures 3a , 3b and 3c ) . a significant negative correlation was also observed between the proportion of west african ancestry and bone mineral density ( bmd ) in the puerto rican sample . proportion west african ancestry and skin pigmentation ( measured as melanin index ) in individuals is also correlated in african americans from washington dc and african caribbeans from the uk , but not in european americans from state college , pennsylvania ( figure 4 ) . recently , correlations have been observed between proportion west african ancestry and lower insulin sensitivity , higher fasting insulin and acute insulin response to glucose in a combined sample of african - american and european - american children . in a separate sample of african - american females , west african admixture is associated with body mass index , fat mass , fat - free mass and bmd . it is important to keep in mind that ancestry - pheno - type correlations are dependent on both the existence of functional alleles at different frequencies in parental populations , and significant stratification related to admixture . although most admixed populations tested to date are structured , there is variation in the amount of stratification present , and this structure should be tested for explicitly when investigating a new population . each vertex represents a parental population , which for this plot are europeans , west africans and native americans . the three populations shown are hispanics from the san luis valley ( blank circles ) , puerto ricans from new york city ( grey diamonds ) and mexicans from tlapa , mexico ( grey triangles ) ( adapted from bonilla , 2003 ) the relationship between proportional ancestry and skin pigmentation in three hispanic populations . for all populations , proportional ancestry was estimated using the maximum likelihood ( ml ) method ( adapted from bonilla , 2003 ) . ( a ) percent native american ancestry versus lightness index ( l ) in hispanics from the san luis valley , colorado ( ancestry estimated using 22 aims ) . ( b ) percent native american ancestry versus melanin index in mexicans from tlapa , mexico ( ancestry estimates using 29 aims ) . ( c ) percent african ancestry versus melanin index ( m ) in puerto ricans from new york city ( ancestry estimated using 35 aims ) the relationship between percent african ancestry and skin pigmentation in three populations . percent african ancestry ( obtained using 34 aims and calculated by the maximum likelihood ( ml ) method ) and the melanin index ( m ) are shown for three populations , european americans from state college , pennsylvania ( diamonds ) , african americans from washington , dc and state college , pennsylvania ( squares ) and african caribbeans from britain ( triangles ) . theoretical and experimental studies have explored the parameters that characterise and affect admixture studies . the acronym mald was proposed to designate the mapping method proposed originally by chakraborty and weiss , which exploited the long range allelic associations created through ald . parameters critical for mald include the genetic distance between markers and disease locus ( ) ; number of generations since admixture ( t ) ; proportion of admixture ( m ) from one parental population ; the allele frequency differential ( ) between parental populations ; and sample size ( n ) . simulation studies suggest that sample sizes of 200 - 300 patients , typed for 200 - 300 evenly spaced markers , each having allele frequency differentials > 0.3 , have a > 95 per cent chance of locating the causative gene , when there has been no new admixture from the parental population in the last four generations and no other sources of population structure or sample heterogeneity . other approaches proposed for using admixture include a method based on the transmission disequilibrium test ( tdt ) that assesses excess transmission of alleles derived from high - risk ancestors to affected offspring of parents who are heterozygous at the marker locus , containing one allele from each of two ancestral populations . a second tdt - based likelihood approach was developed that compared the transmission of haplotypes with non - transmission in affected offspring in an admixed population following a multipoint method . it obtained a likelihood statistic to determine the significance of various models under different scenarios . one fundamental limitation of mald as initially described and in its early extensions , is the effects of stratification on causing false - positive association . marker data at all loci are combined to estimate ancestry of alleles at each locus . when allelic ancestry at marker loci is known , this approach is analogous to a linkage analysis , hence the term am is more appropriate than mald for describing this method and to distinguish it from ld approaches . the underlying variation in ancestry of chromosomes of mixed descent is modelled to extract all of the information about linkage that is generated by admixture . for example , where a locus is assumed to account for variation in skin pigmentation between two parental groups , eg west africans and europeans , individuals can be classified according to whether they have 0 , 1 or 2 alleles of west african descent at this locus . by comparing these three groups for mean pigmentation level , holding all other factors constant , variation in pigmentation can be observed depending upon the number of alleles of west african ancestry in an individual . controlling for parental admixture eliminates association of the trait with ancestry at unlinked loci . by removing the background effects of ancestry , it is possible to observe the locus - specific effects on a trait / disease . allelic ancestry at a locus is inferred from the marker by using the conditional probability of each allelic state given the ancestry - specific allele frequencies . a complex hierarchical model with many nuisance parameters this is implemented using the admixmap program ( at http://www.lshtm.ac.uk/eph.eu/geneticepidemiologygroup/htm ) , which follows a bayesian approach with markov chain simulation , and incorporates the admixture of each individual 's parents and the random variation of ancestry on chromosomes inherited from each of the parents in the model . variation in individual admixture introduces population stratification , which in turn can inflate the number of significant associations that are observed and is a potential confounder in association studies [ 29,57 - 59 ] . various statistical approaches have been developed to detect and control for stratification within a population sample [ 14,15,17,42,60 - 62 ] . for example , the dt / d0 test examines the relationship between the observed ld and the predicted ald between unlinked marker pairs for detecting structure within the sample . using individual ancestry as a conditioning variable in analysis of variance tests , it is possible to eliminate association of the trait with unlinked alleles . offer an advantage over classical maximum likelihood based methods by allowing for missing genotype and ancestry data and modelling admixture hierarchically . methods have been developed to control for parental admixture and to account for uncertain bga estimation . several theoretical and practical studies indicate that am approaches promise to be suitable for identifying genes causing complex diseases . methodological advancements have been made to offset the potential problems arising from association between unlinked loci by conditioning on parental admixture , and to detect and correct for population stratification . use of bayesian am can take into consideration various uncertainties , including missing data values for estimating admixture proportions , and can overcome problems arising out of mis - specification of parental allele frequencies and promises to be an effective tool for admixture studies . this method , which is different from the classical disequilibrium - based approach that is more commonly used , is perhaps more suitable for disease gene mapping in admixed populations and has already been successfully used for mapping . table 2 summarises recent studies showing associations between ancestry and phenotypes / diseases and instances where am was used to identify genes . currently , the primary impediment to exhaustive am genome scans is the lack of verified aim panels . sufficient numbers of markers are available as candidate aims , but effort and resources are required to confirm these markers and to generate accurate parental allele frequencies . it seems inevitable that more such studies will be carried out in the near future to utilise the immense potential of this approach . diseases showing ancestry - phenotype correlation a = r ; b = risk ratio ; c = rank - order correlation . we thank dr paul mckeigue and dr esteban parra for helpful discussions on the subject . we also acknowledge helpful comments from an unknown reviewer . this work was supported in part by grants from nih

admixture is an important evolutionary force that can and should be used in efforts to apply genomic data and technology to the study of complex disease genetics . admixture linkage disequilibrium ( ald ) is created by the process of admixture and , in recently admixed populations , extends for substantial distances ( of the order of 10 to 20 cm ) . the amount of ald generated depends on the level of admixture , ancestry information content of markers and the admixture dynamics of the population , and thus influences admixture mapping ( am ) . the authors discuss different models of admixture and how these can have an impact on the success of am studies . selection of markers is important , since markers informative for parental population ancestry are required and these are uncommon . rarely does the process of admixture result in a population that is uniform for individual admixture levels , but instead there is substantial population stratification . this stratification can be understood as variation in individual admixtures and can be both a source of statistical power for ancestry - phenotype correlation studies as well as a confounder in causing false - positives in gene association studies . methods to detect and control for stratification in case / control and am studies are reviewed , along with recent studies showing individual ancestry - phenotype correlations . using skin pigmentation as a model phenotype , implications of am in complex disease gene mapping studies are discussed . finally , the article discusses some limitations of this approach that should be considered when designing an effective am study .

Introduction Admixture studies and their use in disease gene mapping Marker choice for admixture mapping Admixed populations and admixture proportions Ancestry-phenotype correlations; phenotype and complex disease gene mapping Methods developed for admixture analyses/study design Recent studies and future directions Acknowledgements

acyl - coa thioesterases ( acots ) represent a group of enzymes that metabolise acyl - coa esters to their corresponding non - esterified fatty acid and coenzyme a ( coash ) . in addition to ' acyl - coa thioesterases ' , these enzymes also have been described as ' acyl - coa hydrolases ' , ' acyl - coa thioester hydrolases ' and ' deacylases ' . the reaction catalysed by acot enzymes is very important during fatty acid metabolism . as such acyl - coa thioesterase expression is widespread and activity is detectable within many tissues and cell types . acot activity helps to regulate cellular pools , as well as proper ratios , of activated fatty acyl - coas ( acyl - coa esters ) , free fatty acids ( non - esterified fatty acids ) and coash . these enzymes are found within a number of subcellular locations , including peroxisomes , mitochondria and the cytosol . peroxisomal and mitochondrial acots are thought to play a major role in fatty acid degradation via -oxidation , which provides energy through the citric acid cycle , as well as in ketone body formation . in order for a fatty acid to be transported into the peroxisome for -oxidation , it must first be activated to its coenzyme a ester by acyl - coa synthetases . some acyl - coa esters undergo -oxidation much more slowly than others -- which can effectively sequester large amounts of coash and limit the activity of acyl - coa synthetases and the rate of subsequent fatty acid -oxidation . under these conditions , acots may act to increase the levels of free coash , thereby restoring fatty acid -oxidation . mammalian acots are capable of metabolising a wide variety of compounds , including short- , medium- and long - chain acyl - coas . they can also metabolise polyunsaturated fatty acyl - coas , branched - chain acyl - coas and aromatic - acyl - coas . these compounds are not only utilised during -oxidation and fatty acid degradation , but also have roles in inflammation , ion channel opening , signal transduction , lipid biosynthesis and gene regulation . ten human acot genes have been identified to date , compared with 13 in mice and 12 in rats . these genes can be divided into two groups , based upon differences in sequence and structure . these enzymes are members of the /-hydrolase fold enzyme superfamily , which includes other esterase - activity - exhibiting enzymes such as carboxylesterases and lipases . type ii acots make up the second group ; these enzymes are members of the ' hot dog ' fold enzyme superfamily , which includes a wide variety of functionally diverse proteins . despite catalysing the same reaction , type i and type ii enzymes share no similarity in terms of structure or sequence this observation is reminiscent of the cytochrome p450 ( cyp ) and nitric oxide synthase ( nos ) gene families . studies with purified nos proteins revealed that they possess p450-like activity ; however , detailed alignment analyses showed < 15 per cent amino acid identity , thereby confirming that these two gene families were not evolutionarily related . it was thus concluded that this was an example of convergent evolution . in other words , during the evolution of enzyme activities needed at a particular subcellular location ( probably more than 1 billion years ago ) mother nature needed a ' p450-like activity ' and assigned that task to the nearest protein , which happened to be an nos . the revised nomenclature for human , mouse and rat acyl - coa thioesterase genes was established by hunt et al . and has been accepted by the hugo gene nomenclature committee . in recent years , a number of human acots have been recombinantly expressed and enzymatically characterised , although the exact physiological function and importance of many of these enzymes remain unknown . to date , a total of ten genes have been identified within the human genome , all of which exhibit acyl - coa thioesterase activity ( table 1 ) . these ten genes can be sub - divided into two distinct groups , type i and type ii , based on sequence and structural homology . type i acots comprise the smaller group , which contains only four genes : acot1 , acot2 , acot4 and acot6 . the larger group includes six genes , and these members are referred to as type ii . there is no sequence homology between the two groups , and the translated proteins have very different domain architecture , indicating two distinct , evolutionarily unrelated , origins ( figure 1 ) and , most likely , an example of convergent evolution . list of all human ( acot ) with full gene name , aliases , chromosomal location , isoforms , ncbi refseq mrna accession number , ncbi refseq protein accession number and total amino acid number ( aa # ) domain organisation of human type i and type ii acyl - coa thioesterases ( acots ) . abbreviations : acth , acyl - coa thioester hydrolase domain ; baat , bile acid coa : amino acid n - acyltransferase ; el , esterase - lipase domain ; hd , ' hot dog ' fold domain ; start , steroidogenic acute regulatory protein ( star)-related lipid transfer ( start ) domain ; them , thioesterase superfamily member . the type i proteins , acot1 , acot2 , acot4 and acot6 , share a high degree of sequence homology ( table 2 ) . acot1 and acot2 are the most closely related , having 98 per cent amino acid sequence identity . acot6 is the least homologous , but still shares 54 - 57 per cent sequence identity with the other members . the four type i genes form an 80 kilobase gene cluster on chromosome 14q24.3 , indicating that they most likely have arisen as the result of gene duplication . amino acid sequence identity between type i and type ii human acyl - coa thioesterases ( acots ) . homology was considered not significant and left blank if the observed identity was less than 15 per cent abbreviations : baat , bile acid coa : amino acid n - acyltransferase ; them , thioesterase superfamily member a similar situation is observed within the mouse and rat orthologues acot1 , acot2 , acot3 , acot4 , acot5 and acot6 , which are clustered on chromosomes 12 d3 and 6q31 , respectively . a cladogram depicting the evolutionary relationships observed between human , mouse and rat type i proteins is shown in figure 2a . this cladogram clearly shows that , prior to the mammalian radiation approximately 75 million years ago ( mya ) , there was a single ancestral acot1 ( or -2 ) gene ; after the mammalian radiation but before the mouse - rat split at approximately 15 mya , this single ancestor divided twice to become four rodent acot functional genes ( now named acot1 , acot3 , acot4 and acot5 ) . by contrast , after the mammalian radiation , the human single ancestor duplicated into a second functioning gene , leading to acot1 and acot2 . on the other hand , acot4 and acot6 existed before the mammalian radiation and cladograms depicting the evolutionary relationships between human , mouse ( m ) and rat ( r ) orthologous protein sequences for type i ( a ) and type ii ( b ) acots . human carboxylesterase 1 ( ces1 ) is an unrelated esterase that was added simply to ' ground ' the tree . in the mouse , the acot3 , acot4 and acot5 enzymes each exhibit specific expression patterns and enzyme substrate specificity . the lack of a human orthologue to mouse acot3 and acot5 was suggested to be the result of convergent evolution ; it was hypothesised that convergent , functional evolution led to a reduced need for multiple enzymes in humans . the protein basic local alignment search tool ( blastp ) analysis of all non - redundant protein sequences , however , failed to identify orthologues for acot3 or acot5 in non - rodent species . this finding suggests that acot4 was an ancestral gene encoding an acot4 enzyme with broad substrate recognition , and that gene duplication events led to acot1 ( or -2 ) , and that further duplication events led to the mouse acot3 and acot5 genes , encoding the acot3 and acot5 enzymes , which have similar substrate specificity to that of mouse acot4 and human acot4 . analysis of the genomic acot6 sequence indicates transcription of a complete mrna containing three exons ; however , a full - length mrna is not detectable . subsequent translation begins at a methionine located within the second exon , producing a truncated protein that lacks the n - terminal acyl - coa thioester hydrolase domain found within other type i proteins . the truncated protein contains all residues required for catalysis and could therefore still produce an active acyl - coa thioesterase . the blast results indicate that bile acid coa : amino acid n - acyltransferase ( baat ) is the only enzyme to share significant sequence homology with type i acots . baat is known to participate in the transfer of bile acids from the acyl - coa thioester to either glycine or taurine , which are used for bile salt secretion . baat contains the same domain architecture and acot1 , acot2 and acot4 ; however , the catalytic triad found within the esterase - lipase domain contains a cysteine , instead of the highly conserved serine residue . this substitution may explain the difference in activity ( ie transferase versus hydrolase ) observed between the two enzymes . despite sharing catalytic domains , other /-hydrolase fold enzyme superfamily esterases , such as carboxylesterase 1 ( ces1 ) and acetylcholinesterase ( ache ) , share very little sequence homology with their acyl - coa thioesterase relatives . ces1 has thus been added as an evolutionarily unrelated gene to figure 2a simply to ' ground ' the dendrogram . none of the type ii proteins shares more than 25 per cent identity and many do not share any significant sequence similarity ( table 2 ) . unlike the type i genes , type ii acots are widely distributed throughout the genome . with the exception of acot13 , all type ii proteins contain double ' hot dog ' domains , which may have evolved as a gene duplication event that allowed for the accommodation of bulky substrates . acot7 shares 19 per cent identity with acot9 , 22 per cent with acot11 and 24 per cent identity with acot12 . acot11 and acot12 share 51 per cent sequence identity and both enzymes contain a c - terminal steroidogenic acute regulatory protein ( star)-related lipid transfer ( start ) domain , which seems to be specific to mammalian evolution . the n - terminal region of both acots is unrelated to any of the other start - containing enzymes , suggesting that these proteins may be the result of a gene fusion event between an acyl - thioesterase and start domain - containing sequences . like acot8 , blast results did identify 22 per cent sequence identity with thioesterase superfamily member 4 ( them4 is the official name ) -- also known as akt c - terminal modulator protein ( ctmp ) . this small degree of homology suggests that the two proteins may share common evolutionary ties . another protein , thioesterase superfamily member 5 ( them5 ) shares more than 37 per cent identity with them4 but lacks significant sequence homology with acot13 . similar to acot13 , both them4 and them5 contain a single ' hot dog ' domain . furthermore , type ii proteins , them4 and them5 do not share sequence homology with other ' hot dog ' domain superfamily members . the ' hot dog ' fold - containing proteins possess highly conserved structural elements that are created by highly divergent sequences . this high degree of divergence makes evolutionary comparisons difficult without three - dimensional structures , because conservation of structural interactions is not directly associated with residue conservation . a cladogram depicting the evolutionary relationships observed between human , mouse and rat type ii protein is shown in figure 2b . the mouse genome contains an additional type ii acot , acot10 , which shares approximately 95 per cent mrna nucleotide identity with acot9 . unlike acot3 and acot5 , however , which are found in both mouse and rat , acot10 has only been identified in mice , indicating that the gene duplication event occurred less than 17 mya -- that is , after the mouse - rat split . in mouse , acot9 is located at chromosomal location x f3 and acot10 is found on 15 a2 . the amplification and movement of genes to a new chromosomal location is often facilitated by transposable elements , as opposed to duplications occurring within close proximity on a chromosome , which are attributed to errors during meiosis . the remaining seven type ii acot genes are highly conserved among human , mouse and rat , confirming that these genes were all present in the ancestor prior to the mammalian radiation . type i and type ii acyl - coa thioesterases represent two structurally distinct enzyme groups . an unrooted dendrogram -- created using protein sequences from both type i and type ii enzymes , closest - related homologues ( baat , them4 and them5 ) , distant /-hydrolase fold homologues ( ces1 , ache ) and -actin ( actb ) as an unrelated sequence -- exemplifies how dissimilar the two groups are from one another ( figure 3 ) . it also illustrates how the type ii proteins must have originated from a common , evolutionarily distant , predecessor ( as reflected in individual branch proximity to the origin of the tree ) . bile acid acetyl co - a : amino acid n - acyltransferase ( baat ) , thioesterase superfamily members 4 and 5 ( them4 and them5 ) are the closest - related homologues to type i ( baat ) and type ii sequences ( them4 and them5 ) . type i acot branches are highlighted in blue , whereas type ii are in red . acot1 , acot2 and acot4 contain an n - terminal acyl - coa thioester hydrolase domain ( pfam 04775 ) . this domain does not participate directly in catalysis , but studies indicate that the n - terminus plays an important role in regulating enzyme activity . all type i proteins also contain a c - terminus esterase - lipase superfamily domain . all residues required for catalysis are located within this domain , which is also known as the /-hydrolase fold . proteins that share this common structural element make up the functionally diverse /-hydrolase fold superfamily of enzymes . within this family are a number of other distantly related proteins that exhibit esterase activity , including carboxylesterases , ache and lipases . the superfamily has diverged greatly and many members share very little sequence homology but are considered evolutionarily linked , based on structural homology . the enzyme activity is dependent on a catalytic triad composed of highly conserved serine , histidine and aspartate residues found within the active site . the crystalline structure for acot2 indicates that the enzyme is most likely biologically active as a monomer . the structure also reveals that the n - terminal acyl - coa thioester hydrolase domain is connected to the active site by a long loop ; it is postulated that this contributes substrate specificity , although this has yet to be verified experimentally . genomic analysis suggests that acot6 should encode a similar 3 exon transcript ; however , the only detectable transcript is truncated and utilises a start codon located within the 3 ' end of exon two , resulting in the synthesis of a truncated protein . in addition to the four protein - coding genes , there is one intronless pseudogene , referred to as acot4p , located on chromosome 19q13.12 . acotp4 is most likely the result of retroviral action that has inserted this dna segment elsewhere in the genome . acot1 is cytosolic and primarily responsible for the metabolism of long - chain acyl - coas . acot2 and acot1 can metabolise a similar range of substrates ; however , acot2 contains an n - terminal mitochondrial targeting signal which directs its transport to mitochondria . acot4 contains a c - terminal peroxisomal targeting signal , directing transport to peroxisomes , where it exhibits high activity against succinyl - coa , as well as a wide variety of short - chain dicarboxylic - coas . as previously mentioned , acot6 lacks the n - terminal acyl - coa thioester hydrolase domain . although studies have yet to confirm the enzymatic activity of acot6 , the fact that it contains a complete c - terminal esterase - lipase domain supports its role as a functional esterase . acot6 is predicted to be cytosolic , due to a missense mutation which alters the peroxisomal targeting signal that is found in the acot6 mouse ortholog . the subcellular localisation and substrate specificity for each enzyme suggest that each plays an important , non - redundant function within the cell . the type ii enzymes are more distantly related than type ii acots , as indicated by the relatively low degree of sequence conservation observed between proteins . despite the low degree of sequence homology , the enzymes are structurally related and capable of metabolising similar substrates . acot7 , formerly called brain acyl - coa hydrolase ( bach ) , has been extensively characterised , owing to recent studies that indicate arachidonoyl - coa as a preferred substrate . the metabolism of arachidonoyl - coa by acot7 gives rise to arachidonic acid and coenzyme a. arachidonic acid serves as a precursor for a number of compounds associated with inflammation , including prostaglandins , leukotrienes and thromboxanes . acot8 is found in peroxisomes , where it metabolises a diverse range of acyl - coa compounds . acot8 also has high activity towards branched- and methyl - branched chain acyl - coas . acot9 contains a mitochondrial - targeting signal and metabolises medium- to long - chain acyl - coas . acot9 exhibits the highest activity towards myristoyl - coa , which can then be used by n - myristoyltransferase to modify proteins posttranslationally . expression of mouse acot11 , formerly referred to as brown fat - inducible thioesterase ( bfit ) , increases after cold shock . this protein was found to play a role in regulating thermogenesis , which helps to maintain body temperature . the start domain in animals is commonly found within homeodomain transcription factors , where lipid - binding regulates transcription . the binding of lipids to the start domain in acot11 or acot12 acts as a mechanism for regulating activity and for allowing for rapid enzyme activation . acot13 , previously called thioesterase superfamily member 2 ( them2 ) , differs from the other type ii acots , in that it contains a single ' hot dog ' domain . were recently determined using recombinant protein ; results indicate that acot13 prefers medium- to long - chain - acyl - coa thioesters . this finding suggests that the start domains , identified in acot11 and acot12 , could have a similar effect on enzyme activity . recent in vitro kinetic activity studies revealed that them4 is a broad - range , high - activity acyl - coa thioesterase . similar to acot13 , them4 preferentially metabolises medium- to long - chain acyl - coa thioesters . as such , we recommend that the enzyme be renamed acot14 according to the accepted nomenclature established by hunt et al . . acyl - coa thioesterase activity has not been verified for them5 ; however , activity is probably based on the high degree of sequence identity with them4 , but the exact role that this protein might play in fatty acid metabolism has yet to be determined . the human acyl - coa gene family represents a diverse group of enzymes that catalyse the hydrolysis of acyl - coa thioesters to their corresponding free fatty acids and coenzyme a. these enzymes are involved in a number of cellular processes but are primarily thought to play an important role in lipid metabolism . despite catalysing the same enzymatic reaction , the acyl - coa thioesterase family members can be divided into two distinct groups , type i and type ii . the two groups do not share common structural elements or sequence homology and are therefore considered analogous , rather than homologous , enzymes ; this is probably another example of convergent evolution . type i proteins belong to the /-hydrolase fold superfamily , whereas type ii proteins fall into the ' hot dog ' fold superfamily . to date , four type i and six type ii genes have been identified within the human genome . there are two additional genes , them4 and them5 , which gene products share both structural and sequence homology with other type ii proteins . them4 was identified as having acyl - coa thioesterase activity and should be considered as a new family member . in the future , perhaps them5 should also be considered as a type ii acot family member . recent studies have greatly expanded the current understanding of acyl - coa thioesterases ; however , many precise physiological functions are not completely understood and warrant further investigation . we would like to thank our colleagues for valuable discussion and critically reviewing this manuscript . this work was supported , in part , by the following nih grants ; r01ey17963 and r21aa017754 ( v.v . ) and p30es06096 ( d.w.n . ) .

the acyl - coa thioesterase gene ( acot ) family encodes enzymes that catalyse the hydrolysis of acyl - coa thioester compounds , also known as activated fatty acids , to their corresponding non - esterified ( free ) fatty acid and coenzyme a ( coash ) . these enzymes play a very important role in lipid metabolism by maintaining cellular levels and proper ratios of free and activated fatty acids , as well as coash . within the acyl - coa family there are two distinct subgroups , type i and type ii . despite catalysing the same reaction , the two groups are not structurally similar and do not share sequence homology , strongly suggesting convergent evolution . this suggestion is further supported if one compares the human with the mouse and rat acot gene families . to date , four human type i acots have been identified which belong to the /-hydrolase fold enzyme superfamily . type ii acots fall into the ' hot dog ' fold superfamily . there are currently six human type ii genes ; however , two homologous proteins , thioesterase superfamily members 4 ( them4 ) and 5 ( them5 ) share common type ii structural features and , in the case of them4 , acyl - coa thioesterase activity -- suggesting that the family may be larger than previously realised . although recent studies have greatly expanded the current understanding of these proteins and their physiological importance , there are a number of members whose functions are relatively unexplored and which warrant further investigation .

Introduction Human Type I ACOT structures Type II ACOT structures Conclusions Acknowledgements

a colony was established from soybean and cowpea field collections of n. viridula in stoneville , ms , during the spring and the summer of 2010 . this colony has been maintained in culture for 5 years at the usda - ars national biological control laboratory . after approximately 40 generations in culture , three orange morph adult males were detected in the colony . the orange morph males were paired with green females at a two females per male sex ratio to represent a parent population ( f0 ) , and allowed to reproduce using the rearing methods described by rojas and morales - ramos ( 2014 ) . cages constructed from clear plastic boxes ( l 320 w 260 h 100 mm , part no . 048-c , pioneer packing , dixon , ky ) were modified with ten windows ( 27 mm dia . ) on the sides and four windows ( 65 mm dia . ) on the top all covered with nylon screen ( mesh 500 m ) . all adults in this study were reared in an environmentally controlled room at 26 1 c , 50 5% rh , and 14 h photophase and provisioned with peanuts , broccoli , a diet supplement , and fresh bean pods and a diet supplement that was replaced at 3 days intervals . eggs were collected daily and hatching nymphs ( f1 ) were allowed to develop under same conditions . the number of males and females of each coloration type was recorded for each generation in this study . resulting f1 adults were paired and reared at the same conditions to obtain the f2 generation . resulting f2 adults consisted of green females and green and orange males . only f2 orange males ( app . 25% of the total numbers ) were retained to mate with f2 females ( at two females per male sex ratio ) to produce the f3 . eggs were collected daily and enclosing nymphs were reared to the adult stage under the same conditions described above . because the f3 colony had become considerably larger , only resulting f3 adults from the first three emerging dates were counted and sexed . phenotype and sex frequencies observed in each generation were compared with frequencies expected , assuming that the orange color phenotype was determined by a single sex - linked recessive allele as reported by follett et al . the test was used to compare observed versus expected frequencies within each generation cross . resulting f3 adults were divided to form two different colonies : orange type and green type . the pure orange - type colony was established using solely f3 orange - type adults . the f4 pure orange - type colony was used to generate a pure orange - type f5 . the reproductive potential of the f5 pure orange colony was compared with that of the pure green stock ( parental ) colony . newly emerging f5 orange - type adults were collected and grouped in cohorts of the same emerging date . a total of three cohorts were obtained from the green - type stock colony ( 209 adults total ) and four cohorts from the f5 orange type ( 168 adults total ) . adults were reared as described above and were monitored for oviposition and mortality , which were recorded daily . egg masses collected each day were recorded , labeled , and allowed to develop for 3 days before counting the eggs . embryos in developing eggs at 3 days old showed noticeable red eye coloration , which made them visible and allowed discrimination between fertilized and unfertilized eggs . the fertility ratio was calculated as fertile eggs / total eggs and fertility ratio was compared between color types using the z test . the mean number of eggs per mass was compared between treatments using anova and the student s t test . fertility tables were produced for each type using the methods reported by portilla et al . the f1 generation resulted in only green morphs ( 590 and 684 ) as expected from a recessive orange allele of single gene character . however , the f2 generation produced 672 green females , 351 green males , 298 orange males , and no orange females . these ratios were not significantly different to those expected for a recessive allele of a sex - linked gene ( table 1 ) and were consistent with follett et al . the first three cohorts of the f3 generation consisted of 345 green females , 346 green males , 100 orange females , and 85 orange males . the difference between observed and expected ratios were significant ( =8.067 , df = 3 , p = 0.046 ) ( table 1 ) , but this difference could be explained by significant deviations from the expected sex ratio of the f3 orange types ( |z| = 2.38 , p = 0.0087 ) resulting in a slight bias of 1.17 females per male . a similar female bias sex ratio was observed by follett et al . ( 2007 ) and this bias was explained as higher mortality in males occurring during their development . table 1.expected progeny ratios and observed frequencies in resulting f1 , f2 , and f3 generations after cross of orange males and green females in parent generation ( f0).expected phenotype ratios in progenyobserved phenotype frequencies in progenygeneration : parent and ( progeny)assumed genotypesg g o o g g o o f0 ( f1)xx x xy0.50.500590684006.936f1 ( f2)xx x xy0.50.2500.2567235102984.653f2 ( f3)0.5 xx x xy + 0.5 xx x xy0.3750.3750.1250.125345346100858.067*degrees of freedom ( df ) = 3 ; values with * show significant deviation from expected ratios.f2 green males were removed leaving only orange males to cross with green females . expected progeny ratios and observed frequencies in resulting f1 , f2 , and f3 generations after cross of orange males and green females in parent generation ( f0 ) . degrees of freedom ( df ) = 3 ; values with * show significant deviation from expected ratios . mean oviposition rate ( sem ) measured as eggs per female per day was significantly higher in green - type stock colony females ( 2.76 0.29 ) as compared with f5 orange - type females ( 1.17 0.27 ) ( |t| = 2.456 ; df = 222 ; p = 0.0148 ) . there was no significant difference in the number of eggs per egg mass between the parental stock green ( 27.14 1.26 ) and f5 orange ( 26.39 1.62 ) types . also , the proportion of fertile eggs was not significantly different between green ( 0.718 0.0094 ) and orange ( 0.726 0.0058 ) types . however , green - type females produced significantly higher number of egg masses per day ( 2.09 0.16 ) than orange type females ( 0.71 0.15 ) ( |t| = 6.14 ; df = 222 ; p < 0.0001 ) . the reproductive output of green - type females was higher as measured by the ro value ( 20.67 ) compared with orange - type females ( 13.71 ) ( fig . in addition to the reduced expected phenotypic ratio in natural populations ( consisting mostly of males due to the sex linked and recessive nature of the orange allele ) , the lower reproductive output by orange types may be an explanation for their rarity in natural populations . 1.cumulative lxmx ( reproductive output ) from fertility tables of green and pure orange types of nezara viridula . cumulative lxmx ( reproductive output ) from fertility tables of green and pure orange types of nezara viridula .

nezara viridula adult coloration can vary , including a rare orange - colored type ( i.e. , n. viridula f. aurantiaca ) . in november 2015 , three nezara viridula males displaying orange coloration were found in an established colony in stoneville , ms . the objectives of this study were to determine if alleles of these orange types conformed to the allele characteristics previously reported for n. viridula f. aurantiaca and to determine if there were any differences in reproductive output compared with the green - colored type . the three orange - type males were crossed with green - type females to produce a hybrid f1 . the f1 progeny was allowed us to cross to produce an f2 . the f2 progeny consisted of 672 green females , 351 green males , 298 orange males , and 0 orange females . these ratios did not differ significantly from the expected 50:25:25:0 ratios for a single recessive sex linked allele for color phenotype . the f2 cross of green females and orange males produced an f3 consisting of 345 green females , 346 green males , 100 orange females , and 85 orange males . these ratios also conformed to the expected ratios ( 0.375:0.375:0.125:0.125 ) with the exception of orange males , which numbers were slightly lower than expected . the pure orange type n. viridula produced significantly less egg masses ( 0.71 0.15 ) per day than green types ( 2.09 0.16 ) and their reproductive output , measured as net reproductive rate ( ro ) , was lower in orange ( 13.71 ) compared with green ( 20.67 ) types .

Materials and Methods Results and Discussion

peritoneal dialysis ( pd ) therapy has increased in popularity since the end of the 1970s . the method was developed as an alternative to hemodialysis ( hd ) presenting a patient survival rate equivalent to hd and better preservation of residual renal function . currently , the two principal causes of technique failure in order of importance are ( a ) peritonitis , this important medical problem can also represent nearly 16% of the causes of death ; ( b ) ultrafiltration failure , a multifactorial complication that can affect up to 40% of patients after 3 years of therapy . these proinflammatory stimuli can induce lymphokine secretion by macrophages , which in turn , activate fibroblasts . fibroblast activation has been associated with structural alterations in the peritoneal membrane of varying intensity . these alterations can be seen in figure 1 which was extracted from a submitted study of our group . in this prospective controlled study in 20 nonuremic wistar rats , peritoneal fibrosis occurs after exposure to glucose - based pd solutions and regardless the use of simvastatin . eps is a clinical syndrome that leads to persistent or recurrent intestinal obstruction , with or without inflammatory parameters of peritoneal thickening , sclerosis , calcification , and encapsulation , and can be inferred by clinical symptoms and radiology , but confirmed only by direct visualization with laparotomy [ 2 , 3 ] . incidence of eps is heterogenous and has been reported to vary from 6 to 20% in eight years depending on the region . causes of inflammation in peritoneal dialysis range from traditional factors to those related to chronic kidney disease per se as well as from the peritoneal dialysis treatment itself . uremia is a factor present in all pd patients and generates an inflammatory state causing stress on the peritoneum due to the formation of carbonyl products . it accelerates the formation of advanced glycation end products ( ages ) that induces an upregulation of the receptors of advanced glycation end products ( rage ) . the peritoneal dialysis catheter is the first proinflammatory factor associated to pd with which the patient comes into contact . after implantation in the peritoneum , the catheter can induce an inflammatory reaction as was demonstrated by flessner et al . . in addition , the catheter can occasionally be the site of bacterial biofilm formation . several pd solutions are available on the market today , and all are , to varying degrees , associated with peritoneal inflammation . such inflammation is generated by several characteristics of these solutions , varying from low ph , presence of lactate , hyperosmolality , increased glucose concentration , presence of glucose degradation products ( gdp ) and advanced glycation end products ( ages ) , and icodextrin metabolites , among others [ 6 , 7 ] . currently available glucose - based pd solutions the glucose load offered daily by a traditional pd prescription usually ranges from 120 g to 400 g. the majority of pd solutions prescribed today markedly acidify ph to nearly 5.7 in approximately 2 to 3 minutes . this ph decreases viability of neutrophils and mesothelial cells , thus decreasing cytokine production and phagocytosis capacity . its bioincompatibility with the peritoneal membrane is well known as well as its capacity to stimulate the production of fibroblast growth factors contributing to peritoneal fibrosis . some studies have associated the osmotic agent with eps development , while others have shown it to be distinct , confirming its safety even with long - term utilization . even experimental studies with rats addressing this question are compromised by the increased -amylase activity in these animals . the presence of this enzyme in plasma and in the peritoneal cavity provokes a rapid drop in peritoneal icodextrin concentration . chronic exposure to high glucose load in traditional pd solution induces significant inflammation of the peritoneal membrane . these solutions induce several proinflammatory factors such as pga , vascular endothelial growth factors ( vegfs ) , fibroblast growth factor ( tgf-1 ) , ages , and upregulation of rages . glucose degradation products ( gdps ) , such as methylglyoxal , glyoxal , and 3-deoxyglucosone generated during the heat sterilization process , increase inflammation by inducing oxidative stress , which thus causes damage to mesothelial cells and leads to apoptosis and mesothelial denudation . substituting traditional solutions for more biocompatible solutions it has been suggested for some years that the pathway of transforming growth factor 1/smad plays a part in the development of peritoneal fibrosis . the latter is recognized as playing a role in angiogenesis , a histological characteristic that allows for differentiation from simple peritoneal fibrosis to eps . the endothelial system is another known factor with potent profibrotic characteristics and plays a role in the development of peritoneal fibrosis . this system can be activated by two receptors , endothelial receptors a and b. however , endothelial receptor b apparently does not play a role in peritoneal membrane thickening in experimental studies inducing deficiency of endothelial receptor b. finally , and of extreme importance , infectious peritonitis is an obvious cause of peritoneal inflammation and is associated with eps development . gram - positive organisms remain as the more prevalent peritonitis agents over the past decades representing up to 60% of cases followed by gram - negative organisms . however , the prevalence of peritonitis due gram - negative organisms is growing fast with the development of efficient strategies to control gram - positive infections . despite all efforts made over the past decades , all the above - mentioned factors contribute to the release of proinflammatory cytokines such as interleukin 1 ( il 1 ) , tumor necrosis factor ( tnf- ) , il-6 , and il-18 . thickening and cubic transformation of mesothelial cells occurs and is more accentuated in the parietal peritoneum . human peritoneal mesothelial cells ( hpmcs ) also suffer structural alterations and prominent transdifferentiation of hpmc to myofibroblasts occurs . histological alterations of the peritoneal membrane observed in eps cases are nonspecific and are masked by the alterations commonly observed in patients with ultrafiltration failure and infectious peritonitis over the long term . the most common findings are fibrin deposition , fibrous capsule formation , perivascular bleeding , interstitial fibrosis , and the presence of tissue granulation with vascular proliferation . submesothelial tissue thickening also occurs with an increase in deposition of mesothelial conjunctive tissue [ 19 , 20 ] . fibrosis is characterized by the accumulation of extracellular matrix ( ecm ) , resulting in disequilibrium between synthesis and degradation . expression of collagen types 1 and 3 is significantly increased as well as collagen type 4 . one of these clinical manifestations is ultrafiltration ( uf ) failure and can occur in up to 30% of patients on pd after five years of treatment . one of the presentations of uf failure occurs due to the increase in pores in the peritoneal membrane , which in turn accelerates small - solute transport dissipating the osmotic gradient necessary to maintain adequate fluid balance . this increase in vascular surface is observed in conjunction with an increase in density of interstitial fibers . these findings help justify the increase in transport of small molecules , while the alterations in the uf coefficient are only moderate . in addition to uf failure , clinical manifestations such as severe malnutrition , subocclusion or intestinal occlusion , and ascites suggest the presence of eps even after discontinuation of pd . prescribing more hypertonic glucose solutions is a common strategy to counter this drop in uf , primarily where there is no available icodextrin . this intensifies and perpetuates inflammatory disturbances , with a direct impact on dialysis adequacy and fluid balance . the final consequence is the inevitable transfer to hd . despite all damage to the peritoneal membrane with therapies performed today , large observational studies have shown an important evolution in pd patient survival when compared to hd over the past years . pd initiation increases inflammatory stimuli for the chronic kidney patient such as the presence of the peritoneal catheter , use of bioincompatible solutions , and possible infectious peritonitis . these manifestations are frequently observed and can range from difficulties in obtaining an adequate fluid balance until the dreaded encapsulant peritoneal sclerosis . an understanding of the mechanisms involved in peritoneal inflammation is fundamental for the development of new strategies . this knowledge can provide not only a better technique survival , but also improvements in patient survival and a better quality of life .

peritoneal dialysis therapy has increased in popularity since the end of the 1970s . this method provides a patient survival rate equivalent to hemodialysis and better preservation of residual renal function . however , technique failure by peritonitis , and ultrafiltration failure , which is a multifactorial complication that can affect up to 40% of patients after 3 years of therapy . encapsulant peritoneal sclerosis is an extreme and potentially fatal manifestation . causes of inflammation in peritoneal dialysis range from traditional factors to those related to chronic kidney disease per se , as well as from the peritoneal dialysis treatment , including the peritoneal dialysis catheter , dialysis solution , and infectious peritonitis . peritoneal inflammation generated causes significant structural alterations including : thickening and cubic transformation of mesothelial cells , fibrin deposition , fibrous capsule formation , perivascular bleeding , and interstitial fibrosis . structural alterations of the peritoneal membrane described above result in clinical and functional changes . one of these clinical manifestations is ultrafiltration failure and can occur in up to 30% of patients on pd after five years of treatment . an understanding of the mechanisms involved in peritoneal inflammation is fundamental to improve patient survival and provide a better quality of life .

1. Introduction 2. Causes of Inflammation in PD 3. Structural Consequences of Inflammation of PD 4. Functional Consequence of Inflammation in PD 5. Conclusion

the incidence of this cancer varies throughout the world with the highest rates in developed communities ( 1 ) . the gold standard for detection and surveillance of bladder cancers are cystoscopy and urine cytology ( 2 ) . despite its benefit , cytology has low sensitivity , particularly for detection of low grade cancers and cystoscopy is a costly , invasive procedure . hence , there is a need to develop other simple , non - invasive diagnostic methods that can be used to prolong the intervals between cystoscopies and for disease monitoring ( 3 ) . an ideal bladder cancer screening and monitoring test should be non - invasive , easy to perform , and have high sensitivity and specificity . in recent years various markers these include bladder tumor antigen ( bta ) , nuclear matrix protein 22 ( nmp22 ) , survivin , telomerase , and cytokeratins ( cks ) ( 2 , 4 ) . cks are a family of more than 20 intermediate filament proteins expressed in cells of epithelial origin as well as endothelial cells . cks1 - 8 are the type ii group that comprise neutral to basic proteins whereas cks 9 - 20 are a type i group that include acidic proteins ( 5 ) . epithelial tumors largely maintain the features of specific ck expression of their normal epithelial origin ; therefore , these molecules have been extensively used as immunohistochemical markers in diagnostic tumor pathology ( 6 ) . these three markers are expressed by most types of carcinomas , including breast , prostate , lung , colon and ovarian ( 6 , 7 ) . their main use as immunohistochemical markers is to monitor treatment , evaluate response to therapy , and provide early prognostic information on tumor progression and metastasis formation ( 8).circulating cks result from release of intact proteins from rapidly proliferating tumor or dead cells ( 9 ) . several monoclonal anti - cks antibodies are available that recognize ck8 , ck18 and ck19 ( 10 ) . the presence of ck18 fragments in cancerous epithelial cells has been reported ( 5 , 11 ) . termed m30 , a specific monoclonal antibody is available , as well as another one , m65 that recognizes total soluble ck18 fragments . by using these monoclonal antibodies it is possible to determine different circulating forms of ck18 in plasma or serum ( 12 , 13).the m30 antibody detects ck18 fragments that contain a neo - epitope at positions 387 - 396 generated by the action of caspases 3 , 7 and 9 which are activated during the early stages of apoptosis . the m65 antibody also detects cleaved fragments , but can not distinguish between the full - length protein and its fragments ( 14 ) . thus , the m65 enzyme - linked immunosorbent assay ( elisa ) theoretically measures both caspase cleavage ( apoptosis ) and cellular release of intact ck18 ( necrosis ) . the potential diagnostic and prognostic significance of circulating m30 and m65 has been investigated in patients with non - small cell lung cancer ( nsclc ) and in colon cancer ( 15 , 16 ) . their levels correlated with disease stage and recurrence . in other malignancies such as lung cancer ( 17 ) and pancreatic cancer ( 18 ) , high pretreatment ck18 levels indicated a larger tumor burden and less favorable prognosis . a number of clinical trials on breast ( 19 , 20 ) , prostate ( 21 ) , small cell lung ( 22 ) and testicular ( 23 ) cancers used both m30 and m65 as biomarkers of cell death from a variety of different chemotherapeutic agents . it has been claimed that the m30 assay has both predictive value of drug response ( 24 ) and prognostic value for survival ( 17 , 25 ) . m30 and m65 have been used as markers of host tissue toxicity in a number of different clinical conditions such as acute myocardial infarction ( 26 ) , chronic liver disease ( 27 ) and hepatitis c ( 28 ) . however , there is scant information on the prognostic application of these markers in bladder cancer . the current study aims to characterize baseline levels and post - surgical changes of circulating serum m30 and m65 levels in patients with tcc , and to examine their correlation with various clinical features . this study enrolled 60 tcc patients ( 49 male and 11 female ) aged 35 - 85 years who referred to nemazi hospital , shiraz , iran . tumor staging was determined according to the tumor - node - metastases ( tnm ) classification and grading was confirmed by a pathologist . all protocols were approved by the ethics committee at shiraz university of medical sciences and informed consent was obtained from the patients . blood samples were collected before and 7 - 10 days after operation and the obtained sera were stored at -70c until analyzed . the level of ccck18 and total ck18 in sera were determined using the m30- apoptosense elisa and m65-elisa assay kits ( peviva , sweden ) , respectively , according to the protocols described by the manufacturer . briefly , 25 l of standard solutions , low and high controls , and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m5 as catcher antibody . then , 75 l of the diluted horseradish peroxidase ( hrp)-conjugated monoclonal antibody m30 as detector was added . the samples were then incubated for 4 hours at room temperature with constant shaking , after which excess unbound conjugate was removed by five washing steps . color development was then achieved by the addition of 200 l of tmb ( 3 , 3 , 5 , 5-tetramethyl benzidine ) solution , and incubation for 20 minutes in the dark . the reaction was stopped by the addition of 50 l of stop solution and the absorbance measured in elisa reader ( anthos 2020 , australia ) at 450 nm . total level of ccck18 in the samples was measured through plotting a standard curve of known concentrations of the antigen against absorbance . with respect to m65elisa , monoclonal m5 and m6 antibodies directed against two different epitopes of ck18 in both its intact and caspase - cleaved forms was used . twenty five l of standard solution , controls and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m6 , followed by addition of diluted hrp - conjugated monoclonal antibody m5 as detector . the procedure was continued as described for m30 elisa method and then ck18 quantity was calculated through plotting the standard curve . correlation between ccck18 ( m30 ) and ck18 ( m65 ) levels and clinical characteristics of the patients were evaluated . statistical analysis was performed using graphpad prism version5.00 for windows ( graphpad software , san diego , ca , usa ) . relationship between m30 or m65 levels in pre- and post - operative sera were analyzed by paired t - test two - tailed with wilcoxon signed rank test . spearman correlation test was used when determining an association between either m30 or m65 and age , and mann - whitney test was used for the relation between m30 or m65 level or m30:m65 ratio with gender or when comparing the markers between two groups . when there were more than two groups , the nonparametric kruskal - wallis was performed to test for significant differences among the groups . to compare m30 and m65 pre - operative and post - operative levels , unpaired t - test with welch s correction was performed . this study enrolled 60 tcc patients ( 49 male and 11 female ) aged 35 - 85 years who referred to nemazi hospital , shiraz , iran . tumor staging was determined according to the tumor - node - metastases ( tnm ) classification and grading was confirmed by a pathologist . all protocols were approved by the ethics committee at shiraz university of medical sciences and informed consent was obtained from the patients . blood samples were collected before and 7 - 10 days after operation and the obtained sera were stored at -70c until analyzed . the level of ccck18 and total ck18 in sera were determined using the m30- apoptosense elisa and m65-elisa assay kits ( peviva , sweden ) , respectively , according to the protocols described by the manufacturer . briefly , 25 l of standard solutions , low and high controls , and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m5 as catcher antibody . then , 75 l of the diluted horseradish peroxidase ( hrp)-conjugated monoclonal antibody m30 as detector was added . the samples were then incubated for 4 hours at room temperature with constant shaking , after which excess unbound conjugate was removed by five washing steps . color development was then achieved by the addition of 200 l of tmb ( 3 , 3 , 5 , 5-tetramethyl benzidine ) solution , and incubation for 20 minutes in the dark . the reaction was stopped by the addition of 50 l of stop solution and the absorbance measured in elisa reader ( anthos 2020 , australia ) at 450 nm . total level of ccck18 in the samples was measured through plotting a standard curve of known concentrations of the antigen against absorbance . with respect to m65elisa , monoclonal m5 and m6 antibodies directed against two different epitopes of ck18 in both its intact and caspase - cleaved forms was used . twenty five l of standard solution , controls and samples were added to wells pre - coated with mouse monoclonal ck18 antibody m6 , followed by addition of diluted hrp - conjugated monoclonal antibody m5 as detector . the procedure was continued as described for m30 elisa method and then ck18 quantity was calculated through plotting the standard curve . correlation between ccck18 ( m30 ) and ck18 ( m65 ) levels and clinical characteristics of the patients were evaluated . statistical analysis was performed using graphpad prism version5.00 for windows ( graphpad software , san diego , ca , usa ) . relationship between m30 or m65 levels in pre- and post - operative sera were analyzed by paired t - test two - tailed with wilcoxon signed rank test . spearman correlation test was used when determining an association between either m30 or m65 and age , and mann - whitney test was used for the relation between m30 or m65 level or m30:m65 ratio with gender or when comparing the markers between two groups . when there were more than two groups , the nonparametric kruskal - wallis was performed to test for significant differences among the groups . to compare m30 and m65 pre - operative and post - operative levels , unpaired t - test with welch s correction was performed . sera from 60 patients with tcc of the bladder were assessed for m30 and m65 serum levels . abbreviation : nd , not determined . a wide range of m30 values ( 25 - 724 u / l ) was observed in total patients , with a mean of 148 16 u / l , median : 118 u / l ) and females ( 152 50 u / l , median : 122 u / l ) . patients were categorized into three groups according to age : 35 - 49 , 50 - 69 and 70 years . the levels of the soluble m30 and m65 molecules was determined in patients sera pre- ( n = 60 ) and post - operation ( n = 26 ) by elisa method . differences between pre- and post - operative m30 and pre- and post - operative m65 were not significant ( ns ) . m65 levels also showed a wide range of values ( 80 - 1375 u / l ) with a mean of 318 34 the m65 level in males was 305 35 u / l compared to females ( 376 108 u / l ; p = 0.53 ) . the level of this molecule significantly correlated with age ( p = 0.03 , spearman r = 0.27 ) and appeared to increase with age . as shown in table 1 , the majority of patients had stages t1 and t2 ( 61.7 % ) and grades iii and iv ( 55% ) tumors . figure 2 shows the results of m30 and m65 analyses in patients according to tumor stage . as shown , there were no significant differences between m30 levels in patients with different tumor stages . similarly , the level of m30 showed no significant difference in patients with various tumor grades ( figure 3 ) . in contrast to m30 , m65 levels showed a significant relation to tumor stage and grade . as shown in figure 2 , the quantity of this molecule in patients with t3/t4 stages ( 350 42 u / l ) was higher than patients with t1/t2 stages ( 293 45 u / l ; p < 0.038 ) . patients with tumor grades iii / iv had higher levels of m65 ( 383 58 u / l ) compared to those with grades i / ii tumors ( 231 26 u / l ; p = 0.04 ; figure 3 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor stages ( p= 0.038 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor grades ( p= 0.04 ) . as shown in figure 4 , this ratio for all patients was 0.54 0.04 . there was no significant difference in ratio between males ( 0.55 0.05 ) and females ( 0.46 0.09 ) . this ratio was 0.84 0.20 for patients aged 35 - 49 years , 0.53 0.04 for 50 - 69 year - old patients and 0.41 0.04 for patients 70 years of age ( p < 0.02 ) . this ratio was 0.61 0.04 in patients with stage t1 , which decreased to 0.260.008 in patients with stage t4 disease ( p < 0.002 ) . as shown in figure 5 , the m30:m65 ratio was lower in patients with t3/t4 stages ( 0.38 0.04 ) compared to those with t1/t2 stages ( 0.59 0.06 ; p < 0.002 ) . similarly , we observed a lower m30:m65 ratio in patients with grades iii / iv ( 0.48 0.07 ) compared to grades i /ii tumors ( 0.64 0.04 ; p < 0.01 ; figure 5 ) . data presented the mean m30:m65 ratio standard error in all patients according to sex and age . a significant higher m30:m65 ratio in younger group of patients ( 30 - 49 years ) vs. older ones ( 70 years ) was observed ( p = 0.02 ) . data presented the mean m30:m65 ratio standard error in patients with different stages and grades . a significant lower m30:m65 ratio in patients with higher stages ( p = 0.002 ) and grades of tcc ( p = 0.01 ) was observed . as shown in figure 1 , the m30 level in patients after surgery was 133 19 u / l which showed no significant difference with its pre - operative value ( 148 30u / l ) in this group of patients . the corresponding value for post - operative m65 levels was 240 21 u / l compared to its pre - operative level ( 319 63 there was a correlation between pre - operative m30 and m65 ( p < 0.0001 , spearman r = 0.51 figure 6a ) and post - operative m30 and m65 values ( p < 0.02 , spearman r = 0.45 ; figure 6b ) . however , we found no significant difference in m30 or m65 levels before and after surgery . the m30:m65 ratios before and after surgery showed a significant correlation in older patients ( 70 years ; p = 0.009 ) . a significant correlation between pre - operative m30 and m65 levels ( p < 0.0001 , spearman r = 0.51 ) and post - operative values ( p = 0.023 , spearman r = 0.45 ) was observed . a wide range of m30 values ( 25 - 724 u / l ) was observed in total patients , with a mean of 148 16 u / l , median : 118 u / l ) and females ( 152 50 u / l , median : 122 u / l ) . patients were categorized into three groups according to age : 35 - 49 , 50 - 69 and 70 years . the levels of the soluble m30 and m65 molecules was determined in patients sera pre- ( n = 60 ) and post - operation ( n = 26 ) by elisa method . differences between pre- and post - operative m30 and pre- and post - operative m65 were not significant ( ns ) . m65 levels also showed a wide range of values ( 80 - 1375 u / l ) with a mean of 318 34 u / l compared to females ( 376 108 u / l ; p = 0.53 ) . the level of this molecule significantly correlated with age ( p = 0.03 , spearman r = 0.27 ) and appeared to increase with age . as shown in table 1 , the majority of patients had stages t1 and t2 ( 61.7 % ) and grades iii and iv ( 55% ) tumors . figure 2 shows the results of m30 and m65 analyses in patients according to tumor stage . as shown , there were no significant differences between m30 levels in patients with different tumor stages . similarly , the level of m30 showed no significant difference in patients with various tumor grades ( figure 3 ) . in contrast to m30 , m65 levels showed a significant relation to tumor stage and grade . as shown in figure 2 , the quantity of this molecule in patients with t3/t4 stages ( 350 42 u / l ) was higher than patients with t1/t2 stages ( 293 45 u / l ; p < 0.038 ) . patients with tumor grades iii / iv had higher levels of m65 ( 383 58 u / l ) compared to those with grades i / ii tumors ( 231 26 u / l ; p = 0.04 ; figure 3 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor stages ( p= 0.038 ) . the m65 but not m30 levels was significantly higher in group of patients with higher tumor grades ( p= 0.04 ) . we determined the ratio of m30 to m65 for each patient . as shown in figure 4 , this ratio for all patients was 0.54 0.04 . there was no significant difference in ratio between males ( 0.55 0.05 ) and females ( 0.46 0.09 ) . this ratio was 0.84 0.20 for patients aged 35 - 49 years , 0.53 0.04 for 50 - 69 year - old patients and 0.41 0.04 for patients 70 years of age ( p < 0.02 ) . this ratio was 0.61 0.04 in patients with stage t1 , which decreased to 0.260.008 in patients with stage t4 disease ( p < 0.002 ) . as shown in figure 5 , the m30:m65 ratio was lower in patients with t3/t4 stages ( 0.38 0.04 ) compared to those with t1/t2 stages ( 0.59 0.06 ; p < 0.002 ) . similarly , we observed a lower m30:m65 ratio in patients with grades iii / iv ( 0.48 0.07 ) compared to grades i /ii tumors ( 0.64 0.04 ; p < 0.01 ; figure 5 ) . data presented the mean m30:m65 ratio standard error in all patients according to sex and age . a significant higher m30:m65 ratio in younger group of patients ( 30 - 49 years ) vs. older ones ( 70 years ) was observed ( p = 0.02 ) . data presented the mean m30:m65 ratio standard error in patients with different stages and grades . a significant lower m30:m65 ratio in patients with higher stages ( p = 0.002 ) and grades of tcc ( p = 0.01 ) was observed . as shown in figure 1 , the m30 level in patients after surgery was 133 19 u / l which showed no significant difference with its pre - operative value ( 148 30u / l ) in this group of patients . the corresponding value for post - operative m65 levels was 240 21 u / l compared to its pre - operative level ( 319 63 there was a correlation between pre - operative m30 and m65 ( p < 0.0001 , spearman r = 0.51 figure 6a ) and post - operative m30 and m65 values ( p < 0.02 , spearman r = 0.45 ; figure 6b ) . however , we found no significant difference in m30 or m65 levels before and after surgery . the m30:m65 ratios before and after surgery showed a significant correlation in older patients ( 70 years ; p = 0.009 ) . a significant correlation between pre - operative m30 and m65 levels ( p < 0.0001 , spearman r = 0.51 ) and post - operative values ( p = 0.023 , spearman r = 0.45 ) was observed . in the present study we have examined sera from tcc patients for levels of circulating m30 and m65.during apoptosis ck18 is cleaved by caspases and subsequently released into the extracellular environment and blood ( 9 , 29 ) . these fragments can be detected by elisa using the m30 monoclonal antibody which recognizes the ck18asp396 neo - epitope . the m65 assay is based on two antibodies , m5 and m6 , directed against two different epitopes of ck18 . all ck18 fragments that contain epitopes in the 300 to 390 amino acid region of the protein are recognized . in addition to apoptosis , m65 is thought to measure intact ck18 released during cell necrosis ( 30 - 32 ) . therefore , ck18 levels can be considered as a surrogate marker of cell death activity in tumors and non - tumor conditions . determining its level in patients can be useful for diagnosis of tumor recurrence , prognosis and monitoring . additionally , in some experiments circulating ck18 has been used to assess the efficiency of different anticancer drugs during chemotherapy ( 14 ) . olofsson et al . suggested that the ck18 marker could be useful for early prediction of the response to chemotherapy in breast cancer and a useful biomarker for clinical trials ( 33 ) . circulating ck18 was considered as a biomarker of chemotherapy - induced cell death in testicular cancer ( 23 ) . post - surgical plasma ck18 levels showed a correlation with tumor recurrence and presence of residual disease in colorectal cancer ( 34 ) . the first report of using ck18 as a diagnostic value in tcc patients was in 2002 by ramazan sekeroglu et al . these researchers used a solid - phase two - site chemiluminescence assay to measure ck18 levels . the results suggested that serum ck18 could not be a diagnostic or screening tool in early stages of bladder cancer , but was helpful in diagnosis of higher tumor stages . they determined that a significant relationship between urinary nmp 22 , a tumor marker of bladder cancer , and ck18 levels existed which suggested that nmp22 and ck18 were useful markers for diagnosis and monitoring of tcc . levels of urinary ck18 significantly differed according to pathological grade and stage of patients tumors ( 36 ) . in the phase i study of intravesical adenoviral transduction of human bladder cells with human interferon- ( ad - ifn- ) treatment in patients with bladder cancer ( 37 ) , significant apoptosis and necrosis in the patients tumors this study was the first to suggest that analysis of urinary m30 and m65 levels might be used as a potential surrogate biomarker for tumor cell death and prognosis after treatment of non - muscle invasive bladder cancer with any therapeutic agent . however , to the best of our knowledge no studies examined both m30 and m65 levels in serum of tcc patients . in the current study , we evaluated m30 and m65 serum levels in a group of iranian patients with tcc . we sought to have an insight regarding the relationship of these markers to patient characteristics and prognostic factors such as tumor stage and grade . moreover , we measured the changes in the quantity of these markers in a number of our patients after surgery to determine their value for disease monitoring . the results of the study showed a significant correlation between m30 and m65 levels in patients prior to surgery . the levels of m65 , but not m30 , were significantly related to stage and grade of patients tumors which emphasized the importance of cell necrosis in tcc biology . higher levels of m65 in patients with greater stages and grades might suggest the relation of this biomarker with tumor progression . these results were consistent with results obtained by ramazan sekeroglu et al . who reported that serum ck18 could be helpful in the diagnosis of higher stage tumors ( 35 ) . in previous studies , serum levels of m30 and/or m65 significantly correlated with tumor stage in breast ( 24 ) and colorectal cancers ( 38 ) . unlike these studies , ozturk et al . observed no difference in serum m30 and m65 levels between patients with stages iii and iv of locally advanced head and neck tumors ( 39 ) . . showed that in colorectal cancer patients , despite the tendency for m65 to decrease with increasing tumor grade , differences between the groups did not reach statistical significance as withm30 ( 34 ) . we measured post - operative m30 and m65 levels to determine a possible relation of these marker levels with tumor burden . ( 38 ) on colorectal cancer patients that showed good correlation with m30 and m65 levels in the plasma of patients before and after surgical resection . in addition , we measured m30 and m65 levels only once after surgery ; possibly , by measuring these markers at different time intervals and in a higher number of patients , different results would be obtained . various studies conducted on different tumors compared the extent of apoptosis to total cell death by calculating the m30:m65 ratio . this ratio might be an important factor to select an appropriate treatment strategy for patients . the m30:m65 ratio decreased in endometrial cancer stages iii and iv when compared with stage ii , which indicated less apoptosis and/or more necrosis during tumor progression ( 14 ) . in colorectal cancer ( 38 ) our results showed a relationship between this ratio and age , tumor stage and grade . this finding was in line with the positive correlation obtained in this study between m65 levels and age which might suggest a predominance of apoptosis in younger patients versus necrosis in older patients . the pre - operative m30:m65 ratio has shown a tendency to decrease with increase in tumor stage and tumor grade . in this regard , because m30 is considered an indicator of apoptosis , it can be assumed that in tcc patients with more aggressive tumors the rate of apoptosis may be lower than those with less aggressive tumors and cell death is mostly due to necrosis in these types of tumors . we did not find a significant difference between the pre- and post - operative m30:m65 ratio in total patients ( p = 0.08 ) . however , a correlation between the pre - and post - operative ratio in patients 70 years was observed . in conclusion , the serum levels of m65 , but not m30 , showed a significant correlation with stage and grade of patients tumors . the pre - operative m30:m65 ratio has shown a tendency to decrease with increase in tumor stage and tumor grade . the significantly decreased ratio after surgery in the older group of patients may imply the importance of this ratio for tumor monitoring in this group of patients . further studies on a larger number of patients along with follow - up of the patients for tumor recurrence and presence of residual disease will determine the exact value of these markers for tcc monitoring .

backgroundvarious markers are suggested for diagnosis and monitoring of transitional cell carcinoma of the bladder ( tcc ) , including cytokeratins ( cks).objectivesin the present study , the circulating ck18 ( m65 ) and its caspase - cleaved form , ccck18 ( m30 ) , have been investigated in a group of patients with tcc.patients and methodsserum samples were obtained from 60 patients before surgical resection , among which the samples of 26 patients after resection were also included . we measured the levels of soluble m30 and m65 molecules by enzyme - linked immunosorbent assay . the relation between these markers and patients clinical characteristics was evaluated.resultsm30 and m65 in total patient sera were 148 16 u / l and 318 34 u / l , respectively . a correlation existed between pre - operative m30 and m65 levels ( p < 0.0001 , spearman r = 0.51 ) . m65 , but not m30 , showed a significant relation to tumor stage and grade . the m65 quantity in patients with t3/t4 tumor stages ( 350 42 u / l ) was higher than that of patients with t1/t2 stages ( 293 45u / l ; p < 0.038 ) . patients with tumor grades iii / iv also showed higher levels of m65 compared to patients with tumor grades i / ii ( p < 0.04 ) . the m30:m65 ratio in all patients was 0.54 0.04 . there was a lower m30:m65 ratio in patients with t3/t4 stage tumors and those with tumor grades iii / iv ( p < 0.02 ) . the m30 ( 133 19 u / l ) and m65 levels ( 240 21 u / l ) after surgery did not significantly differ compared to their pre - operative values . however , a correlation between the pre- and post - operative m30:m65 ratio in patients 70 years was seen ( p = 0.009).conclusionsthese data suggested a relationship of both m65 and the m30:m65 ratio to tumor progression which might imply their importance in tcc monitoring .

1. Background 2. Objectives 3. Patients and Methods 3.1. Patients 3.2. Patients Samples 3.3. ELISA Assays 3.4. Statistical Analysis 4. Results 4.1. Pre-Operative Serum M30 and M65 Levels 4.2. Pre-Operative M30:M65 Ratio 4.3. Post-Operative Serum M30 and M65 Levels 5. Discussion

spinal dural arteriovenous fistula ( sdavf ) is a rare neurological disease with an estimated frequency of 510 cases per million per year . it is caused by a subdural shunt between a radiculomeningeal artery and a radicular vein , leading to impaired venous drainage from the spinal cord and the arterialization of the vein due to increased venous pressure . the time to diagnosis is often delayed due to unspecific neurological complaints to as long as 1015 years after the onset of symptoms . therefore , 40% of patients are severely disabled at the time of diagnosis . the most common location of an sdavf is in the lower thoracic and lumbar region and it is accompanied by complaints in the lower extremities . sdavf at cervical level ( c - sdavf ) is extremely rare , accounting for approximately 2% of the sdavf patients and can lead to quadriplegia and respiratory insufficiency . early clinical features are nonspecific and include gait claudication , worsened by exercise . by the time of the diagnosis , the triad consisting of ( 1 ) motor deficits ( leg / arm weakness , muscle cramps ) , ( 2 ) sensory disturbances ( paresthesia , numbness , pins and needles sensation , back pain ) , and ( 3 ) cauda syndrome ( micturition problems , erectile dysfunction , anal sphincter disturbances ) are present in 70% of the patients . characteristic mri features include ( 1 ) engorged perimedullary veins , ( 2 ) central medullary edema , and ( 3 ) swelling of the caudal segments of the spinal cord . in the absence of the findings , diagnosis is difficult to make solely based on clinical symptoms and is usually erroneous . differential diagnoses may include medullary pathology ( myelitis , medullary tumors , disc herniation ) or peripheral pathology ( sensory polyneuropathy , chronic inflammatory demyelinating polyneuropathy ) ( table 1 ) . evidence of bladder or bowel dysfunction which share no evident relation with the site of medullary pathology could be very useful in suggesting the diagnosis . the association of cauda symptoms should be considered a red flag in sdavf and can guide the physician toward the correct diagnosis . the treatment involves either superselective catheterization with endovascular embolization of the fistula or neurosurgical occlusion of the arterialized vein . if successful , the treatment usually stops the progression of symptoms or leads to an improvement of neurological deficits . therefore , it is of utmost importance that the diagnosis is established as soon as possible in order to avoid permanent handicap . a 56-year - old man complained of sensory symptoms ( pins and needles sensation ) and pain in both legs and the right arm , which progressively worsened in about 4 months . the patient had difficulty performing his daily work as a bus driver and noticed impaired foot sensation and arm weakness during changing gears and pressing the pedals . the patient 's past medical history was unremarkable except for hypertension and hypercholesterolemia , both treated with statins and oral antihypertensive drugs . neurological examination showed generalized weakness in both lower limbs and the right arm ( medical research council grade 4 ) with hyperreflexia and bilateral plantar reflexes . sensory exam showed generalized sensation loss ( involving both dorsal columns and the spinothalamic tract ) and subjective pins and needles sensations in both feet and the right arm . given the association of both lower and upper limb involvement and hyperreflexia , a cervical lesion was suspected . the patient underwent diagnostic brain and spinal cord mri , which showed an elongated intramedullary hyperintensity from the foramen magnum to c4 on t2-weighted images ( fig 1 ) with a small contrast enhancement at the junction between the brainstem and the cervical spinal cord . transverse myelitis was suspected and the patient was treated with a high intravenous corticosteroid dose [ 7 , 8 , 9 , 10 ] . the patient developed quadriplegia with severe generalized weakness and sensation loss in both legs and both arms , inspiratory muscle weakness , dysphonia , and high urinary retention . an allergic reaction was excluded and the patient was transferred to an intensive care unit for assisted breathing . a cervical mri was performed showing varicose dilatations , irregular cervical veins , and central medullary edema ( fig 2 ) characteristic of a cervical sdavf [ 4 , 5 , 6 ] . superselective angiography confirmed the presence of an sdavf and the patient successfully underwent open spine surgery with ligation and coagulation of the arterialized communicating vein . ten days after the surgical treatment , he almost completely regained neurological functions and was further referred to an intensive rehabilitation program in a specialized clinic . by the time of discharge from the neurology ward 2 weeks after surgery , he could walk more than 30 m with rollator support and only had mild numbness in the feet and some difficulty voiding . sdavf may present with a wide range of clinical features such as motor and sensory deficits often associated with a cauda equina syndrome . neurological deficits are due to perimedullary venous congestion leading to intramedullary edema or ischemia in severe cases or direct compression of the nerve roots at the level of the fistula . abnormal communication between a radiculomeningeal artery and a radicular vein leads to increased venous pressure in the dural sleeve , impairing normal venous return from the intramedullary capillaries from and below the level of the fistula , causing medullary edema . furthermore , decreased pressure gradient at capillary level accounts for decreased gas exchange and medullary hypoxia . an acute increase in venous hypertension due to sudden volume or pressure overload may exacerbate neurological deficits by hampering the already decreased venous drainage at the level of the sdavf causing transitory medullary ischemia . previous publications report clinical worsening related to an acute increase in intrathoracic and venous pressure such as during singing , valsalva maneuvers , or abdominal muscle compression during exercise . in addition to the current paper , several publications report rapid worsening of symptoms in patients receiving corticosteroids intravenously for other presumed diagnoses ( most commonly myelitis ) [ 12 , 13 , 14 , 15 , 16 ] . although the exact mechanism which leads to worsening of spinal cord dysfunction is not yet understood , it is possible that both corticosteroid and intravenous saline in which these are administered could play a role . in addition to anti - inflammatory effects , corticosteroids can also play a role in regulating water and mineral metabolism through their mineralocorticoid effects , causing fluid retention . this can compromise the already fragile venous return into the dural sac , leading to medullary hypoxia and further worsening the sdavf symptoms . furthermore , steroids administered via an intravenous line placed in the upper extremity eventually drain via the cephalic veins into the brachiocephalic veins . the azygos and semiazygos veins , which collect venous blood from the spinal cord , are also tributaries of the brachiocephalic veins . therefore , a rapid rise in venous return from the upper extremities into the brachiocephalic vein could also directly diminish the venous return from the medullary level and exacerbate symptoms . this theory is supported by the mri findings before and after corticoid treatment in our patient . the cervical mri before intravenous treatment shows no evident perimedullary venous abnormalities , whereas after treatment , engorged veins can clearly be identified in the subdural space , as evidence of acute venous hypertension in the subdural sac ( fig 3 ) . we would like to conclude this study by stressing out the importance of early diagnosis and treatment in patients with sdavf . therefore , the presence of red flags can be extremely useful in guiding the physician toward the correct diagnosis . acute neurological worsening after intravenous steroid administration for any presumed diagnosis should be considered such a red flag and raise the suspicion of an sdavf .

subdural arteriovenous fistula ( sdavf ) is a rare condition characterized by clinical manifestations ranging from mild bilateral sensory deficits to quadriplegia . the diagnosis is often delayed due to unspecific neurological symptoms , initially diagnosed as polyneuropathy or myelopathy . the diagnosis can be delayed for as long as 115 years . the following report describes a cervical sdavf case initially misdiagnosed as myelitis transversa and treated with intravenous steroids . a 56-year - old male presented with sensory deficits and mild leg and right arm weakness . cervical mri showed a central medullary hyperintense lesion with contrast enhancement . after metabolic , infectious , and malignant causes were excluded , myelitis transversa was presumed and the patient was treated intravenously with methylprednisolone . shortly after that , he developed quadriplegia . cervical mri imaging showed engorged cervical perimedullary vessels , which were not visible on the initial mri . the diagnosis was revised and a sdavf identified . prompt surgical treatment led to a complete recovery . the effect of intravenous steroids in sdavf is controversial . acute clinical worsening after steroid administration is previously reported in several publications ; however , due to the paucity of clinical studies on sdavf , this effect remains mostly overlooked or unknown . the findings in this patient support the causative relation between sdavf clinical worsening and steroid administration . we propose that acute clinical worsening under steroids in patients initially diagnosed with myelitis should raise suspicion of an sdavf .

Introduction Clinical Case Discussion Conclusion Statement of Ethics Disclosure Statement

the world health organization ( who ) has estimated that about 17 million people die annually from cardiovascular disease worldwide , which accounts for approximately 33% of all deaths . in addition , 9.4 million deaths are attributed to complications of hypertension . according to who statistics in 2008 , hypertension was diagnosed in almost two - fifths of people aged 25 years and older worldwide . if current trends continue , the number of patients with hypertension will increse by 500 million and reach a total of 1.56 billion in 2025 . there are about 639 million hypertensive people , which equals almost 75% of the population in developing countries with limited resources . these hypertensive persons also have a very low awareness of their condition , as well as poor blood pressure ( bp ) control . non - optimal bp control accounts for 66.6% of all strokes and 50% of coronary artery disease cases . furthermore , hypertension is known to be one of the main risk factor of deaths in south asia . according to a study conducted between december 2011 and march 2012 in kabul , afghanistan the prevalence of hypertension was 46.2% for the age group of 40years and older . several epidemiological studies have reported an association between proteinuria and a poor prognosis in hypertensive patients . the burden of proteinuria and its association with hypertension have not been studied in the adult hypertensive population of afghanistan . therefore , this study was aimed at determining the prevalence of proteinuria , and the association of different variables with proteinuria , among adult hypertensive patients attending an outpatient clinic in afghanistan . five hundred fifty - five patients with a high bp recorded at an outpatient clinic in andkhoy , afghanistan from december 2014 to may 2015 . the case subjects included patients aged 18 years and older , who were receiving treatment for newly diagnosed arterial hypertension , defined as seated systolic / diastolic hypertension 140/90 mm hg at rest on the day of the study visit . patients who had engaged in strenuous physical activity in the preceding 24 h , as well as female participants who were pregnant or menstruating , were ineligible because of the likely presence of false - positive results . the subjects were evaluated during their clinic visit , and the study protocol included assessment of data obtained from each patient , including demographic characteristics , body mass index ( bmi ; weight in kilograms divided by height in meters squared ) , bp pattern , cardiovascular history , presence of cardiovascular risk factors , comorbidity and current drug - therapy . the study was approved by the scientific review committee of balkh regional hospital . for this study , diabetes was defined as a fasting blood sugar level of 126 mg / dl , at the visit or self - reported use of hypoglycaemia medications . the results were obtained by using the reagent tests strips of the accu - trend plus kit for cholesterol / glucose ( roche diagnostic usa ) . the definitions of obesity varies across studies , even among from the same country . for this study , overweight was defined as a bmi of 25 to < 30 kg/ m and obesity as bmi 30 . dipstick screening for proteinuria and glycosuria were performed with the reagent strips ( roche diagnostic usa ) . the urinary protein levels of dipstick urinary analyses were recorded as follows ; negative ( ) , trace ( + / ) or proteinuria ( 1 + , 2 + , 3 + or 4 + ) . cigarette smoking was assessed through a face to face interview . patients were grouped into three categories : ( i ) patients who had smoked 100 cigarettes in his or her lifetime and currently smokes cigarettes ( current smoker ) ; ( ii ) patients who had smoked at least 100 cigarettes in his or her lifetime but had stopped smoking at the time of the interview ( past smokers ) ; ( iii ) patients who have never smoked , or who had smoked < 100 cigarettes in his or her lifetime ( non - smoker ) . other factors that were considered were regular aerobic physical activity a minimum of 30 min for 5 days / week or vigorous physical activity for a minimum of 20 min for 3 days / week . the results from this measurement were entered onto each patient s case report form . univariate and multivariate logistic regression analyses were performed to observe the association between proteinuria and the risk factors as well as target organ lesions . odds ratios ( ors ) with 95% confidence intervals ( cis ) were estimated by using a logistic regression model . all the prognostic valuables with p < 0.05 in a univariate logistic regression analysis were entered into a multivariate logistic regression analysis in order to determine independent predictors . a p value of < 0.05 was considered statistically significant . all analyses were performed with the spss 20.0 software package ( spss , chicago , il , usa ) . five hundred fifty - five patients with a high bp recorded at an outpatient clinic in andkhoy , afghanistan from december 2014 to may 2015 . the case subjects included patients aged 18 years and older , who were receiving treatment for newly diagnosed arterial hypertension , defined as seated systolic / diastolic hypertension 140/90 mm hg at rest on the day of the study visit . patients who had engaged in strenuous physical activity in the preceding 24 h , as well as female participants who were pregnant or menstruating , were ineligible because of the likely presence of false - positive results . the subjects were evaluated during their clinic visit , and the study protocol included assessment of data obtained from each patient , including demographic characteristics , body mass index ( bmi ; weight in kilograms divided by height in meters squared ) , bp pattern , cardiovascular history , presence of cardiovascular risk factors , comorbidity and current drug - therapy . the study was approved by the scientific review committee of balkh regional hospital . for this study , diabetes was defined as a fasting blood sugar level of 126 mg / dl , at the visit or self - reported use of hypoglycaemia medications . the results were obtained by using the reagent tests strips of the accu - trend plus kit for cholesterol / glucose ( roche diagnostic usa ) . the definitions of obesity varies across studies , even among from the same country . for this study , overweight was defined as a bmi of 25 to < 30 kg/ m and obesity as bmi 30 . dipstick screening for proteinuria and glycosuria were performed with the reagent strips ( roche diagnostic usa ) . the urinary protein levels of dipstick urinary analyses were recorded as follows ; negative ( ) , trace ( + / ) or proteinuria ( 1 + , 2 + , 3 + or 4 + ) . cigarette smoking was assessed through a face to face interview . patients were grouped into three categories : ( i ) patients who had smoked 100 cigarettes in his or her lifetime and currently smokes cigarettes ( current smoker ) ; ( ii ) patients who had smoked at least 100 cigarettes in his or her lifetime but had stopped smoking at the time of the interview ( past smokers ) ; ( iii ) patients who have never smoked , or who had smoked < 100 cigarettes in his or her lifetime ( non - smoker ) . other factors that were considered were regular aerobic physical activity a minimum of 30 min for 5 days / week or vigorous physical activity for a minimum of 20 min for 3 days / week . the results from this measurement were entered onto each patient s case report form . univariate and multivariate logistic regression analyses were performed to observe the association between proteinuria and the risk factors as well as target organ lesions . odds ratios ( ors ) with 95% confidence intervals ( cis ) were estimated by using a logistic regression model . all the prognostic valuables with p < 0.05 in a univariate logistic regression analysis were entered into a multivariate logistic regression analysis in order to determine independent predictors . a p value of < 0.05 was considered statistically significant . all analyses were performed with the spss 20.0 software package ( spss , chicago , il , usa ) . the mean age of the patients was 57.9 13.3 years ( range 1892 years ) . most of the patients ( 59.1% ) were aged between 40 and 65 years , 222 were men ( 40.0% ) and 333 were women ( 60.0% ) . the prevalence of current smokers among female patients ( 18.6% ) was significantly ( p < 0.001 ) lower than that among male patients ( 37.8% ) . the prevalence of proteinuria was 67.2% and considerably higher in male than in female patients ( p < 0.001 ) . among all patients , 14.9% had a family history of ischemic heart disease , 14.8% had family history of diabetes , and 53.4% had a family history of hypertension . of these patients , 26.3% were current smokers , 33.7% had a bmi of 25 , 78.2% had uncontrolled bp , and 59.2% had regular physical exercise . fequency distribution of sociodemographic characteristics of study participants ( n=555 ) the mean age of patients in the proteinuria and non - proteinuria groups was 62.0 13.6 years and 57.7 13.4 years , respectively . the male - to - female ratio in the proteinuria group was 1.15 and that in the nonproteinuria group was 2.81 . smokers had higher rates of proteinuria than non - smokers 30.7% vs. 17.5% ( p = 0.03 ) . patients with proteinuria had substantially increased levels of both systolic bp and diastolic bp ( 155.0 24.8 vs.146.2 24.0 mm hg , and 94.6 13.8 vs. 88.6 10.8 mm hg ; p < 0.001 , respectively ) . the fasting blood glucose levels were 115.1 34.9 versus 104.5 23.7 ; p = 0.001 ) . the prevalence of proteinuria was significantly higher among diabetic patients than non - diabetic patients ( 14.7% vs. 3.8% ; p < 0.001 ) . patients with myocardial infarction had significantly higher proteinuria levels than those without myocardial infarction ( 25.7% vs.13.2% ; p = 0.001 ) . proteinuria was more common in patients with heart failure ( 15.2% vs. 6.6% ; p = 0.04 ) . physical activity was inversely associated with proteinuria ( 45.0% vs. 25.9% ; p < 0.001 ) . there was also a considerable difference with regard to use of angiotensin - receptor blockers ( arbs ) , angiotensin - converting enzyme ( ace ) inhibitors , calcium channel blockers ( ccbs ) , and beta blockers ( bbs ) between patients with and those without proteinuria ( table 2 ) . comparison of factors associated with proteinuria among hypertensive patients multivariate logistic regression analysis was used to determine the association between proteinuria and smoking , heart failure , myocardial infarction , diabetes mellitus , stroke , hypercholesterolemia , and regular physical activity . the odds of proteinuria among smokers were 1.88 times greater than those in non - smokers ( 95%ci 1.102.95 ) . patients with heart failure had a more than two - fold increase in the odds of proteinuria than those without heart failure ( 95%ci 1.134.45 ) . patients aged 65 years showed a 1.02 times increase of in the odds of proteinuria than those < 65 years ( 95%ci 1.001.04 ) . the odds of proteinuria among diabetic patients were 3.41 times higher than those in patients without diabetes ( 95%ci 1.497.80 ) . myocardial infarction , regular physical activity and diabetes mellitus were other variables that had a significant association with proteinuria in the univariate logistic regression model , however , in the multivariate model , this association was not significant ( table 3 ) . there has been a lack of data about the prevalence of proteinuria in afghanistan , which is important information on the evaluation of hypertensive patients . the presence of proteinuria helps in identifying patients who are at high risk and need to receive aggressive treatment for risk factors . the prevalence of proteinuria among patients with hypertension in the northern part of afghanistan was 67.2% . a similar prevalence , 67.8% was reported in morocco , as part of an i - search ( survey for evaluating microalbuminuria routinely by cardiologists in patients with hypertension ) study , in which the data were collected from 40 cardiology centers with a population of 476 patients . in another i - search study performed in turkey in an international multi - center the study was designed to evaluate the frequency of microalbuminuria , which was observed in a large number of outpatient clinics , in a total of 21,050 patients that were receiving treatment for hypertension in 26 countries . however , in our study , the prevalence of proteinuria was higher than that seen in another study from a tertiary hospital in uganda ( 39.5% ) . these differences are most likely associated with the different criteria used in patient selection , such as age , severity of hypertension , race , and coexisting cardiovascular risk factors ( e.g. , diabetes mellitus , renal disease , dyslipidemia , and obesity ) , as well as the technique used for proteinuria determination . moreover , in a study conducted in 26 countries , the highest rates of microalbuminuria were observed in asia . the prevalence of smoking among female patients was lower than that among male patients . a similar this low prevalence of smoking among female patients may due to social factors that discourage smoking in women . in our study , we found a significant association between proteinuria and the male which is comparable with the results of others studies . this discrepancy is most probably due to a combination of risk factors ( i.e. , diabetes , smoking , and obesity ) and the fact that ischemic heart disease is more frequent in men than in women . in the present investigation , we found the association between high systolic bp and diastolic bp and proteinuria . a similar result was obtained by ya - pan et al . who found that an increase in systolic bp and diastolic bp was related to a prevalence of proteinuria , however , al - saffar et al . the likely reason might be due to a substantial reduction in renal perfusion , which leads to proteinuria . according to our study there was a substantial statistically difference between proteinuria and patient age , which is in agreement with other studies . this could be explained by relevant studies that found advancing age to be a risk factor for a higher prevalence of proteinuria . therefore , the early detection of proteinuria and screening of hypertensive patients might prevent complication such as renal and cardiovascular diseases . the prevalence of proteinuria is higher in smoking hypertensive patients than in those who do not smoke . this could be explained by the presence of endothelial dysfunction , which involves an imbalance between the contracting and relaxing substances produced by the endothelium . for example , the plasma concentration of endothelin-14 is higher in smokers than in nonsmokers . the present study has shown an independent relationship between proteinuria and the presence of concomitant diseases such as diabetes , congestive heart failure , and myocardial infarction . this observation is in agreement with other studies that found a relationship between albuminuria and cardiovascular risk . this relationship could be explained by other relevant studies in which microalbuminuria was found to indicate a generalized dysfunction of the vascular endothelium with permeability changes , which leads to a leakage of albumin through the glomerular membrane . furthermore , the finding that the prevalence of proteinuria was higher among patients with diabetes is most likely attributable to the fact that these patients had diabetes nephropathy . the result of our study also showed that physical activity decreased the risk of proteinuria in hypertensive patients . this association has been previously demonstrated by reports that showing physical activity was inversely associated with microalbuminuria . physical activity has protective effects on the vascular endothelium among patients with cardiac diseases . we found a significant association between the use of ccbs and proteinuria risk in the present study . this can be explained by the dilating effect of ccbs on the efferent glomerular arterioles . the present study also shows that the use of bbs is associated with proteinuria . however , kozan et al . could not find a significant association between the use of bbs and microalbuminuria risk . the reason for this association was illustrated by other observations in which different mechanisms were identified to explain the beneficial effect of ace inhibitors and arbs on proteinuria . second , this group of drugs can reduce the permeability of the basement membrane of the glomerulus . both hemodynamic and non - hemodynamic effects of ace inhibitors and arbs on renal activity lead to a reduction in proteinuria among patients with hypertension . the samples consisted of patients who were referred to an outpatient clinic for treatment . in addition , the data source was a single center and the study design was an observational analysis . patients were not randomly selected in our study ; therefore , there might be a selection bias . furthermore , the examination of proteinuria by using our data did not demonstrate the proportion of patients who had positive or negative results based on retesting . moreover , concerning the study sample , the patients were selected on a convenience basis . finally , the study only included the uzbek - turkmen ethnic group and the findings may not be generalizable to the general population . we conclude that our results confirm the presence of a high incidence of proteinuria among hypertensive outpatients in a clinic in andkhoy , afghanistan , especially in those with a high cardiovascular risk . therefore , more frequent screening for proteinuria might be imperative for the evaluation this project was supported in part , by the non - profit organization epidemiological and clinical research information network ( ecrin ) . we would like to thank dr.mirwais rabi , and dr.ahmad arsalam karimi for their cooperation for this study .

abstractproteinuria in hypertension is an early marker of renal disease and a predictor for the progression of end stage renal disease , and cardiovascular diseases . this study was designed to determine the prevalence of proteinuria and its association with cardiovascular risk factors among adult hypertensive patients in afghanistan . five hundred fifty - five patients with a high blood pressure recorded in an outpatient clinic in andkhoy , afghanistan from december 2014 to may 2015 , were included in this study . data obtained from each patient , included demographic characteristics , body mass index , blood pressure patterns , cardiovascular history , cardiovascular risk factors , comorbidity , and current drug - therapy . dipstick screening for proteinuria was performed with reagent test strips . the mean age of the patients was 57.9 13.3 years , and a female predominance was observed ( n = 333 , 60% ) . the prevalence of proteinuria was 67.2% . the predictors of proteinuria were found to be age 65 years ( odds ratio [ or ] 1.02 , 95% confidence interval [ ci ] 1.001.04 ) , smoking ( or 1.88 , 95% ci 1.173.02 ) , heart failure ( or 2.23 , 95% ci 1.134.41 ) , and diabetes mellitus ( or 3.41 , 95% ci 1.497.81 ) . in conclusion , this study shows that proteinuria is highly prevalent among hypertensive outpatients in an outpatient clinic in andkhoy , afghanistan , especially in those with high cardiovascular risk .

INTRODUCTION MATERIALS AND METHODS Subjects Statistical analysis RESULTS DISCUSSION ACKNOWLEDGEMENT CONFLICTS OF INTEREST

the vast majority of cancers in the oral cavity are squamous cell carcinomas ( scc)its evolution is influenced by host immune response cells ( e.g. , cd8 t , cd4 t , natural killer cells - nk , dendritic cells - dc , macrophages , and eosinophils ) . the eosinophils are considered as destructive effector leukocytes with cytotoxic activities mainly implicated in parasitic infections ( e.g. , helminthic infections ) and allergic diseases ( e.g. , bronchial asthma , allergic dermatitis , etc . ) . however , studies have shown that they can also be involved in tissue remodelling and in innate and acquired immunity response modulation [ 2 , 3 ] ( figure 1 ) . under diverse stimuli ( e.g. , infections , tumours , etc . ) , the eosinophils are able to release different substances , such as , eosinophil cationic protein ( ecp ) , major basic protein ( mbp ) , eosinophil peroxidise ( epo ) , eosinophil - derived neurotoxin ( edn ) , il-1 , il-2 , il-4 , il-5 , il-6 , il-8 , il-10 , il-12 , il-13 , il-18 , interferon ( inf)- , tumor necrosis factor ( tnf)- , transforming growth factor ( tgf)- , tgf- , chemokines ( rantes , endotaxin-1 ) , platelet - activating factor ( paf ) , leukotriene c4 ( ltc4 ) , neuromediators , and indoleamine 2,3-dioxygenase ( ido ) [ 4 , 5 ] . these substances may cause cell death and induction of inflammatory symptoms as well as contribute to tumour progression or regulation . furthermore , the eosinophils present membrane receptors ( e.g. , il-1 , 3 , 4 , 5 , 8 , 10 , 12 , 13 , granulocyte monocyte colony - stimulating factor gm - csf , ifn- , tnf- , and macrophage inflammatory protein-1 ) that confer a survival and recruitment capacity of eosinophils themselves . in oral scc , several studies have shown that eosinophils can be associated with an improved prognosis , but there are other studies however , showing their association with a poor prognosis as well [ 6 , 7 ] . in this short review , we summarize briefly the role of the eosinophils in the general context of immunoregulation and its relation with oral squamous cell carcinoma . under a specific stimulus , the nave cd4 t and cd8 t cell can be differentiated , respectively , into th2 and tc2 cells and secrete mainly cytokines involved with humoral immunity such as il-4 , il-5 , il-10 , and il-13 [ 8 , 9 ] . the initial recruitment and activation of eosinophils towards the tumour microenvironment is principally related with th2 response , although necrotic cells can stimulate both the migration and the activation of eosinophils [ 10 , 11 ] . according to the literature , th2 response may eliminate the cancer on the dependence of eosinophils and macrophages [ 8 , 1214 ] . il-4 and il-13 are potent inducers of eotaxin chemokines that can explain the eosinophilia associated with th2 responses . in oral squamous cell carcinoma , the eotaxin expressed by tumour cells and eosinophils were involved in the mechanisms of eosinophil chemotaxis to the tumour . . il-13 has also been involved with the antitumour immune response mediated mainly by neutrophils ( gr-1 ) and macrophages ( mac-3 ) . however , il-13 can also inhibit the ifn- secretion and cd8 cytotoxic t lymphocyte ( ctl ) activity and compromise the anti - tumour immunity response . studies have also shown that eosinophils can process and present histocompatibility complex ii ( mhc - class ii ) molecules and polarize the th2 response . upon stimulation with ifn- , il-3 , and gm - csf , human eosinophilc cell line differentiated with dibutyryl cyclic amp ( deol-1 ) and human peripheral blood ( pb ) eosinophils were able to respond to the lymphoid chemokines ( e.g. , ccl11 , ccl21 , and ccl25 ) . in addition , cytokine - stimulated deol-1 cells expressed human leukocyte antigen ( hla)-dr and costimulatory molecules such as cd80 , cd86 and cd40 . therefore , eosinophils can migrate to the lymphoid chemokine microenvironment and express antigen - presenting cells ( apcs)- related costimulatory molecules . however , some experimental models have suggested that eosinophils are inefficient antigen - presenting cells when compared to macrophages or dendritic cells . the eosinophil - derived neurotoxin stored in eosinophils granules is able to induce dc maturation and activation through up - regulation of mitogen - activated protein ( map ) kinases , nuclear factor - kappa b ( nf kappa b ) and cd83 , cd86 costimulatory molecules , and mhc class ii expression [ 27 , 28 ] . edn - treated human dcs stimulated th2 immune response via toll - like receptor ( tlr)2-myd88 signal . they may express ifn- as well as il-4 , il-5 and il-10 suggesting a subpopulation of human eosinophils that expresses th1 or th2 cytokines , respectively . it has been shown that not only the th1 response is able to eradicate the tumour through the cellular immunity response but also th2 response via tumour necrosis . contrarily , eosinophils are capable to downregulate the antitumour immunity , through mainly il-10 and ido production . il10 is a potent inhibitor of mhc complex and cd80 and cd86 expression on dc as well as it suppresses the dc differentiation . contrarily , il-10 can play a role in the b cell activation and survival . through indoleamine this enzyme catalyzes the amino acid tryptophan to kynurenine which is able to cause cycle arrest and apoptosis from uncommitted cd4 t cells as well as maintenance of th2 response . the ido was correlated with the poor prognosis in the non - small cell lung cancer . it has been shown that the eosinophils are capable of producing various substances ( e.g. , vascular endothelial growth factor - vegf , fibroblast growth factor - fgf , tnf- , gm - csf , nerve growth factor - ngf , tgf- , and il-8 ) that can promote angiogenesis and produce collagenous fibers . in head and neck scc , it has been shown that the th1 response is mainly associated with a better prognosis than those with the th2 response [ 38 , 39 ] . according to argarwal and colleagues , early stage of oral scc expressed mainly inf- and il-2 genes ( th1 responses ) , whereas the advanced stage tumours presented il-4 and il-10 expression ( th2 response ) . in addition , advancing lesions of the tongue squamous cell carcinoma , induced by 4-nitroquinoline-1-oxide , have downregulated th1-type and upregulated th2-type cytokine production . regarding studies with eosinophils in the blood , the presence of eosinophils and th2 cells can be related with tumour progression and poor prognosis [ 43 , 44 ] . peripheral blood analyses of intraoral squamous cell carcinoma with a history of tobacco use presented enhanced expression of th2 cytokine . however , patients with malignant disease ( e.g. , acute lymphoblastic leukemia , acute myelogenous leukemia ) that underwent stem cell transplantation or patients with cervical cancer treated with whole - pelvic irradiation presented increased blood eosinophilia and better overall survival . tumour - associated tissue eosinophilia ( tate ) has been reported in diverse sites [ 14 , 35 , 4753 ] including the head and neck region [ 6 , 7 , 5465 ] ( figure 2 ) . in the head and neck region , some studies have shown that the tate has a favourable prognosis , suggesting that eosinophils may play a protective role against epithelial tumours [ 6 , 55 , 60 , 62 ] . other studies however , suggest that eosinophils may play a role in promoting epithelial tumour growth accounting for a poor prognosis [ 7 , 57 , 58 ] or even no effect on tumour evolution [ 54 , 56 , 59 , 63 ] . with regard to good prognosis , it has been shown that oral scc patients with tate presented higher overall survival and less incidence of distant metastasis in head and neck tumours . nevertheless , tadbir and colleagues have shown that tate was not associated with vascular , perineural , muscle invasion , and locoregional metastasis in scc of the oral cavity . similar results were observed in nasopharyngeal carcinoma and oral ssc where the eosinophils were not associated with local recurrence , distant metastases , or survival . in another study of laryngeal squamous cell carcinoma , tate was associated only with the age - tate - positive patients presented between 50 and 60 years of age , whereas the tate - negative patients were between 60 and 70 years of age . according to oliveira and colleagues and cormier and colleagues , the presence of eosinophis in damaged striated muscular fibers of oral cancer or in capsule of b16-f10 melanoma cell - derived tumours , respectively , could be related with tissue remodelling . on the other hand , the presence of eosinophils has also been related with a poor prognosis . in oral scc , eosinophilic infiltration and hla - dr expression in tumour cells were related to unfavourable prognosis . in the experimental studies of oral carcinoma , the depletion of tate with anti - il-5 mab was associated with a delayed development of the tumours . according to falconieri and colleagues and oliveira and colleagues , the eosinophil presence in the scc of the oral cavity similar results have been observed in head and neck carcinoma [ 57 , 58 ] and cervix where eosinophils infiltrate has increased the suspicion of invasion . the presence of > 3 eosinophils / high - power field ( hpf ) , 5/hpf , and 10/10 hpf in cervical incisional biopy and excisional specimens were associated with invasion . according to dorta and colleagues , the prognosis related with tate is controversial due to the different methodology utilized to count tumour - associated tissue eosinophila , thereby prejudicing a comparison of the results . for this reason alkhabuli and high suggested the counting of eosinophils through a density method , which utilizes the highest density of eosinophils per surface area , in preference to the classic method that counts eosinophils / hpf . in general , the presence as well as the state of activation of immunologic cells plays an important role in tumour cell progression . further studies on eosinophils and their state of activation are necessary in order to elucidate these findings .

the eosinophil cell has been related as a prognostic indicator for cancers . however , its exact function in tumour behaviour is still not clearly defined . in the oral cavity the presence of eosinophils can be a favourable prognostic indicator as well as it may be associated with a poor prognosis . in this short review , we briefly summarize the role of the eosinophils in the general context of immunoregulation and its relation to oral squamous cell carcinoma .

1. Introduction 2. The Eosinophil and Its Diverse Functions in the Context of Immunoregulation 3. Prognostic Value of Eosinophils in Oral Squamous Cell Carcinoma 4. Conclusion

the kcps is a prospective cohort study of korean government employees , public and private school teachers , and their dependents who were insured by the korean medical insurance corporation , the former national health insurance corporation ( 16,17 ) . the cohort includes 1,329,525 koreans ( 846,907 men and 482,618 women ) aged 3095 years ; 784,870 ( 59% ) subjects were enrolled in 1992 , 367,903 ( 27.7% ) were enrolled in 1993 , 98,417 ( 7.4% ) were enrolled in 1994 , and 78,335 ( 5.9% ) were enrolled in 1995 . follow - up began on 1 january 1993 , so 904 people enrolled in 1992 and who died in that year were excluded . to avoid confounding of the association between fasting serum glucose and the risk of death by preexisting disease , 88,420 who reported having cvd , liver disease , cancer , respiratory disease , and diabetes diagnosed at or before the initial study visit were excluded . in addition , 42,817 people with missing information on fasting serum glucose , total cholesterol , systolic blood pressure , and alcohol intake and those with extremely low bmi ( < 16.0 kg / m ) , or those with exceptionally short stature ( < 130 cm ) were also excluded . people insured by the korean medical insurance corporation were required to have biennial medical examinations conducted at designated hospitals or clinics nationwide by medical staff following guidelines provided by the korean medical insurance corporation . participants were asked to provide their medical history and to respond to a lifestyle questionnaire that included items on smoking , alcohol drinking , and performance of regular exercise . the examination included height , weight , and blood pressure measurements , urinalysis , blood cell count , and routine blood chemistries after overnight fasting . incident events were determined from diagnoses on the discharge summaries of inpatient hospital records . in korea , certified medical chart recorders review and abstract the medical chart and assign discharge diagnoses in a standardized form using the international classification of diseases , 10th revision ( world health organization 1992 ) . vital status and cause of death were determined from computerized searches of death certificate data from the korean national statistical office . study outcomes were defined as hospitalization or mortality attributable to ischemic heart disease ( international classification of diseases , 10th revision , codes i20i25 ) , stroke ( codes i60i69 ) , and atherosclerotic cvd , which included ischemic heart disease ( codes i20i25 ) , stroke ( codes i60i69 ) , hypertensive heart disease ( codes i10i15 ) , other forms of heart disease likely related to atherosclerosis ( codes i44i52 ) , disease of arteries ( codes i70i74 ) , and other sudden death with cause unknown ( code r96 ) . cardiovascular outcomes included hemorrhagic stroke ( i60i62 ) and ischemic stroke ( i63i66 ) . for those who had more than one event during follow - up an ischemic heart disease event validation study was conducted in collaboration with the korean society of cardiology through the formation of the event validation committee ( july 2008may 2009 ) . for 673 participants with an ischemic heart disease event , review of individual hospital records showed that 73% of myocardial infarction diagnoses were valid ( 18 ) . proportional hazards models were used to evaluate the association between baseline fasting serum glucose levels and atherosclerotic cvd . fasting serum glucose levels were categorized as < 70 , 7084 , 8599 , 100109 , 110125 , 126139 , and 140 mg / dl . we used the group with fasting glucose level of 8599 mg / dl as the reference category . all analyses were conducted separately for men and women and were adjusted for the following covariates : age at enrollment ; bmi ; systolic blood pressure ; total cholesterol ; alcohol intake ( five categories based on grams consumed per day : 0 , 124 g , 2549 g , 5099 g , and 100 g ) ; participation in regular physical activity ( yes or no ) and smoking status ( never smoker , former smoker , or current smoker ) ; and the number of cigarettes smoked daily among current smokers ( 19 , 1019 , and 20 ) . for more detailed analyses of the dose - response trends , we also used restricted quadratic spline models with knots at fasting glucose plasma levels of 70 , 85 , 100 , 110 , 126 , and 140 mg / dl . the kcps is a prospective cohort study of korean government employees , public and private school teachers , and their dependents who were insured by the korean medical insurance corporation , the former national health insurance corporation ( 16,17 ) . the cohort includes 1,329,525 koreans ( 846,907 men and 482,618 women ) aged 3095 years ; 784,870 ( 59% ) subjects were enrolled in 1992 , 367,903 ( 27.7% ) were enrolled in 1993 , 98,417 ( 7.4% ) were enrolled in 1994 , and 78,335 ( 5.9% ) were enrolled in 1995 . follow - up began on 1 january 1993 , so 904 people enrolled in 1992 and who died in that year were excluded . to avoid confounding of the association between fasting serum glucose and the risk of death by preexisting disease , 88,420 who reported having cvd , liver disease , cancer , respiratory disease , and diabetes diagnosed at or before the initial study visit were excluded . in addition , 42,817 people with missing information on fasting serum glucose , total cholesterol , systolic blood pressure , and alcohol intake and those with extremely low bmi ( < 16.0 kg / m ) , or those with exceptionally short stature ( < 130 cm ) were also excluded . people insured by the korean medical insurance corporation were required to have biennial medical examinations conducted at designated hospitals or clinics nationwide by medical staff following guidelines provided by the korean medical insurance corporation . participants were asked to provide their medical history and to respond to a lifestyle questionnaire that included items on smoking , alcohol drinking , and performance of regular exercise . the examination included height , weight , and blood pressure measurements , urinalysis , blood cell count , and routine blood chemistries after overnight fasting . incident events were determined from diagnoses on the discharge summaries of inpatient hospital records . in korea , certified medical chart recorders review and abstract the medical chart and assign discharge diagnoses in a standardized form using the international classification of diseases , 10th revision ( world health organization 1992 ) . vital status and cause of death were determined from computerized searches of death certificate data from the korean national statistical office . study outcomes were defined as hospitalization or mortality attributable to ischemic heart disease ( international classification of diseases , 10th revision , codes i20i25 ) , stroke ( codes i60i69 ) , and atherosclerotic cvd , which included ischemic heart disease ( codes i20i25 ) , stroke ( codes i60i69 ) , hypertensive heart disease ( codes i10i15 ) , other forms of heart disease likely related to atherosclerosis ( codes i44i52 ) , disease of arteries ( codes i70i74 ) , and other sudden death with cause unknown ( code r96 ) . cardiovascular outcomes included hemorrhagic stroke ( i60i62 ) and ischemic stroke ( i63i66 ) . for those who had more than one event during follow - up , we used only the first event for this analysis . an ischemic heart disease event validation study was conducted in collaboration with the korean society of cardiology through the formation of the event validation committee ( july 2008may 2009 ) . for 673 participants with an ischemic heart disease event , review of individual hospital records proportional hazards models were used to evaluate the association between baseline fasting serum glucose levels and atherosclerotic cvd . the assumption of proportional hazards was tested and met . fasting serum glucose levels were categorized as < 70 , 7084 , 8599 , 100109 , 110125 , 126139 , and 140 mg / dl . we used the group with fasting glucose level of 8599 mg / dl as the reference category . all analyses were conducted separately for men and women and were adjusted for the following covariates : age at enrollment ; bmi ; systolic blood pressure ; total cholesterol ; alcohol intake ( five categories based on grams consumed per day : 0 , 124 g , 2549 g , 5099 g , and 100 g ) ; participation in regular physical activity ( yes or no ) and smoking status ( never smoker , former smoker , or current smoker ) ; and the number of cigarettes smoked daily among current smokers ( 19 , 1019 , and 20 ) . for more detailed analyses of the dose - response trends , we also used restricted quadratic spline models with knots at fasting glucose plasma levels of 70 , 85 , 100 , 110 , 126 , and 140 mg / dl . on enrollment , the average age of study participants was 45.0 years for men and 49.4 years for women , and the average bmi was 23.2 kg / m for both sexes . the average fasting glucose values were 91.1 mg / dl and 94.4 mg / dl in men and women , respectively . the percentages of study participants with fasting serum glucose < 100 , 100125 , and 126 mg / dl were 76.8 , 19.3 , and 3.9% in men and 82.0 , 14.8 , and 3.2% in women . among men , 60.2% were current smokers and only 20.8% were never smokers ; among women , the corresponding figures were 4.1 and 93.9% , respectively . in the cross - sectional baseline data , men and women with higher fasting glucose were more likely to be older , to have a higher bmi , and to have higher systolic and diastolic blood pressure and total cholesterol levels ( supplementary tables 1 and 2 ) . participants in higher fasting glucose categories were also more likely to report that they were physically active . in men , the prevalence of current smoking was inversely , but weakly , associated with fasting glucose level , but positively and also weakly positively associated with glucose level in women . we separately assessed the association of fasting serum glucose for risk of various cvd outcomes in men and women , finding similar patterns of association by sex for fatal and nonfatal events combined ( tables 1 and 2 ) . the risks for the various cvd outcomes were lowest in both sexes for those with serum glucose in the range of 70109 mg / dl , and increased above and below this range . fasting glucose levels < 70 mg / dl were associated with slightly increased risk of atherosclerotic cvd in men ( hazard ratio [ hr ] 1.04 , 95% ci 1.011.08 ) and in women ( hr 1.06 , 1.021.10 ) . fasting glucose levels < 70 mg / dl were associated with increased risk of all stroke in men ( hr 1.06 , 1.011.11 ) in women ( hr 1.11 , 1.051.17 ) . for categories > 100109 mg / dl group , the hrs increased progressively with increasing serum glucose , except for hemorrhagic stroke . the results were similar after exclusion of the first 5 years of follow - up ( data not shown ) . fasting serum glucose levels at enrollment and risk of cardiovascular diseases in male participants of the korean cancer prevention study 19932010 fasting serum glucose levels at enrollment and risk of cardiovascular diseases in female participants of the korean cancer prevention study 19932010 for cardiac outcomes and thrombotic stroke , the general patterns of association were quite comparable . for participants having a glucose level in the range of 110125 mg / dl the hr was increased by 1020% , and those in the highest category experienced a doubling or more of the risk of having a cvd event . in restricted quadratic spline models , the risks of cardiac outcomes and thrombotic stroke were lowest at fasting serum glucose levels of 90 mg / dl and increased sharply above these levels in both men and women ( figs . 1 and 2 ) . hrs for cardiovascular outcomes by fasting serum glucose levels in male participants of the kcps . to convert glucose from mg / dl to mmol / l , hrs for cardiovascular outcomes by fasting serum glucose levels in female participants of the kcps . to convert glucose from mg / dl to mmol / l , we repeated the analyses for fatal and nonfatal events separately ( supplementary tables 36 ) . for men , the results for the fatal and nonfatal events were generally comparable . for women , the risks associated with low blood glucose were greater for fatal compared with nonfatal events . we also assessed potential effect modification of the risk for all cvd outcomes by other risk factors , age , and sex . in general , we found little indication of substantial effect modification ( supplementary table 7 ) . there were significant overall interactions for hypertension ( present versus absent ) and age ( younger than 55 years versus 55 years or older ) , but the differences in hrs were small . for both men and women , the hrs changed in a similar pattern with fasting glucose concentration across the two strata of age . in a large cohort of korean men and women , we found that fasting glucose level was associated with higher risk for major cvd outcomes , increasing from a level of 90 mg / dl after controlling for other risk factors . the dose - response curves showed progressive increments in the hrs from this value at both higher and lower levels ; the increased risk was greatest for stroke . the patterns of association were similar in men and women , but the associations were stronger in women . experimental studies show that abnormal glucose metabolism impairs normal endothelial function , accelerates atherosclerotic plaque formation , and contributes to plaque rupture and thrombosis . epidemiological studies provide complementary evidence . in the rotterdam study , among elderly participants with a fasting blood glucose < 110 mg / dl and without diabetes , those with higher blood glucose levels had higher levels of arterial stiffness ( 20 ) . the cathay study found that higher levels of glycemia ( 102124 mg / dl ) were associated with arterial endothelial dysfunction and intima - media thickening ( 21 ) . in a biomarker study in italy , a number of cvd biomarkers showed positive dose - response relationships with fasting glucose across three strata : < 100 ; 100109 ; and 110125 mg / dl ( 22 ) . our study adds to the increasing evidence that ifg is an independent risk factor for incident cvd , including ischemic heart disease and stroke ( 7 ) . in addition , the effects of other cvd risk factors may be enhanced by abnormal glucose metabolism ( 2325 ) . in 1997 , the american diabetes association expert committee introduced the ifg category , defined at the time as fasting plasma glucose level of 110 to 125 mg / ml or a 2-h value on the oral glucose tolerance test of 140199 mg / dl ( 26 ) . ifg is not a clinical entity in itself but is a risk category for development of diabetes and cvd ( 10 ) . in 2003 , the american diabetes association expert committee lowered the cut - point for ifg to 100 mg / dl to align the proportion of the population that would be included in the category using fasting glucose and the oral glucose tolerance test , and to indicate that glucose levels < 110 mg / dl were predictive of diabetes development ( 26,27 ) . although previous studies also have identified an increased risk of cvd associated with ifg ( 100125 mg / dl ) ( 3,5,28,29 ) , the size of kcps provides a much more precise characterization of the up - turn in risk in relation to blood glucose level , placing it at 90 mg / dl . as a consequence , our findings support lowering the current american diabetes association ifg cut - off of 100 mg / dl , and they also open the possibility that low fasting glucose levels may identify people at increased risk for cvd . severe hypoglycemia in diabetes is known to increase risks of vascular events ( 3032 ) . low fasting glucose levels in the nondiabetic population were associated with increased mortality in several studies ( 5,15 ) . in our study , the lowest risk for cvd mortality occurred in the interval of 8599 mg / dl , with a nadir at 90 mg / dl , suggesting that the range of fasting glucose levels associated with the lowest risk for cvd is narrow . in a prospective cohort study of > 40,000 people , the lowest total mortality was associated with fasting plasma glucose of 79109 mg / dl ( 15 ) . in the study by balkau et al . ( 33 ) , the interval of lowest total mortality was 94103 mg / dl , and in the decode study it was 8190 mg / dl ( 34 ) . the pooled analysis performed by the emerging risk factors collaboration described a nonlinear relationship between fasting glucose and coronary heart disease that had the same configuration as in the kcps analysis ( 3 ) . in the atherosclerosis risk in communities ( aric ) study , selvin et al . ( 35 ) found a j - shape relationship of glycated hemoglobin with risk for death attributable to coronary heart disease . fasting blood glucose level < 100 mg / dl was not associated with coronary heart disease death ( 35 ) . because different studies have used different categories for fasting glucose , further and perhaps pooled analyses of data from large studies should be performed using flexible models for dose - response analysis to confirm our findings of a narrow range of fasting glucose levels with lowest mortality . the basis of the association between low fasting glucose levels and risk for cvd in people without diabetes is unclear . wei et al . ( 15 ) hypothesized that long - term exposure to low fasting plasma glucose may serve as a risk factor for cvd mortality , perhaps through abnormal cardiac activity and thrombosis , particularly in patients with atherosclerosis . ( 36 ) reported a j - shape relationship between fasting plasma glucose and incident ischemic cerebrovascular events in patients with preexisting atherothrombotic disease and suggested that hypoglycemia or rapid changes in plasma glucose may lead to elevations of counter - regulatory hormones , such as epinephrine and norepinephrine , and these increases induce vasoconstriction and platelet aggregation ( 36 ) . in a pooled analysis of data from 97 cohorts involving people without vascular disease on enrollment , an increase in risk for vascular disease death was observed in those with blood glucose level < 70 mg / dl on enrollment ( 7 ) . because cvd risk factors and development of cvd are usually associated with metabolic abnormalities that increase fasting plasma glucose , additional studies need to be conducted to understand if low fasting plasma glucose levels are a consequence of impending disease ( i.e. , reverse causation ) or if they have a role in precipitating acute cvd events . limitations of our findings need to be considered . first , single fasting glucose measurements are subject to substantial within - person variation and we did not perform 2-h glucose tolerance testing . measurement error and between - study variability may have attenuated our results , adding to the identification of a narrow range of fasting glucose levels for optimal survival . additionally , we measured serum glucose , not the currently recommended plasma glucose ( 37,38 ) . however , because all measurements were of serum glucose , our findings should not be biased . second , we lacked information on the evolution of fasting glucose levels and incidence of diabetes during follow - up , and we can not identify if the increased risk associated with ifg depends on future evolution to diabetes or on other pathways . third , we lacked data on other metabolic abnormalities associated with overweight and obesity and fat distribution . fourth , the outcomes were obtained from admission records and death certificates . because the outpatient records were not included , the incidence of cvd morbidity in our study may be lower than actual incidence . with regard to generalizability of findings , the kcps population differs from those of other investigations in several ways . of course , participants were asian and the population was also leaner than those in the other studies of blood glucose and cvd risk . fasting glucose is readily and widely measured for early detection and prevention of diabetes . beyond risk for diabetes , it also conveys information about cvd risk . additionally , the findings suggest that the optimal glucose level may be below the current cut - offs used to identify people at risk for cvd . health care providers should recognize the j - shape relationship between fasting blood glucose and cvd risk in interpreting and communicating clinical data .

objectivealthough diabetes increases the risk of cardiovascular disease ( cvd ) and mortality , the dose - response relationship between fasting glucose levels below those diagnostic of diabetes with cardiovascular events has not been well characterized.research design and methodsa prospective cohort study of more than one million koreans was conducted with a mean follow - up of 16 years . a total of 1,197,384 korean adults with no specific medical conditions diagnosed were classified by baseline fasting serum glucose level . associations of fasting glucose level with cvd incidence and mortality , stroke incidence and mortality , and all - cause mortality were analyzed using multivariate proportional hazards regression.resultsthe relationships between fasting glucose levels and cvd risks generally followed j - shape curves , with lowest risk in the glucose range of 8599 mg / dl . as fasting glucose levels increased to > 100 mg / dl , risks for cvd , ischemic heart disease , myocardial infarction , and thrombotic stroke progressively increased , but risk for hemorrhagic stroke did not . fasting glucose levels < 70 mg / dl were associated with increased risk of all stroke ( hazard ratio 1.06 , 95% ci 1.011.11 ) in men and ( hazard ratio 1.11 , 1.051.17 ) in women.conclusionsboth low glucose level and impaired fasting glucose should be considered as predictors of risk for stroke and coronary heart disease . the fasting glucose level associated with the lowest cardiovascular risk may be in a narrow range .

RESEARCH DESIGN AND METHODS Study population Data collection Statistical analysis RESULTS CONCLUSIONS

in october 2011 , the u.s . national science advisory board for biosecurity ( nsabb ) was asked to review two papers for their potential as dual - use research of concern ( durc ) . these papers contained results on the adaptation of the highly pathogenic avian influenza a / h5n1 virus to mammalian hosts such that it could be transmitted via respiratory droplets from animal to animal . we found that this work had great potential for harm or misuse and recommended that the general conclusions highlighting the novel outcome be published , but that the manuscripts not include the methodological and other details that could enable replication of the experiments by those who would seek to do harm the recommendation not to publish scientific results was highly unusual and the first such recommendation by the nsabb membership . we are primarily a group of actively practicing basic research scientists , and we have consistently advocated for open publication practices . as per our advisory nature to the u.s . there was agreement by nsabb voting members for these recommendations , though the rationale of individual members as they arrived at the same conclusions varied . we had to judge the beneficial attributes of these research results against their potential to cause harm . over the last 7 years , nsabb has studied the issues associated with dual - use research , including risk / benefit assessments , and developed principles and tools to guide the deliberative process . much of this has been formalized in a series of reports and recommendations that are available at a public website ( http://oba.od.nih.gov/biosecurity/biosecurity.html ) . despite this experience and carefully crafted guidance , there are points in the deliberations where uncertainties and even contradictory information necessitate subjective decisions . is there a clear and bright line to be crossed or is this a more nebulous and fuzzy region of yes or no for this research ? i will present only my personal rationale and how i came to the strong conclusion that this work had the potential to be very dangerous and that its communication should be restricted at this time . i heard from members of the influenza research community and reviewed the world health organization ( who ) data indicating that this avian virus had a very high mortality rate in humans . while the influenza a / h5n1 virus rarely infects humans , when it does it causes catastrophic disease . we are all aware of the rapid global spread of human - adapted influenza both on a yearly basis and during less common pandemics . the documented devastation of the 1918 influenza pandemic , even with its lower mortality rate , was a testament to the powerful potential of influenza . the thought of combining the high human mortality of influenza a / h5n1 with a highly transmissible human - adapted phenotype was sobering . a pandemic by such a pathogen could reasonably be concluded to cause such devastation that it should be prevented at all costs . i carefully considered how restricting the information would compromise scientific research progress and even how it would hinder public health efforts to prevent such a horrific pandemic . i know from firsthand experience that the free flow of information is part of the best and most productive research endeavors and that any restrictions burden the progress . the conclusion that this virus could be adapted to mammal - to - mammal respiratory transmission was , in my mind , the foremost beneficial part of the research . with this firm conclusion in hand , policy makers , granting agencies , public health officials , and vaccine and drug developers should have both the motivation and a compelling argument to move forward to improve our influenza - fighting infrastructure . the details of the research , on the other hand , would add little to this short - term effort and could enable someone to replicate the work in a short period of time . the short - term negative consequences of restricting experimental details seemed small in contrast to the large consequences of facilitating the replication of these experiments by someone with nefarious intent . current public health surveillance and public health responses would be enhanced little by these details . this comes not only from my own professional experience in globally tracking dangerous pathogens but also from personally watching the 2009 h1n1 influenza pandemic spread globally . it was impossible to contain , and i believe that the same would be true for an h5n1 influenza pandemic . we were lucky in that the h1n1 virus has low virulence , but the best current data suggest that this would not be the case for the h5n1 virus . publishing a detailed experimental protocol on how to produce a highly transmissible h5n1 virus in a highly regarded scientific journal is a very bad idea . since our recommendations were announced in mid - december , there has been considerable response from scientists , policy makers , funding agencies , and global health organizations . there have been criticisms that restriction of the publications was insufficient and that even performing such experiments should be restricted . the debate has touched upon both biosafety and biosecurity aspects , with some calling for the destruction of the virus or for moving all such research to the highest safety level , biosafety level 4 ( bsl-4 ) . the nsabb has not yet offered specific recommendations concerning these statements , and my personal opinions are relatively unimportant . what is gratifying and essential is that the debate is occurring ; it is occurring on an international stage , and it is occurring rapidly . in the midst of nsabb deliberations and formulation of our recommendations , the need for a global debate to develop policy has always been in our discussions . why should the nsabb be telling the world what to do ? why has not the world already had these discussions and debates ? how could the nsabb stimulate the process such that global leaders in science , policy , and public health engage in a broad - based conversation on these issues ? the specific nsabb recommendations seem to have been accepted and are being implemented by two research groups and two scientific journals ; more importantly , the research issue of adapting an avian virus to mammals , potentially humans , is a topic that is being widely discussed . the who has agreed to participate and facilitate in policy development . and the u.s . research and public policy will be developed from this global engagement process , a process that should increase the public s confidence in the scientific endeavor , in scientists ethical behavior , and in the transparency that a free research environment embraces .

abstractthe national science advisory board for biosecurity ( nsabb ) has recommended that two scientific papers concerning the laboratory adaptation of avian h5n1 influenza virus to mammal - to - mammal respiratory transmission restrict their content to prevent others from replicating their work . after hearing from experts in the field of influenza research and public health , the benefits of the research were deemed less important than the potential negative consequences . the evaluation followed established nsabb procedures and prior policy recommendations for identifying dual use research of concern ( durc ) . this recommendation was received by the united states government , endorsed and forwarded to the research teams and scientific journals involved with the publications .

Commentary

pneumococcal infections are especially dangerous for children and adults with immunodeficiencies , such as hiv , or with illnesses such as chronic cardiac disease and diabetes.1 pneumococcal vaccination has therefore been recommended for persons at high risk of severe illness or complications . asthma was first identified as an independent risk factor for pneumococcal disease ( pd ) in 2005 when talbot et al2 reported that asthma was associated with a 2.4-fold increased odds of pd among persons 249 years old enrolled in tennessee s medicaid program between 1994 and 2002 . several studies conducted since then have provided additional evidence that asthma is associated with an increased risk of pd in adults,35 but no study has provided convincing evidence that asthma is associated with an increased risk of pd in children.6 currently , pneumococcal vaccination is recommended for asthmatic adults , but it is not specifically recommended for asthmatic children . asthma is the most common chronic disease in children,7 and the prevalence of asthma is increasing worldwide.812 as the prevalence of asthma increases , so does the importance of understanding whether children with asthma are at increased risk of pd . the aim of the current study was to evaluate the association between asthma and the development of pd among danish children born between 1994 and 2007 . the study population included all singleton live births in denmark from january 1 , 1994 through december 31 , 2007 . routine childhood pneumococcal vaccination with 7-valent pneumococcal conjugate vaccine ( pcv7 ) did not begin in denmark until october 2007,13 so few children in the study population were expected to be vaccinated . we used the unique civil personal registry number assigned to all danish citizens at birth and to residents upon immigration , which has been used in public records since 1968 , to identify all live births from the danish civil registration system . this continually updated national registration system includes information about date and place of birth , immigration , sex , marital status , citizenship , emigration , and vital status.14 all study children were followed from birth until the diagnosis of pd , removal from the danish civil registration system due to any cause , or december 31 , 2007 , whichever came first . the danish data protection agency provided permission to use these data ( record number : 1 - 16 - 02 - 1 - 08 ) . because this study was based on data extracted from registries , it was exempt from human subjects review , and members of the study population did not have to provide informed consent . to ascertain pd , we used each subject s unique civil personal registry number to link their danish civil registration system data to the danish national registry of patients . the danish national registry of patients began in 1977 and includes inpatient diagnoses made at nonpsychiatric hospitals and , beginning in 1995 , diagnoses made at outpatient specialist clinics and emergency room visits.15 we used the danish version of international classification of diseases ( icd)-10 codes g00.1 , a40.3 , and j13.9 to ascertain pneumococcal meningitis , pneumococcal septicemia , and pneumococcal pneumonia , respectively . diagnosis of pneumococcal pneumonia using these icd codes was validated in a prior study in which 64% of icd-10-identified cases had microbiologic , radiologic , and clinical evidence consistent with pd , and the remaining 36% were classified as having probable pd.16 we used icd-10 codes ( j45 and j46 ) recorded in the danish national registry of patients to ascertain asthma . registry - based asthma diagnoses have been previously validated in denmark and determined to be of high quality.17,18 we classified study subjects as having asthma if they had at least one diagnosis code indicating asthma hospitalization , emergency , or outpatient visits at any time before the diagnosis of pd , or before the end of follow - up for children who did not develop pd . information obtained from the danish medical birth registry included place of birth , gestational age , fetal presentation , mode of delivery , birth weight , 5-minute apgar score , maternal place of birth , maternal age at the time of delivery , maternal cohabitation status , and maternal parity.19 maternal smoking status during pregnancy was based on self - report during the first antenatal visit and was classified as yes or no , and presence or absence of congenital malformations or selected underlying comorbidities was ascertained using icd-10 codes recorded in the danish national registry of patients ( table s1 ) . children accumulated unexposed person - time from birth until an asthma diagnosis , if any , and exposed person - time thereafter . we calculated the frequency and proportion of children with and without asthma within categories of demographic variables and birth outcomes , as well as age - specific incidence rates of pd . we used poisson regression to estimate crude and adjusted incidence rate ratios ( irrs ) and 95% confidence intervals ( cis ) associating childhood asthma and pd . we included covariates that changed the crude association between asthma and pd by more than 10% in adjusted models ; child s sex was retained in the adjusted models regardless of its impact on the unadjusted measures . because underlying comorbidity is known to substantially increase the risk of pd in children,20 and because it was strongly associated with childhood asthma exposure in our study population , we evaluated the impact of comorbidity on the association between asthma and pd in stratified analyses . the impact of congenital malformations was also assessed . for these analyses , we used poisson regression to calculate crude and adjusted irrs and 95% cis stratified by the presence or absence of congenital malformations or selected underlying comorbidities . we further evaluated any observed effect measure modification for evidence of biologic interaction using standard measures ( supplementary materials).21,22 we conducted all statistical analyses using sas / stat software , version 9.2 ( sas institute inc . , cary , nc , usa).23 the study population included all singleton live births in denmark from january 1 , 1994 through december 31 , 2007 . routine childhood pneumococcal vaccination with 7-valent pneumococcal conjugate vaccine ( pcv7 ) did not begin in denmark until october 2007,13 so few children in the study population were expected to be vaccinated . we used the unique civil personal registry number assigned to all danish citizens at birth and to residents upon immigration , which has been used in public records since 1968 , to identify all live births from the danish civil registration system . this continually updated national registration system includes information about date and place of birth , immigration , sex , marital status , citizenship , emigration , and vital status.14 all study children were followed from birth until the diagnosis of pd , removal from the danish civil registration system due to any cause , or december 31 , 2007 , whichever came first . the danish data protection agency provided permission to use these data ( record number : 1 - 16 - 02 - 1 - 08 ) . because this study was based on data extracted from registries , it was exempt from human subjects review , and members of the study population did not have to provide informed consent . to ascertain pd , we used each subject s unique civil personal registry number to link their danish civil registration system data to the danish national registry of patients . the danish national registry of patients began in 1977 and includes inpatient diagnoses made at nonpsychiatric hospitals and , beginning in 1995 , diagnoses made at outpatient specialist clinics and emergency room visits.15 we used the danish version of international classification of diseases ( icd)-10 codes g00.1 , a40.3 , and j13.9 to ascertain pneumococcal meningitis , pneumococcal septicemia , and pneumococcal pneumonia , respectively . diagnosis of pneumococcal pneumonia using these icd codes was validated in a prior study in which 64% of icd-10-identified cases had microbiologic , radiologic , and clinical evidence consistent with pd , and the remaining 36% were classified as having probable pd.16 we used icd-10 codes ( j45 and j46 ) recorded in the danish national registry of patients to ascertain asthma . registry - based asthma diagnoses have been previously validated in denmark and determined to be of high quality.17,18 we classified study subjects as having asthma if they had at least one diagnosis code indicating asthma hospitalization , emergency , or outpatient visits at any time before the diagnosis of pd , or before the end of follow - up for children who did not develop pd . information obtained from the danish medical birth registry included place of birth , gestational age , fetal presentation , mode of delivery , birth weight , 5-minute apgar score , maternal place of birth , maternal age at the time of delivery , maternal cohabitation status , and maternal parity.19 maternal smoking status during pregnancy was based on self - report during the first antenatal visit and was classified as yes or no , and presence or absence of congenital malformations or selected underlying comorbidities was ascertained using icd-10 codes recorded in the danish national registry of patients ( table s1 ) . children accumulated unexposed person - time from birth until an asthma diagnosis , if any , and exposed person - time thereafter . we calculated the frequency and proportion of children with and without asthma within categories of demographic variables and birth outcomes , as well as age - specific incidence rates of pd . we used poisson regression to estimate crude and adjusted incidence rate ratios ( irrs ) and 95% confidence intervals ( cis ) associating childhood asthma and pd . we included covariates that changed the crude association between asthma and pd by more than 10% in adjusted models ; child s sex was retained in the adjusted models regardless of its impact on the unadjusted measures . because underlying comorbidity is known to substantially increase the risk of pd in children,20 and because it was strongly associated with childhood asthma exposure in our study population , we evaluated the impact of comorbidity on the association between asthma and pd in stratified analyses . the impact of congenital malformations was also assessed . for these analyses , we used poisson regression to calculate crude and adjusted irrs and 95% cis stratified by the presence or absence of congenital malformations or selected underlying comorbidities . we further evaluated any observed effect measure modification for evidence of biologic interaction using standard measures ( supplementary materials).21,22 we conducted all statistical analyses using sas / stat software , version 9.2 ( sas institute inc . , cary , nc , usa).23 there were 890,681 singleton live births during the study period . after excluding 2,026 records , records were excluded if a child had 0 days of follow - up ( number [ n ] = 1,228 ) , birth weight < 500 g ( n=90 ) , gestational age < 25 completed weeks or > 45 completed weeks ( n=510 and n=5 , respectively ) , implausible gestational age and birth weight combinations ( n=70 ) , or if they met < 1 exclusion criteria ( n=123 ) . the mean and median follow - up periods were both 8.0 years ( interquartile range : 4.411.6 years ) . among the 888,655 children in the study population , 6.0% ( n=53,024 ) received an asthma diagnosis before the end of follow - up . the mean age of asthma diagnosis was 31 months ( standard deviation : 31.0 months ) , and the median was 18.7 months ( interquartile range : 10.739.6 months ) . a total of 6,641 children had a recorded comorbidity ; cardiac disease was the most common comorbidity ( 32.8% ) , followed by renal disease ( 21.5% ) and type 1 diabetes ( 20.3% ) . compared with children without asthma , children with an asthma diagnosis were more likely to be male , to have been born preterm ( < 37 weeks ) , to have a low birth weight ( < 2,500 g ) , and to be born to a mother who reported smoking at the first prenatal visit ( table 1 ) . in addition , asthmatic children had more congenital malformations ( 6.7% versus 4.4% , respectively ) and selected underlying comorbidities ( 2.4% versus 0.6% , respectively ) compared with nonasthmatic children . a total of 2,253 children were diagnosed with pd during the follow - up period , and most of them were admitted as inpatients ( 96.3% ) . pneumonia accounted for the majority of cases ( 72.9% ) , followed by septicemia ( 14.6% ) and meningitis ( 12.5% ) . most pd diagnoses occurred between 6 months and 24 months after birth ( n=1,180 ; 52.7% ) and the fewest occurred at more than 60 months after birth ( n=255 ; 11.3% ) . there was no trend toward increasing or decreasing incidence throughout the study s calendar period . the rate of pd was highest among children 6 to < 24 months old ( 91.2 cases per 100,000 child - years ) followed by children 0 to < 6 months old ( 78.8 cases per 100,000 child - years ) and children 24 to < 60 months old ( 21.5 cases per 100,000 child - years ) . children in the oldest age group had the lowest pd rates ( 8.0 cases per 100,000 child - years ) . pd incidence rates in asthmatic children were consistently higher than in nonasthmatic children , with the exception of children aged 0 to < 6 months . the unadjusted irr associating asthma with incident pd among all children was 2.4 ( 95% ci : 2.1 , 2.6 ) . age - specific measures of association were confounded by year of birth , birth weight , congenital malformations , and underlying comorbidities . the adjusted irr associating asthma with incident pd among all children was 2.2 ( 95% ci : 2.0 , 2.5 ) ; in order of increasing age strata , the adjusted irrs were 0.4 ( 95% ci : 0.2 , 0.8 ) in children 0 to < 6 months old , 2.1 ( 95% ci : 1.8 , 2.5 ) in children 6 to < 24 months old , 4.1 ( 95% ci : 3.3 , 5.1 ) in children 24 to < 60 months old , and 2.3 ( 95% ci : 1.6 , 3.2 ) in children 60 months old ( table 2 ) . restricting the study population to children without selected comorbidities or malformations did not substantially change the irrs associating asthma with incident pd in any age strata . restricting the study population to children who did have selected comorbidities or malformations , however , revealed that comorbidity was an effect modifier of the association between asthma and pd in children aged 24 to < 60 months and 60 months ( table 3 ) . evaluation of biologic interaction between asthma and comorbidity on the incidence of pd in these age groups showed that the rate of pd was greater among those with a recorded diagnosis of both asthma and comorbidity compared with the rate that would be expected based on the independent effects of asthma or comorbidity alone . figure 1 shows that among children aged 24 to < 60 months old , the unadjusted incidence rate of pd in children with both asthma and comorbidity was 7.5 times the rate in children with asthma alone , and that after adjusting for confounding variables , 55% ( 95% ci : 31 , 79 ) of pd cases among asthmatic children could be attributed to the presence of both asthma and comorbidity at the same time . among children aged 60 months old , the unadjusted irr of pd in children with both asthma and comorbidity was 14 times the rate in children with asthma alone , and the adjusted percentage of pd cases attributable to biologic interaction increased to 73% ( 95% ci : 52 , 93 ) . there were 890,681 singleton live births during the study period . after excluding 2,026 records , records were excluded if a child had 0 days of follow - up ( number [ n ] = 1,228 ) , birth weight < 500 g ( n=90 ) , gestational age < 25 completed weeks or > 45 completed weeks ( n=510 and n=5 , respectively ) , implausible gestational age and birth weight combinations ( n=70 ) , or if they met < 1 exclusion criteria ( n=123 ) . the mean and median follow - up periods were both 8.0 years ( interquartile range : 4.411.6 years ) . among the 888,655 children in the study population , 6.0% ( n=53,024 ) received an asthma diagnosis before the end of follow - up . the mean age of asthma diagnosis was 31 months ( standard deviation : 31.0 months ) , and the median was 18.7 months ( interquartile range : 10.739.6 months ) . a total of 6,641 children had a recorded comorbidity ; cardiac disease was the most common comorbidity ( 32.8% ) , followed by renal disease ( 21.5% ) and type 1 diabetes ( 20.3% ) . compared with children without asthma , children with an asthma diagnosis were more likely to be male , to have been born preterm ( < 37 weeks ) , to have a low birth weight ( < 2,500 g ) , and to be born to a mother who reported smoking at the first prenatal visit ( table 1 ) . in addition , asthmatic children had more congenital malformations ( 6.7% versus 4.4% , respectively ) and selected underlying comorbidities ( 2.4% versus 0.6% , respectively ) compared with nonasthmatic children . a total of 2,253 children were diagnosed with pd during the follow - up period , and most of them were admitted as inpatients ( 96.3% ) . pneumonia accounted for the majority of cases ( 72.9% ) , followed by septicemia ( 14.6% ) and meningitis ( 12.5% ) . most pd diagnoses occurred between 6 months and 24 months after birth ( n=1,180 ; 52.7% ) and the fewest occurred at more than 60 months after birth ( n=255 ; 11.3% ) . there was no trend toward increasing or decreasing incidence throughout the study s calendar period . the rate of pd was highest among children 6 to < 24 months old ( 91.2 cases per 100,000 child - years ) followed by children 0 to < 6 months old ( 78.8 cases per 100,000 child - years ) and children 24 to < 60 months old ( 21.5 cases per 100,000 child - years ) . children in the oldest age group had the lowest pd rates ( 8.0 cases per 100,000 child - years ) . pd incidence rates in asthmatic children were consistently higher than in nonasthmatic children , with the exception of children aged 0 to < 6 months . the unadjusted irr associating asthma with incident pd among all children was 2.4 ( 95% ci : 2.1 , 2.6 ) . age - specific measures of association were confounded by year of birth , birth weight , congenital malformations , and underlying comorbidities . the adjusted irr associating asthma with incident pd among all children was 2.2 ( 95% ci : 2.0 , 2.5 ) ; in order of increasing age strata , the adjusted irrs were 0.4 ( 95% ci : 0.2 , 0.8 ) in children 0 to < 6 months old , 2.1 ( 95% ci : 1.8 , 2.5 ) in children 6 to < 24 months old , 4.1 ( 95% ci : 3.3 , 5.1 ) in children 24 to < 60 months old , and 2.3 ( 95% ci : 1.6 , 3.2 ) in children 60 months old ( table 2 ) . restricting the study population to children without selected comorbidities or malformations did not substantially change the irrs associating asthma with incident pd in any age strata . restricting the study population to children who did have selected comorbidities or malformations , however , revealed that comorbidity was an effect modifier of the association between asthma and pd in children aged 24 to < 60 months and 60 months ( table 3 ) . evaluation of biologic interaction between asthma and comorbidity on the incidence of pd in these age groups showed that the rate of pd was greater among those with a recorded diagnosis of both asthma and comorbidity compared with the rate that would be expected based on the independent effects of asthma or comorbidity alone . figure 1 shows that among children aged 24 to < 60 months old , the unadjusted incidence rate of pd in children with both asthma and comorbidity was 7.5 times the rate in children with asthma alone , and that after adjusting for confounding variables , 55% ( 95% ci : 31 , 79 ) of pd cases among asthmatic children could be attributed to the presence of both asthma and comorbidity at the same time . among children aged 60 months old , the unadjusted irr of pd in children with both asthma and comorbidity was 14 times the rate in children with asthma alone , and the adjusted percentage of pd cases attributable to biologic interaction increased to 73% ( 95% ci : 52 , 93 ) . this study provides evidence that asthma is an important risk factor for the development of pd in children , and it provides new insight about a potentially meaningful interaction between asthma and comorbidity on the risk of pd . consistent with data presented by talbot et al,2 we observed a twofold increased rate of pd among all children < 18 years old following a childhood asthma diagnosis compared with person - time before an asthma diagnosis or in children who never had an asthma diagnosis after adjusting for confounding factors . the highest rate ratio occurred among children 24 to < 60 months old , which was twice as large as the rate ratios observed among children 6 to < 24 months old and 60 months old . we also observed a rate ratio less than 1 among children 0 to < 6 months old , but emphasize that we do not believe that this indicates that asthma is protective against pd in this age group . instead , we suspect that this observation is due to the difficulty of reliably diagnosing asthma in very young children.24 the evaluation of a biological interaction between childhood asthma and comorbidity on the incidence of pd revealed that the combined effect of these two exposures was synergistic in older children , and that a high proportion ( 55% in children aged 24 to < 60 months , and 73% in children aged 60 months ) of the pd incidence among asthmatic children with comorbidities was attributable to this interaction . no synergy between childhood asthma and comorbidity was observed in children less than 2 years old . these results suggest that children more than 2 years old who have underlying comorbidities are more sensitive to the effect of asthma on pd than children more than 2 years old without comorbidities . juhn et al3 also identified comorbidity as an effect modifier of the association between asthma and pd among minnesota adults . they reported that the odds ratio ( or ) of the effect of asthma on pd among adults with high - risk conditions was lower ( or : 1.2 ; p=0.86 ) than the or among those without high - risk conditions ( or : 2.9 ; p=0.04 ) . similar results were observed in our study in children between 2 years and 5 years old : the association between asthma and incident pd was lower among children with underlying comorbid conditions compared with children without underlying illnesses ( adjusted irr : 2.9 versus 4.2 , respectively ) . the lower rate ratios among children with comorbid conditions probably results from their higher risk of pd , which is sometimes called modification by the baseline risk . juhn et al3 did not assess for biological interaction on the additive scale in their study , and instead reported that there was no statistical interaction between asthma and illnesses based on the addition of an interaction term in a log - linear multivariate model . departure from additivity is a better measure of biologic interaction , and a lack of statistical interaction when measured as an interaction term in a log - linear multiplicative multivariate model can easily be mistaken for a lack of biologic interaction.22 several investigators have identified potential biologic mechanisms that may explain how asthma increases the risk of pd . two studies have identified associations between asthma and increased carriage of streptococcus pneumoniae in the nasopharynx,25,26 suggesting that children with asthma may be at increased risk of pd because they are more likely to be colonized with pneumococci . other proposed mechanisms include asthma - induced pathologic alterations that can impair clearance of pathogenic bacteria from the airway27,28 and chronic airway inflammation leading to impaired respiratory immunity.29,30 several limitations should be considered when interpreting the results from this study . first , the use of registry - based icd-10 codes to identify children with asthma is likely to result in some misclassification . underascertainment of asthma is possible if less severe cases of asthma were missed , most likely due to diagnosis or treatment only by a general practitioner . such underascertainment would bias the irrs describing the association between asthma and pd toward the null because some exposed children would be misclassified as unexposed . overascertainment is also possible if some children with wheezing due to other causes , for example respiratory syncytial virus , were classified as having asthma . although one way to increase sensitivity and specificity of asthma exposure would have been to incorporate the use of prescription asthma medications into a classification scheme for asthma exposure , we did not have access to these data for this study . we were also unable to include a mechanism by which to reclassify asthma - exposed children as unexposed if they grew out of an asthma diagnosis . although imperfect sensitivity and specificity of exposure classification is possible , several studies that have recently evaluated the quality of icd-10-based asthma diagnoses in the danish national registry of patients have found the diagnosis codes to be accurate.17,18 one study that used 3,550 medical records as the gold standard to validate icd-10 inpatient asthma diagnoses recorded in the danish national registry of patients reported 90% sensitivity and 99% specificity of asthma diagnoses among children aged 614 years old.17 another study reported 44% sensitivity and 98% specificity of asthma diagnoses recorded among 18-year - old men reporting for mandatory medical evaluation at the danish military draft board . the authors of this study subsequently demonstrated that the level of nondifferential asthma misclas - sification present in the danish national registry of patients was not sufficient to nullify the association between asthma and various skin cancers that they examined.18 we observed a 6% prevalence of asthma in our study population , which is less than an estimated asthma prevalence of 10%12% based on questionnaire data collected from parents of danish children aged 517 years.31,32 to determine the potential impact of imperfect sensitivity and specificity of exposure ascertainment on our results , we performed a bias analysis by calculating the irr that would have been observed if asthma ascertainment only has a sensitivity equal to 50% and a specificity equal to 97% . this sensitivity is consistent with the differences between the asthma prevalence recorded in this study and that recorded in published reports , and this specificity was the minimum specificity that resulted in no negative cell frequency in the corrected table . we assumed that exposure misclassification was nondifferential and independent for cases and noncases , as the data in our study were prospectively collected . the results from this analysis indicated a minimal impact on the age - specific irrs : the unadjusted irr in children aged 6 to < 24 months old would increase from 2.3 to 2.4 and the irr among children 24 to < 60 months old would increase from 4.8 to 5.0 ; the unadjusted irrs among children 0 to < 6 months old and 60 months old would not change . second , the exclusive use of icd-10 codes to identify pd cases creates the potential for misclassification . some pd cases could have been missed if the resulting illness was mild , if cultures were falsely negative , or due to recordkeeping errors . however , because pd is a serious disease typically requiring medical treatment , icd-10 codes are likely to capture the most important and costly infections . if underascertainment of pd did occur , it was likely to be nondifferential due to the prospective nature of the data which is , in turn , expected to produce unbiased ratio effect estimates33 in the absence of false positives . it is , however , possible that some cases could have been falsely attributed to pd when in fact they were caused by other bacterial infections . although we did not independently verify case status in this study , discharge diagnoses of pd have been found to have high specificity in validation studies conducted by other investigators.16,34 third , although we were able to collect extensive information about pregnancy- and birth - related characteristics , we were not able to capture complete information about some social factors associated with pd . misclassification of exposure to secondary tobacco smoke35 may have occurred in this study because information about smoking was only available from mothers at the beginning of pregnancy , and no information was available from fathers or other childcare providers . despite these limitations , the current study is an important contribution to the current knowledge of the association between asthma and pd . the evidence presented here indicates that asthmatic children are more likely to develop pd compared with nonasthmatic children , thereby providing support for the addition of asthma to the list of pneumococcal vaccine - eligible conditions for older children . these results also indicate that children who have asthma and another underlying comorbidity may be at especially high risk of pd , and should be carefully assessed in the clinic when presenting with bacterial illnesses . effect measure modification observed in stratified analyses was further evaluated for evidence of biologic interaction by using three standard measures1,2 to determine whether the independent effects of asthma and comorbidity summed to the total effect of both factors together . first , the interaction contrast ( ic ) between asthma and comorbidity was calculated by applying the following formula to crude pneumococcal disease ( pd ) incidence rates per 100,000 person years : ic = re+c+re+crec++rec(1)where r represents the rate of disease , e represents exposure to asthma , and c is the modifying covariate which , in this case , was comorbidity . the ic represents the number of cases of disease ( per 100,000 child - years ) that can not be accounted for by baseline factors among children without asthma or comorbidity , asthma only , or comorbidity only , and is therefore presumed to be attributable to a biological interaction between asthma and comorbidity . next , the interaction contrast ratio ( icr ) and 95% confidence interval around the icr was calculated to quantify the excess rate when both asthma and comorbidity were present at the same time relative to the baseline rate of disease that occurred when neither were present , while adjusting for important confounders of the association between asthma and pd:3 icr = icrec=irre+c+irre+cirrec++1(2 ) the icrs in our study were adjusted for sex , birth weight , child year of birth , and congenital malformation . we then used the icr ( to account for confounders ) to calculate the attributable proportion due to interaction ( ap ) , which quantifies the proportion of disease among exposed persons attributable to the interaction between an exposure and a modifying covariate . the ap was calculated by dividing the icr by the irr , comparing children with asthma and comorbidity to children without either of these:3 ap = icrirre+c+=irre+c+irre+cirrec++1irre+c+(3 ) icd-10 codes used to identify asthma , pneumococcal disease , and comorbidities from danish registries abbreviation : icd , international classification of diseases . effect measure modification observed in stratified analyses was further evaluated for evidence of biologic interaction by using three standard measures1,2 to determine whether the independent effects of asthma and comorbidity summed to the total effect of both factors together . first , the interaction contrast ( ic ) between asthma and comorbidity was calculated by applying the following formula to crude pneumococcal disease ( pd ) incidence rates per 100,000 person years : ic = re+c+re+crec++rec(1)where r represents the rate of disease , e represents exposure to asthma , and c is the modifying covariate which , in this case , was comorbidity . the ic represents the number of cases of disease ( per 100,000 child - years ) that can not be accounted for by baseline factors among children without asthma or comorbidity , asthma only , or comorbidity only , and is therefore presumed to be attributable to a biological interaction between asthma and comorbidity . next , the interaction contrast ratio ( icr ) and 95% confidence interval around the icr was calculated to quantify the excess rate when both asthma and comorbidity were present at the same time relative to the baseline rate of disease that occurred when neither were present , while adjusting for important confounders of the association between asthma and pd:3 icr = icrec=irre+c+irre+cirrec++1(2 ) the icrs in our study were adjusted for sex , birth weight , child year of birth , and congenital malformation . we then used the icr ( to account for confounders ) to calculate the attributable proportion due to interaction ( ap ) , which quantifies the proportion of disease among exposed persons attributable to the interaction between an exposure and a modifying covariate . the ap was calculated by dividing the icr by the irr , comparing children with asthma and comorbidity to children without either of these:3 ap = icrirre+c+=irre+c+irre+cirrec++1irre+c+(3 ) icd-10 codes used to identify asthma , pneumococcal disease , and comorbidities from danish registries abbreviation : icd , international classification of diseases .

backgroundalthough asthma has recently been established as a risk factor for pneumococcal disease ( pd ) , few studies have specifically evaluated this association in children.methodswe conducted a nation - wide population - based cohort study of the effect of asthma on childhood pd among all singleton live births in denmark from 1994 to 2007 , before the introduction of the 7-valent pneumococcal conjugate vaccine . all data were abstracted from danish medical registries . because underlying comorbidity substantially increases the pd risk in children , standard methods were used to assess the evidence of biologic interaction between comorbidity and asthma on the risk of pd.resultsthere were 2,253 cases of childhood pd among 888,655 children born in denmark from 1994 to 2007 . the adjusted incidence rate ratio of the effect of asthma on childhood pd was 2.2 ( 95% confidence interval [ ci ] : 2.0 , 2.5 ) . age - stratified incidence rate ratios were 2.1 ( 95% ci : 1.8 , 2.9 ) in children 6 months to < 24 months , 4.1 ( 95% ci : 3.3 , 5.1 ) in children 24 months to < 60 months , and 2.3 ( 95% ci : 1.6 , 3.2 ) in children 60 months . evaluation of the biologic interaction between asthma and comorbidity in older children revealed that 55% ( 24 months to < 60 months ) to 73% ( 60 months ) of cases among asthma - exposed children can be accounted for by the interaction between asthma and comorbidity.conclusionthese results confirm that asthma is an important risk factor for pd in children and suggest that children with underlying comorbidities are more sensitive to the effect of asthma on pd than children without comorbidities .

Introduction Methods Study population Data collection Statistical analyses Results Characteristics of the study population Age-specific PD incidence Association between asthma and PD Discussion Conclusion Supplementary materials Evaluation of observed effect measure modification for evidence of biologic interaction

according to protocol analysis of co - variance reduced - antigen - content diphtheria tetanus and acellular pertussis vaccine filamentous hemagglutinin geometric mean concentration serious adverse event total vaccinated cohort pertussis is a highly infectious disease and remains an important worldwide public health problem , even in countries with sustained high vaccination coverage . despite established infant immunization programmes , pertussis continues to circulate , predominantly due to waning immunity beyond childhood and disease transmission from adolescents and adults to vulnerable infants . the need to maintain antibody levels against pertussis beyond childhood , through booster vaccines is therefore increasingly recognized . reduced - antigen - content combined diphtheria - tetanus - acellular pertussis ( dtpa ) vaccines , such as boostrix [ dtpa ; glaxosmithkline ( gsk ) vaccines ] , have been specifically developed to immunize older children from the age of 4 years , adolescents and adults . boostrix , which was first licensed in 1999 , is currently available in over 70 countries and has a well - established immunogenicity and tolerability profile in populations ranging from school age to the elderly . traditionally , boostrix has been available as a single - dose vial or a prefilled disposable syringe where the tip - cap and plunger stopper contained methylester w1883 . however , following the discontinuation of w1883 production by the manufacturer ( west ) , the syringe presentation has recently been replaced using prefilled syringes from a different manufacturer , wherein the tip - cap component contains fm27 ( a latex - free non - cytotoxic rubber compound ) and the plunger stopper component contains fm457 ( an ultra - low extractable bromobutyl compound ) . although this presentation change has been approved on the basis of a technical variation , this non - inferiority study was conducted to evaluate the impact on safety and immunogenicity , if any , due to the change in the material used in the rubber plunger of the prefilled syringe as required by a regulatory agency ; it compared the immunogenicity and safety of dtpa vaccine injected using the old and new syringe presentations , and thereby support the change with clinical data . this phase iv , randomized , single - blind , parallel group study ( nct01362322 ) , funded by glaxosmithkline biologicals sa , was conducted across 3 centers in chile and mexico between july 2011 and september 2012 . francisco i madero pte s / n y dr e aguirre pequeno , col mitras centro , monterrey , mexico ; subcomite de etica en investigacion , hospital general de ecatepec las america , estado de mexico ; comit de tica en investigacin , facultad de medicina , pontificia universidad catlica de chile ; comit tico cientfico del servicio de salud metropolitano central , santiago ; institute of public health chile ] and adhered to the declaration of helsinki and good clinical practice guidelines . written , informed consent was obtained from parents / guardians and assent from subjects before enrolment . healthy adolescents aged 1015 years who had received 5 or 6 previous doses of dt(p)/dt(pa ) vaccine were randomized ( 1:1 ) to receive dtpa booster via the new ( dtpa - new ) or previous ( dtpa - previous ) syringe presentations . due to visual differences in the presentation of the 2 syringes , this study was conducted in a single - blind manner . each 0.5 ml dtpa vaccine dose contained 2 iu diphtheria toxoid , 20 iu tetanus toxoid , 8 g pertussis toxin ( pt ) , 8 g filamentous hemagglutinin ( fha ) and 2.5 g pertactin ( prn ) . the vaccine was supplied in 2 prefilled syringe presentations : dtpa - previous group had syringes with w1833 tip - caps and plunger stoppers ( lot . no : dc37a005b and expiry date : 31 aug 2013 ) ; dtpa - new group had syringes with fm27 tip - caps and fm457 plunger stoppers ( lot . a single booster dtpa dose was injected intramuscularly into the deltoid region of the non - dominant arm , using a needle 2.54 cm length and 2225 gauge . blood samples ( 5 ml ) were collected from all subjects before , and one month post - booster dosing . antibodies against diphtheria , tetanus and pertussis antigens were measured using standard enzyme - linked immunosorbent assay ( elisa ) . seroprotection against diphtheria and tetanus antigens was defined as an antibody concentration 0.1 iu / ml . a booster response to diphtheria and tetanus antigens was defined as antibody concentrations 4-fold the assay cut - off in initially seronegative subjects or 4-fold increase in pre - vaccination antibody concentrations in initially seropositive subjects . seropositivity against pertussis antigens was defined as an antibody concentration 5 el.u / ml per antigen . a booster response to these antigens was defined as antibody concentrations 4-fold the assay cut - off in initially seronegative subjects ; a 4-fold increase in pre - vaccination antibody concentrations in initially seropositive subjects ( pre - vaccination concentrations 5 to < 20 el.u / ml ) or 2-fold increase in pre - vaccination antibody concentrations in initially seropositive subjects with pre - vaccination concentrations 20 el.u / ml . diary cards were used to assess solicited local ( injection site pain , redness and swelling ) and general ( fatigue , headache , fever [ axillary temperature 37.5c ] and gastrointestinal [ gi ] symptoms ) adverse events for 4 days ( day 03 ) after vaccination . the intensity of symptoms was graded on a 3-point scale : grade 3 redness and swelling : diameter > 50 mm ; grade 3 fever : axillary temperature > 39.0c . for all other symptoms large injection site reactions ( defined as swelling with a diameter > 100 mm , noticeable diffuse swelling or noticeable increase of limb circumference ) were evaluated by the investigator . all other symptoms , including serious adverse events ( saes ) occurring within 31 days of vaccination were recorded . the primary objective of the study was to demonstrate that dtpa - new is non - inferior to dtpa - previous , in terms of immune response to all vaccine antigens , one month after booster vaccination . the criteria for evaluation was that the upper limit ( ul ) of the 95% confidence interval ( ci ) on the gmc ratios [ dtpa - previous over dtpa - new ] for anti - diphtheria , anti - tetanus , anti - pt , anti - fha and anti - prn antibodies was 1.5 ( clinical limit for non - inferiority ) . the 95% cis for the gmc ratio of the 2 study groups was computed using an analysis of co - variance ( ancova ) model including the group and number of previous dt doses ( 5 or 6 ) as fixed effects and the log - transformed pre - vaccination concentration as co - variable . with a minimum of 600 evaluable subjects , the study had 94% power ( bonferroni adjustment of ) to achieve the primary objective . assuming a dropout rate of around 10% , a total of 670 subjects ( 335 subjects in each group ) were to be randomized to ensure a sufficient number of evaluable subjects were available for inclusion in the atp cohort for analysis of immunogenicity . secondary objectives included the evaluation of seroprotection / seropositivity rates , booster response and safety analysis one month after booster vaccination . the primary analysis of immunogenicity was performed on the according - to - protocol ( atp ) cohort , comprising vaccinated subjects who met the eligibility criteria , complied with protocol - defined procedures and for whom immunogenicity data were available . the analysis of safety was performed on the total vaccinated cohort ( tvc ) , which comprised all study participants for whom safety data were available . the safety results are described . of 671 subjects enrolled in the current study [ 376 at pontificia universidad , catolica de chile , santiago ; 93 at hospital universitario , de la uanl , monterrey ; mexico and 202 at hospital general de ecatepec las americas , estado de mexico , mexico ] , 335 received dtpa - new and 336 received dtpa - previous and were included in the tvc . one subject from the dtpa - previous group was eliminated from the atp cohort for safety after receiving a vaccine forbidden in protocol . fourteen subjects were eliminated from the atp cohort in the dtpa - new group due to non - compliance with blood sampling ( 8) and missing serological data ( 6 ) ; 16 were eliminated from the atp cohort in the dtpa - previous group due to protocol violation ( 1 ) , non - compliance with blood sampling ( 8) and missing serological data ( 7 ) . the atp cohort for immunogenicity therefore included 321 and 319 subjects in the dtpa - new and dtpa - previous groups , respectively . the mean age of the subjects in the atp cohort for immunogenicity was 11.9 years ( standard deviation 1.61 ) ; 50.6% subjects were of hispanic origin and 53.0% were female . before booster vaccination , 88.5% subjects in both groups were seroprotected against diphtheria ; 96.9% subjects in both groups were seroprotected against tetanus ( table 1 ) . at least 54.7% were seropositive against pertussis antibodies before the booster dose in both groups ( table 1 ) . table 1.seroprotection/seropositivity rates and gmcs one month after booster vaccination ( atp cohort for immunogenicity)dtpa - newdtpa - previousantibodytimingnnseroprotection ( 95% ci)gmc(95% ci)nnseroprotection ( 95% ci)gmc(95% ci)anti - diphtheriapre32128488.5 ( 84.591.8)0.472 ( 0.4030.553)31928689.7 ( 85.892.8)0.456 ( 0.3920.530)post32132099.7 ( 98.3100)6.784 ( 6.1787.450)319319100 ( 98.9100)6.493 ( 5.9157.128)anti - tetanuspre32131196.9 ( 94.398.5)0.956 ( 0.8351.095)31931498.4 ( 96.499.5)0.899 ( 0.7891.026)post321321100 ( 98.9100)18.937 ( 17.31320.713)319319100 ( 98.9100)18.515 ( 16.85120.342)dtpa - newdtpa - previousantibodytimingnnseropositivity ( 95% ci)gmc(95% ci)nnseropositivity ( 95% ci)gmc ( 95% ci)anti - ptpre32017554.7 ( 49.160.2)7.5 ( 6.68.7)31917554.9 ( 49.260.4)7.2 ( 6.38.2)post31831699.4 ( 97.799.9)140.2 ( 126.0156.1)31831599.1 ( 97.399.8)125.9 ( 112.7140.7)anti - fhapre31631098.1 ( 95.999.3)48.9 ( 43.355.2)31531098.4 ( 96.399.5)49.4 ( 43.656.0)post319319100 ( 98.9100)1080.2 ( 995.21172.5)319319100 ( 98.9100)1013.7 ( 940.01093.2)anti - prnpre32126983.8 ( 79.387.7)14.0 ( 12.315.9)31927285.3 ( 80.989.0)13.4 ( 11.915.0)post321321100 ( 98.9100)652.4 ( 572.1743.9)318318100 ( 98.8100)619.2 ( 546.0702.2)dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation.n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval ; d , diphtheria ; t , tetanus ; pt , pertussis toxin ; fha , filamentous hemagglutinin ; prn , pertactin iu , international unit ; el.u , elisa unit ; gmc , geometric mean concentration calculated on all subjects.*seroprotection = anti - diphtheria and anti - tetanus antibody concentration 0.1 iu / ml.**seropositive = anti - pt , anti - fha and anti - prn antibodies 5 eu / ml . seroprotection / seropositivity rates and gmcs one month after booster vaccination ( atp cohort for immunogenicity ) dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation . n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval ; d , diphtheria ; t , tetanus ; pt , pertussis toxin ; fha , filamentous hemagglutinin ; prn , pertactin iu , international unit ; el.u , elisa unit ; gmc , geometric mean concentration calculated on all subjects . seroprotection = anti - diphtheria and anti - tetanus antibody concentration 0.1 iu / ml . seropositive = anti - pt , anti - fha and anti - prn antibodies 5 eu / ml . one month after booster vaccination , 99.7% subjects in both groups were seroprotected against diphtheria antigens ; all subjects were seroprotected against tetanus antigens ; 99.4% were seropositive against the pertussis antigens ( table 1 ) . as the uls of the 95% ci for the gmc ratios ( dtpa - previous / dtpa - new ) for all antigens were 1.5 ( table 2 ) , non - inferiority of dtpa injected via the new syringe presentation ( dtpa - new ) against the previously used syringe presentation ( dtpa - previous ) table 2.adjusted gmc ratios between groups ( dtpa - previous divided by dtpa - new ) one month after booster vaccination ( atp cohort for immunogenicity)dtpa - previousdtpa - newadjusted gmc ratio ( dtpa - pre group/ dtpa - new group)antibodynadjusted gmcnadjusted gmcvalue ( 95% ci)anti - diphtheria3196.5213216.7650.96 ( 0.851.09)anti - tetanus31918.67232119.1710.97 ( 0.861.10)anti - pt318128.340317138.8320.92 ( 0.821.04)anti - fha3151013.1673141096.8270.92 ( 0.831.03)anti - prn318634.592321645.5040.98 ( 0.851.13)dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation.n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval for the adjusted gmc ratio [ ancova model : adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) pooled variance ] ) ; ll = lower limit , ul = upper limit.adjusted gmc = geometric mean antibody concentration obtained from an ancova model adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) . adjusted gmc ratios between groups ( dtpa - previous divided by dtpa - new ) one month after booster vaccination ( atp cohort for immunogenicity ) dtpa - new = subjects who received boostrix in new syringe presentation ; dtpa - previous = subjects who received boostrix in previous syringe presentation . n = number of subjects with both pre- and post - vaccination results available ; 95% ci = 95% confidence interval for the adjusted gmc ratio [ ancova model : adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) pooled variance ] ) ; ll = lower limit , ul = upper limit . adjusted gmc = geometric mean antibody concentration obtained from an ancova model adjusted for pre - booster concentration and number of previous dt(p)/dt(pa ) doses ( 5 or 6 ) . irrespective of which different syringe presentation was used for vaccine delivery , robust immune responses were observed and booster response rates for all antigens ranged from 79.0% to 99.7% in the 2 study groups ( data not shown ) . during the 4-day post - vaccination follow - up period , 78.8% and 83.0% subjects reported at least one symptom in the dtpa - new and dtpa - previous groups , respectively . injection site pain was the most commonly reported solicited local symptom in 71.8% ( 7.3% grade 3 ) and 75.4% ( 6.1% grade 3 ) of the subjects in the dtpa - new and dtpa - previous groups , respectively . headache , which was reported by 26.7% and 32.8% of the subjects , and fatigue , reported by 25.2% and 26.1% of the subjects , were the most frequently reported solicited general symptoms in the dtpa - new and dtpa - previous groups , respectively ( fig . 1 ) . figure 1.incidence of solicited local and general symptoms reported during the 4-day post - vaccination follow - up period ( total vaccinated cohort ) . incidence of solicited local and general symptoms reported during the 4-day post - vaccination follow - up period ( total vaccinated cohort ) . during the 31-day post - vaccination follow - up period , at least one unsolicited symptom was reported in 13.1% ( 2.4% grade 3 ) and 13.4% ( 2.7% grade 3 ) subjects in the dtpa - new and dtpa - previous groups , respectively . one subject in the dtpa - new group suffered accidental injury 13 days after vaccination ; this sae was considered to be unrelated to vaccination . have been distributed [ data on file ] and the immunogenicity , reactogenicity and safety of the vaccine has been well established in clinical trials and routine practice across a broad age range . owing to a recent technical change in the boostrix syringe presentation , this study was undertaken to compare the immunogenicity and safety of the new and previous syringe presentations , which differed in the nature of the compounds present in the tip - caps and plunger stoppers . in this study , the dtpa vaccine was immunogenic regardless of which syringe presentation was used to administer the vaccine . the results were consistent with previous reports in adolescents and non - inferiority of the new presentation compared to the old presentation was demonstrated . dtpa administered using the new dtpa syringe presentation was also well tolerated and the incidence and nature of adverse events were similar irrespective of the syringe presentation and comparable with previous studies . large swelling reactions , which can be associated with repeated booster doses of dtpa vaccines , were not observed in either study group . due to the resurgence of pertussis in adolescents and adults , the need to maintain antibody levels against pertussis beyond childhood , through booster vaccines although limited by single - blind design and inconsistent vaccination history [ subjects having received either 5 or 6 previous dt(p)/dt(pa ) vaccine doses ] , we demonstrated that a single dtpa booster dose was highly immunogenic and well tolerated in healthy adolescents in chile and mexico , irrespective of which syringe presentation was employed . in conclusion , clinical data from this study support the technical data and the use of the new syringe presentation with fm27 tip - caps and fm457 plunger stoppers to deliver the dtpa vaccine . hhh , sk , gj , kh , yc and al are employees of glaxosmithkline vaccines and hhh , gj , kh and al declare having glaxosmithkline stocks . ka has received grants , personal fees and non - financial support from glaxosmithkline and np - r has received research support from glaxosmithkline . glaxosmithkline vaccines was involved in all stages of the study conduct and analysis ; and also took charge of all costs associated with developing and publishing the manuscript .

reduced - antigen - content diphtheria - tetanus - acellular pertussis ( dtpa ) vaccine , boostrix , is indicated for booster vaccination of children , adolescents and adults . the original prefilled disposable dtpa syringe presentation was recently replaced by another prefilled - syringe presentation with latex - free tip - caps and plunger - stoppers . 671 healthy adolescents aged 1015 years who had previously received 5 or 6 previous dt(p)/dt(pa ) vaccine doses , were randomized ( 1:1 ) to receive dtpa booster , injected using the new ( dtpa - new ) or previous syringe ( dtpa - previous ) presentations . immunogenicity was assessed before and 1-month post - booster vaccination ; safety / reactogenicity were assessed during 31-days post - vaccination . non - inferiority of dtpa - new versus dtpa - previous was demonstrated for all antigens ( uls 95% cis for gmc ratios ranged between 1.03 - 1.13 ) . 1-month post - booster , immune responses were in similar ranges for all antigens with both syringe presentations . dtpa delivered using either syringe presentation was well - tolerated . these clinical results complement the technical data and support the use of the new syringe presentation to deliver the dtpa vaccine .

Abbreviations Introduction Disclosure of Potential Conflicts of Interest Funding Trial Number

to identify the possible barriers to the implementation of pharmaceutical care among community and hospital pharmacists in enugu state using nsukka and enugu metropolis as a case study . the questionnaires were distributed to community and hospital pharmacists from designated areas during one of their quarterly meeting and their practice sites in 2009 . eighty completed questionnaires were collected with 22.8% from community pharmacists , and 77.2% from hospital pharmacists . the important barriers identified were lack of space , enough personnel in pharmacy to handle routine technical tasks , time , need for too much effort , and need for payment for services . the opinions on barriers to pharmaceutical care of pharmacists from community and hospital practice areas in these two metropolises of enugu state are majorly lack of time , space and routine technical task personnel .

objective : to identify the possible barriers to the implementation of pharmaceutical care among community and hospital pharmacists in enugu state using nsukka and enugu metropolis as a case study.method:a semi structured questionnaire was designed to carry out a cross sectional descriptive study . the questionnaires were distributed to community and hospital pharmacists from designated areas during one of their quarterly meeting and their practice sites in 2009.results:eighty completed questionnaires were collected with 22.8% from community pharmacists , and 77.2% from hospital pharmacists . the important barriers identified were lack of space , enough personnel in pharmacy to handle routine technical tasks , time , need for too much effort , and need for payment for services.conclusion:the opinions on barriers to pharmaceutical care of pharmacists from community and hospital practice areas in these two metropolises of enugu state are majorly lack of time , space and routine technical task personnel .

Objective: Method: Results: Conclusion:

we retrospectively evaluated 128 consecutive young adults who were under 40 years of age and who had undergone a ccta evaluation with 64-slice multidetector ct ( mdct ) as a screening test for detecting cad during a general health evaluation from january 2006 to may 2008 . we excluded 16 subjects who had chest pain or discomfort ( n = 15 ) and those with a history of acute coronary syndrome ( n = 1 ) . none of the subjects were excluded due to nondiagnostic ccta image quality . as a result , 112 asymptomatic young adults ( 90 men and 22 women ) were finally enrolled . the mean age of the study population was 35.6 3.7 years ( range : 28 - 40 years ) . all the subjects had conventional coronary risk factors such as diabetes mellitus ( n = 8) , cigarette smoking ( n = 54 ) , hypertension ( n = 36 ) or obesity ( n = 62 ) . there were 77 subjects with a low cad risk , 27 with a moderate risk and eight with a high risk . a single oral dose of 20 mg of a -blocker ( propranolol [ pranol ; daewoong , korea ] ) was administered one hour before mdct for the patients with a heart rate of > 65 beats per minute ( bpm ) . for those with a heart rate that was still greater than 65 bpm , an additional 20 mg of oral propranolol was administered one hour after the administration of the first dose of oral -blocker . ccta was not performed for patients with heart rates more than 70 bpm after repeated drug administration or for those with an intermittent arrhythmia . coronary vasodilatation was achieved by administering 0.6 mg nitroglycerin ( myung moon , seoul , korea ) sublingually before the ccta to obtain the maximum coronary artery opacification . all the examinations were performed using a 64-slice mdct ( somatom sensation 64 , siemens medical solutions , germany ) and the following scan parameters were used : tube voltage 120 kvp , tube current 750 effective mas , detector collimation 64 0.6 mm , gantry rotation 370 msec and pitch 0.24 . a bolus of 70 ml iopamidol ( 370 mg of iodine per milliliter ; iopamiro 370 ; bracco , milan , italy ) was injected intravenously into an antecubital vein at a flow rate of 4 ml / sec , and this was followed by a 40 - 50 ml saline chaser using the bolus tracking technique . the images were initially reconstructed at the mid - diastolic phase ( 60 - 70% of the rr interval ) of the cardiac cycle , and they were then transferred to a computed workstation ( leonardo , siemens medical solutions , germany ) . all the scans were retrospectively analyzed on a three - dimensional workstation by two radiologists ( 14 and 7 years of experience in chest ct , respectively ) , using the multiplanar reformation technique , the maximum intensity projection technique and the volume - rendering technique with a short - axis and the two - chamber and four - chamber views . the coronary artery tree was segmented according to the modified american heart association classification ( 15 segments ) , and these segments were subsequently investigated for the presence and characteristics of coronary plaques ( 6 ) . the degree of stenosis was classified as significant if the patient had more than a 75% area stenosis on the cross - sectional images or more than a 50% diameter stenosis on the longitudinal images . coronary plaques were classified as non - calcified ( plaques without visible calcification ) , mixed ( plaques with non - calcified and calcified components ) or calcified plaques ( completely calcified plaques ) , according to the calcified component of the plaques seen on ccta . plaque densities of more than 130 hu ( hounsfield unit ) on the native scans were classified as calcified ( fig . changes in the coronary artery diameter were measured to determine the remodeling index ( ri ) . the maximum outer diameter at the lesion and at the proximal reference site was measured to calculate the ri ( i.e. , ri = [ maximum outer diameter at the plaque site - maximum outer diameter at the reference vessel ] / maximum outer diameter at the reference vessel ) . positive remodeling was defined as an ri > 0 and negative remodeling as an ri < 0 . the degree of remodeling was calculated by percentage ( i.e. , mean values standard deviations ) . measurements of the vascular diameter were obtained from the manually traced maximal diameters taken from the longitudinal source images ( fig . all the study related data , including the demographics , symptoms , medical histories and laboratory results , was systemically acquired from the hospital database and a chart review . the conventional coronary risk factors such as obesity , cigarette smoking , hypertension , hypercholesterolemia and diabetes mellitus were assessed , and the serum biomarkers such as homocysteine , c - reactive protein and triglyceride were measured . the framingham risk scores , as used by the national cholesterol education program ( ncep ) guidelines , were also calculated ( 7 ) . all the subjects were assigned to one of three different risk groups according to the revised ncep guidelines : the high - risk group ( cad risk equivalents or a 10-year risk > 20% ) , the moderate - risk group ( more than two risk factors and a 10-year risk 20% ) and the low - risk group ( 0 - 1 risk factors ) . of the 112 subjects , 109 underwent abdominal ultrasonography as part of a general health evaluation within one week of ccta , and the presence or absence of a fatty liver was determined . the degree of fatty liver on ultrasonography was classified as follows : 1 ) a mild , minimal diffuse increase of the hepatic echogenicity with normal visualization of the diaphragm and intrahepatic vessel borders ; 2 ) a moderate , moderate diffuse increase of the hepatic echogenicity with slightly impaired visualization of the intrahepatic vessels and diaphragm ; 3 ) a severe , marked increase of the echogenicity with poor visualization or nonvisualization of the hepatic vessels and diaphragm . we also compared the patients with cad to the normal subjects with respect to the coronary risk factors and a fatty liver . the clinical follow - up data ( range : 10 - 39 months ) was obtained from the patients who exhibited cad on ccta , based on the cardiac events , the treatment and the laboratory findings . all the statistical analyses were performed using spss version 17.0 for windows ( spss inc . , the differences between categorical variables were analyzed using chi - square or fisher 's exact tests , and the differences between continuous variables were analyzed using the unpaired student 's t - test or mann - whitney test , as appropriate . we retrospectively evaluated 128 consecutive young adults who were under 40 years of age and who had undergone a ccta evaluation with 64-slice multidetector ct ( mdct ) as a screening test for detecting cad during a general health evaluation from january 2006 to may 2008 . we excluded 16 subjects who had chest pain or discomfort ( n = 15 ) and those with a history of acute coronary syndrome ( n = 1 ) . none of the subjects were excluded due to nondiagnostic ccta image quality . as a result , 112 asymptomatic young adults ( 90 men and 22 women ) were finally enrolled . the mean age of the study population was 35.6 3.7 years ( range : 28 - 40 years ) . all the subjects had conventional coronary risk factors such as diabetes mellitus ( n = 8) , cigarette smoking ( n = 54 ) , hypertension ( n = 36 ) or obesity ( n = 62 ) . there were 77 subjects with a low cad risk , 27 with a moderate risk and eight with a high risk . a single oral dose of 20 mg of a -blocker ( propranolol [ pranol ; daewoong , korea ] ) was administered one hour before mdct for the patients with a heart rate of > 65 beats per minute ( bpm ) . for those with a heart rate that was still greater than 65 bpm , an additional 20 mg of oral propranolol was administered one hour after the administration of the first dose of oral -blocker . ccta was not performed for patients with heart rates more than 70 bpm after repeated drug administration or for those with an intermittent arrhythmia . coronary vasodilatation was achieved by administering 0.6 mg nitroglycerin ( myung moon , seoul , korea ) sublingually before the ccta to obtain the maximum coronary artery opacification . all the examinations were performed using a 64-slice mdct ( somatom sensation 64 , siemens medical solutions , germany ) and the following scan parameters were used : tube voltage 120 kvp , tube current 750 effective mas , detector collimation 64 0.6 mm , gantry rotation 370 msec and pitch 0.24 . retrospective electrocardiogram ( ecg ) gating and ecg - gated dose modulation were used . a bolus of 70 ml iopamidol ( 370 mg of iodine per milliliter ; iopamiro 370 ; bracco , milan , italy ) was injected intravenously into an antecubital vein at a flow rate of 4 ml / sec , and this was followed by a 40 - 50 ml saline chaser using the bolus tracking technique . the images were initially reconstructed at the mid - diastolic phase ( 60 - 70% of the rr interval ) of the cardiac cycle , and they were then transferred to a computed workstation ( leonardo , siemens medical solutions , germany ) . all the scans were retrospectively analyzed on a three - dimensional workstation by two radiologists ( 14 and 7 years of experience in chest ct , respectively ) , using the multiplanar reformation technique , the maximum intensity projection technique and the volume - rendering technique with a short - axis and the two - chamber and four - chamber views . the coronary artery tree was segmented according to the modified american heart association classification ( 15 segments ) , and these segments were subsequently investigated for the presence and characteristics of coronary plaques ( 6 ) . the degree of stenosis was classified as significant if the patient had more than a 75% area stenosis on the cross - sectional images or more than a 50% diameter stenosis on the longitudinal images . coronary plaques were classified as non - calcified ( plaques without visible calcification ) , mixed ( plaques with non - calcified and calcified components ) or calcified plaques ( completely calcified plaques ) , according to the calcified component of the plaques seen on ccta . plaque densities of more than 130 hu ( hounsfield unit ) on the native scans were classified as calcified ( fig . 1 ) . changes in the coronary artery diameter were measured to determine the remodeling index ( ri ) . the maximum outer diameter at the lesion and at the proximal reference site was measured to calculate the ri ( i.e. , ri = [ maximum outer diameter at the plaque site - maximum outer diameter at the reference vessel ] / maximum outer diameter at the reference vessel ) . positive remodeling was defined as an ri > 0 and negative remodeling as an ri < 0 . the degree of remodeling was calculated by percentage ( i.e. , mean values standard deviations ) . measurements of the vascular diameter were obtained from the manually traced maximal diameters taken from the longitudinal source images ( fig . all the study related data , including the demographics , symptoms , medical histories and laboratory results , was systemically acquired from the hospital database and a chart review . the conventional coronary risk factors such as obesity , cigarette smoking , hypertension , hypercholesterolemia and diabetes mellitus were assessed , and the serum biomarkers such as homocysteine , c - reactive protein and triglyceride were measured . the framingham risk scores , as used by the national cholesterol education program ( ncep ) guidelines , were also calculated ( 7 ) . all the subjects were assigned to one of three different risk groups according to the revised ncep guidelines : the high - risk group ( cad risk equivalents or a 10-year risk > 20% ) , the moderate - risk group ( more than two risk factors and a 10-year risk 20% ) and the low - risk group ( 0 - 1 risk factors ) . of the 112 subjects , 109 underwent abdominal ultrasonography as part of a general health evaluation within one week of ccta , and the presence or absence of a fatty liver was determined . the degree of fatty liver on ultrasonography was classified as follows : 1 ) a mild , minimal diffuse increase of the hepatic echogenicity with normal visualization of the diaphragm and intrahepatic vessel borders ; 2 ) a moderate , moderate diffuse increase of the hepatic echogenicity with slightly impaired visualization of the intrahepatic vessels and diaphragm ; 3 ) a severe , marked increase of the echogenicity with poor visualization or nonvisualization of the hepatic vessels and diaphragm . we also compared the patients with cad to the normal subjects with respect to the coronary risk factors and a fatty liver . the clinical follow - up data ( range : 10 - 39 months ) was obtained from the patients who exhibited cad on ccta , based on the cardiac events , the treatment and the laboratory findings . all the statistical analyses were performed using spss version 17.0 for windows ( spss inc . , the differences between categorical variables were analyzed using chi - square or fisher 's exact tests , and the differences between continuous variables were analyzed using the unpaired student 's t - test or mann - whitney test , as appropriate . atheromatous plaques were noted in 15 segments in 12 of 112 subjects ( 11% ) . nine patients had single - vessel disease , and three had two - vessel disease . there were 11 men and one woman with an age range from 31 to 40 years ( mean age : 36.8 2.6 years ) with atheromatous plaques . the patient population included four of 77 subjects ( 5% ) with a low cad risk , six of 27 ( 22% ) with a moderate risk and two of eight ( 25% ) with a high cad risk . the prevalence of cad was significantly higher in the moderate cad risk group than that in the low risk group ( p = 0.018 ) ; however , there was no significant difference between the moderate and high risk groups ( p = 0.604 ) ( fig . the presence of obesity , hypertension , hypercholesterolemia , high triglycerides and fatty liver was significantly higher in the patients with cad than that in the normal individuals . there also tended to be a higher incidence of cigarette smoking , high ldl ( low - density lipoprotein ) cholesterol , low hdl ( high - density lipoprotein ) cholesterol , diabetes mellitus and high c - reactive protein in the patients with cad ; however , any statistical significance was not identified ( table 1 ) . the location and degree of stenosis as well as the type of plaque are shown in table 2 . the coronary plaques were located in the left anterior descending ( lad ) ( n = 11 , 73% ) and the left main ( lm ) ( n = 4 , 27% ) arteries , with the most common location being the proximal lad ( n = 9 , 60% ) ( fig . the types of plaque included non - calcified ( n = 4 , 27% ) , mixed ( n = 7 , 47% ) and calcified ( n = 4 , 27% ) . the changes in the vascular diameter at the cad site were measured in four coronary segments with noncalcified plaque and in seven with mixed plaques . this measure was not evaluated in segments with calcified plaques because the exact measurement of the vascular diameter was unavailable due to beam - hardening and blooming artifacts . positive remodeling was identified in all the segments with noncalcified or mixed plaques , and the degree of positive remodeling tended to be higher in the patients with noncalcified plaques than in those with mixed plaques ( table 3 ) ; however , there was no statistically significant difference between the two groups ( p = 0.471 ) . twelve patients with cad have been free of cardiac events 10 - 39 months after ccta . four of the five patients with hypercholesterolemia started antihypercholesterolemic medication , based on the results of the ccta , and they showed improvement of their hypercholesterolemia on the follow - up tests . early detection of cad provides the opportunity to initiate interventions that will stabilize existing lesions , including ldl - lowering drug therapy ( statins ) , smoking cessation intervention for cigarette smokers , blood pressure lowering for persons with hypertension , and lifestyle intervention for the physically inactive and obese . such interventions can reduce the risk of developing acute coronary syndromes later in life . the current approach for managing the asymptomatic individuals who are at risk for cad is based on traditional clinical risk assessments such as the framingham risk score or the ncep guidelines ( 7 ) . however , there is growing evidence that these traditional risk assessment tools , which are based on risk factor analysis , have substantial limitations when they are used to guide individual therapy ( 8 , 9 ) . in a recently published study by choi et al . ( 10 ) in which cad was evaluated using ccta in 1,000 middle - aged asymptomatic adults ( mean age : 50 years ) , cad was noted in 215 subjects ( 22% ) . this rate is higher than that observed in earlier studies ( 3 - 5% ) , and the results were confirmed by coronary angiography in the asymptomatic individuals ( 11 - 13 ) . to date , there is a paucity of data regarding the prevalence of atherosclerotic plaques as detected on ccta in asymptomatic young adults , and this is largely because these asymptomatic young adults typically do not undergo medical evaluations for cad . in this study that enrolled asymptomatic young adults ( i.e. , < 40 years of age ) who were at risk for cad , coronary plaques were demonstrated in 11% of the subjects , and the prevalence of cad was significantly higher in the subjects with moderate ( 22% ) or high ( 25% ) risk than in those subjects with low risk ( 5% ) . positive remodeling was noted in all the coronary segments that exhibited noncalcified or mixed plaques with non - significant stenosis . this finding may indicate an important cause for underestimating cad on coronary angiography and it may explain the acute coronary syndrome of young patients with angiographically normal coronary arteries ( 14 - 16 ) . positive remodeling occurs in the early stages of coronary plaque development , and it is well known that positive remodeling is related to plaque instability , suggesting it is more prone to rupture and erosion with subsequent coronary events ( 17 - 19 ) . nakamura et al . ( 19 ) reported that positive remodeling was observed more frequently in patients with acute coronary syndrome ( 78 - 82% ) than in patients with stable cad ( 33 - 40% ) , and a significantly higher degree of positive remodeling was noted in patients with acute myocardial infarction ( remodeling index , 1.26 0.15 ) than in elderly patients with unstable angina pectoris ( 1.11 0.10 ) or stable angina pectoris ( 0.94 0.11 ) ( mean age , 63 10 years ) . tanaka et al . ( 18 ) demonstrated that all lipid core plaques showed positive vascular remodeling , which is in contrast to the severely calcified plaques that did not show positive remodeling . furthermore , it is well known from angiographic studies that most myocardial infarctions occur at sites that previously caused only mild to moderate luminal stenosis ( 20 , 21 ) . taken together , these studies demonstrate the clinical significance and importance of early plaques with mild stenosis . choi et al . ( 10 ) also reported that 85% of cad presented at the lad . in our study , plaques were not observed in the left circumflex artery , the peripheral branches of the coronary arteries or the diagonal branches . this may have resulted from the limited temporal resolution of ccta , which would underestimate the small branches of the coronary arteries , or it may also result from a lower prevalence of plaques in those segments in patients with early - stage cad . the coronary calcium score is intimately associated with the total plaque burden ( 22 ) and it is a strong predictor of coronary events , and the coronary calcium score is independent of the traditional coronary risk factors or risk - factor scores ( 23 ) . however , it sometimes fails to predict acute coronary events or significant stenosis because a significant numbers of vulnerable plaques tend to be predominantly noncalcified and nonstenotic lesions ( 20 ) . the absence of coronary calcification has been described in 1% of the male patients with significant coronary stenosis ( 24 ) and in 4% of the patients suffering from an unheralded myocardial infarction ( 25 ) . in our study , 27% of the patients with cad had only noncalcified plaques , which is slightly higher than the 19% reported by choi et al . this may be linked to the observation that the plaques in young patients are probably in the early stages , so they have less calcification than those of older individuals . therefore , the coronary calcium score may underestimate cad in young adults to a greater degree than that in older individuals . obesity , smoking , hypertension and hypercholesterolemia are well known risk factors for cad , and their incidences were significantly higher in the patients with cad than that in the normal subjects of our study . in a recent report , fatty liver was demonstrated to be associated with the risk of cad and early atherosclerosis in middle - aged individuals ( 26 ) . in the current study , we were able to evaluate the incidence of fatty liver because almost all the subjects underwent abdominal ultrasonography and ccta as a part of a general health evaluation . as such , we also found that a fatty liver was significantly more prevalent in the patients with cad ( 83% ) than that in the normal individuals ( 39% ) . the study population was small , it included only koreans living in urban areas and all the subjects were self - referred . therefore , the actual prevalence of cad in the young asymptomatic subjects in various ethnic groups and geographic regions may differ from our results , although this was evaluated in relation to the ncep cad risk categories . in conclusion , the prevalence of occult cad was not negligible in asymptomatic young adults , and the prevalence of cad was significantly higher in those subjects with moderate to high risks than in those with low risk . additionally , most of the patients had single - vessel disease , plaques of various composition , non - significant stenosis and positive vascular remodeling and one fourth of the patients had non - calcified plaques . finally , the most common location for plaques was the proximal lad . this study suggests the importance of management and risk factor modification for the asymptomatic young adults who have a moderate or high risk for cad .

objectivewe aimed at evaluating the prevalence and ct characteristics of occult coronary artery disease ( cad ) in young korean adults under 40 years of age by performing coronary ct angiography ( ccta).materials and methodswe retrospectively enrolled 112 consecutive asymptomatic subjects ( 90 men , mean age : 35.6 3.7 years ) who underwent ccta as part of a general health evaluation . we classified the subjects into three national cholesterol education program risk categories and we assessed the plaque characteristics on ccta according to the number of involved vessels , the location and type of plaques and vascular remodeling.resultstwelve individuals had cad ( 11% , 11 men ) . the prevalence of cad was significantly higher in the subgroups with moderate ( 22% ) or high ( 25% ) risk than that in the low risk subgroup ( 5% ) ( p < 0.05 ) . nine patients had single - vessel disease and three patients had two - vessel disease . the most common location for plaque was the proximal left anterior descending coronary artery ( 60% ) . all the patients had non - significant stenosis and plaque , including the non - calcified ( 27% ) , mixed ( 47% ) and calcified ( 27% ) types . positive vascular remodeling was identified in all the patients with non - calcified or mixed plaques.conclusionthe prevalence of occult cad was not negligible in the asymptomatic young adults with moderate to high risk , and this suggests the importance of management and risk factor modification in this population . all the patients had non - significant stenosis , and one fourth of the plaques did not show calcification .

MATERIALS AND METHODS Study Population Data Acquisition Image Analysis Coronary Artery Disease Risk Assessment and the Clinical Outcomes Statistical Analysis RESULTS DISCUSSION

several risk factors such as contact lens wear , trauma , ocular surface disease , ocular surgery , and systemic disease have been reported to predispose patients to corneal infections . management of microbial keratitis commonly involves obtaining corneal scrapings for microbiological studies , and then , empiric broad - spectrum treatment is typically initiated before culture results are available . appropriate empiric therapy is selected by practitioners on the basis of epidemiological information such as predisposing factors and the spectrum of causative organisms . for example , trauma is a common risk factor for fungal keratitis in developing agricultural countries , whereas contact lens wear is the main risk factor for bacterial keratitis in developed countries . thus , it is essential to establish related information on microbial keratitis , including patient - specific risk factors and the most likely causative organisms , which would facilitate the development of effective strategies for the prevention , diagnosis , and treatment of microbial keratitis . in taiwan , a study from a university hospital reported that contact lens - related pseudomonas keratitis was the most common form of microbial keratitis from 1992 to 2001 . conducting periodic surveys of infectious keratitis is crucial for updating the local information for reference by clinicians because epidemiologic patterns may change over time . therefore , the present study collected data on the predisposing factors , clinical manifestations , spectrum of microorganisms , and treatments for patients with microbial keratitis who were admitted to and treated at chang gung memorial hospital ( cgmh ) , a major teaching hospital in northern taiwan , from 2003 to 2012 . this study was conducted as a retrospective , cross sectional design , and in accordance with the declaration of helsinki and was approved by the institutional research ethics board of cgmh , taiwan ( irb102 - 4073b ) . consent was waived because of the retrospective design of the project and the anonymous analysis of the data . we retrospectively reviewed the medical records of 558 patients with infectious keratitis who were admitted to cgmh from january 1 , 2003 to december 31 , 2012 . we included patients with negative culture results if they presented with an epithelial defect and stromal infiltrate and responded favorably to antimicrobial treatment . admission criteria were primarily severe ( ie , potentially sight - threatening ) keratitis and the need for intensive topical antimicrobials . information on age , sex , predisposing factors , clinical features , microbiological results , treatments , and visual acuity ( if recorded ) was collected . we defined an ulcer as being central if it encroached within 2 mm of fixation , peripheral if it involved a zone within 2 mm from the limbus , and paracentral if it was between the central and peripheral zone . corneal ulcers were defined as being small ( < 2 mm ) , medium ( 26 mm ) , or large ( > 6 mm ) . corneal scrapings were obtained using a surgical blade and directly inoculated into blood agar , chocolate agar , modified sabouraud agar , lowenstein corneal scrapings of patients with clinical characteristics suggestive of acanthamoeba keratitis were inoculated into nonnutrient agar seeded with escherichia coli . the various media were routinely incubated for a week or longer depending on the media before the final culture result was obtained . a positive culture was defined as growth of at least 3 colonies along the line of inoculation on one solid medium on the basis of the criteria in a previous study . before the result of corneal ulcer culture could be obtained , levofloxacin ( 0.5% ) alone or a combination of 2 fortified antibiotics ( 25 mg / ml of cefazolin and 25 mg / ml of amikacin ) was administered topically once per hour . topical natamycin and amphotericin b ( 0.1% ) were applied hourly for mold and yeast infection , respectively . topical polyhexamethylene biguanide ( 0.02% ) was applied for acanthamoeba keratitis . for data presentation , we arbitrarily divided the study years into 2 periods ; the first half was from 2003 to 2007 , and the second half was from 2008 to 2012 . mantel - haenszel linear - by - linear association test was used to detect the trends over the 10-year period . categorical variables were analyzed using a test ; continuous variables were analyzed using analysis of variance ( anova ) . simple linear regression for univariate analysis was used to identify the factors associated with hospital stay . all statistical analyses were performed using spss software , version 22 ( ibm , armonk , ny ) . this study was conducted as a retrospective , cross sectional design , and in accordance with the declaration of helsinki and was approved by the institutional research ethics board of cgmh , taiwan ( irb102 - 4073b ) . consent was waived because of the retrospective design of the project and the anonymous analysis of the data . we retrospectively reviewed the medical records of 558 patients with infectious keratitis who were admitted to cgmh from january 1 , 2003 to december 31 , 2012 . we included patients with negative culture results if they presented with an epithelial defect and stromal infiltrate and responded favorably to antimicrobial treatment . admission criteria were primarily severe ( ie , potentially sight - threatening ) keratitis and the need for intensive topical antimicrobials . information on age , sex , predisposing factors , clinical features , microbiological results , treatments , and visual acuity ( if recorded ) was collected . we defined an ulcer as being central if it encroached within 2 mm of fixation , peripheral if it involved a zone within 2 mm from the limbus , and paracentral if it was between the central and peripheral zone . corneal ulcers were defined as being small ( < 2 mm ) , medium ( 26 mm ) , or large ( > 6 mm ) . corneal scrapings were obtained using a surgical blade and directly inoculated into blood agar , chocolate agar , modified sabouraud agar , lowenstein corneal scrapings of patients with clinical characteristics suggestive of acanthamoeba keratitis were inoculated into nonnutrient agar seeded with escherichia coli . the various media were routinely incubated for a week or longer depending on the media before the final culture result was obtained . a positive culture was defined as growth of at least 3 colonies along the line of inoculation on one solid medium on the basis of the criteria in a previous study . before the result of corneal ulcer culture could be obtained , levofloxacin ( 0.5% ) alone or a combination of 2 fortified antibiotics ( 25 mg / ml of cefazolin and 25 mg / ml of amikacin ) was administered topically once per hour . topical natamycin and amphotericin b ( 0.1% ) were applied hourly for mold and yeast infection , respectively . , we arbitrarily divided the study years into 2 periods ; the first half was from 2003 to 2007 , and the second half was from 2008 to 2012 . mantel - haenszel linear - by - linear association test was used to detect the trends over the 10-year period . categorical variables were analyzed using a test ; continuous variables were analyzed using analysis of variance ( anova ) . simple linear regression for univariate analysis was used to identify the factors associated with hospital stay . all statistical analyses were performed using spss software , version 22 ( ibm , armonk , ny ) . during the 10-year study period , 558 patients were included in this study , of which 285 ( 51.1% ) and 273 ( 48.9% ) were male and female , respectively ( table 1 ) . the mean age of patients was 50.3 22.7 years ( range , 2100 yr ) . the right eye was involved in 287 patients and the left eye was involved in 271 patients . the corneal ulcer was small in 192 eyes ( 37.7% ) , medium in 267 eyes ( 52.5% ) , and large in 50 eyes ( 9.8% ) . the location of the corneal ulcer was central in 238 eyes ( 47.1% ) , paracentral in 206 eyes ( 40.8% ) , and peripheral in 61 eyes ( 12.1% ) . the presence of hypopyon was noted in 153 eyes ( 33.8% ) . except for laterality , trends for demographics and clinical features were not statistically significant . demographics and clinical features of microbial keratitis risk factors for microbial keratitis were identified in 426 patients ( 76.3% ) . the most common risk factor was contact lens wear ( 31.4% ) , followed by systemic and ocular diseases ( 26.3% ) , trauma ( 23.5% ) , and previous ocular surgery ( 12.7% ) ( table 2 ) . a total of 54 patients ( 12.7% ) had at least 2 predisposing factors for corneal ulcers . materials causing ocular trauma included plant ( n = 24 ) , iron ( n = 16 ) , mud ( n = 5 ) , chemical ( n = 4 ) , wood ( n = 3 ) , and unidentified sources ( n = 48 ) . lagophthalmos ( n = 16 ) and dry eye ( n = 14 ) were the most common ocular surface diseases . the trend test showed that during the 10-year study period , the proportion of patients wearing contact lenses and with trauma increased ( p = 0.011 and p = 0.035 , respectively ) , and the rate of previous ocular surgery decreased ( p = 0.027 ) . predisposing factors for microbial keratitis positive culture results were obtained in 353 patients ( 63.3% ) . two hundred thirty - eight ( 42.7% ) patients were administered topical antibiotics before referral , and 88 patients had negative culture results . regarding isolates , 210 bacterial isolates ( 59.9% ) , 62 fungal isolates ( 17.6% ) , 8 nontuberculous mycobacteria ( ntm ) isolates ( 2.3% ) , and 2 acanthamoeba isolates ( 0.6% ) were identified ( table 3 ) . seventy - one patients had polymicrobial infections ( 20.1% ) . among bacterial isolates , gram - negative bacteria ( 37.4% ) were more common than gram - positive bacteria ( 22.1% ) . the most commonly isolated bacterium was pseudomonas aeruginosa ( 28% ) , followed by coagulase - negative staphylococcus ( cns , 5.4% ) and staphylococcus aureus ( 4.5% ) . the percentage of serratia marcescens decreased significantly ( p = 0.033 ) ; the identified organisms did not change over 10 years . isolated organisms in microbial keratitis table 4 lists isolated organisms from patients with different risk factors for microbial keratitis . gram - negative bacteria , particularly p. aeruginosa , mainly accounted for contact lens - related keratitis ( 52.9% ) . keratitis associated with ocular and systemic diseases was mainly caused by bacteria ( 33.8% and 29.4% for gram - positive and gram - negative bacteria , respectively ) . risk factors versus isolated organisms in microbial keratitis all patients were initially treated with empiric antimicrobials , which were adjusted on the basis of the clinical response and the results of the drug susceptibility test . medical treatment was successful for patients with gram - positive bacterial infections ( 62.8% ) , gram - negative bacterial infections ( 78% ) , ntm infections ( 75% ) , acanthamoeba infections ( 100% ) , and polymicrobial infections ( 74.6% ) ( table 5 ) . surgical procedures included amniotic membrane transplantation , patch graft , lamellar keratectomy , penetrating keratoplasty , and evisceration . one patient with fungal keratitis , 2 patients with pseudomonal keratitis , and 3 patients with polymicrobial infections underwent evisceration to eradicate the infections . treatment for microbial keratitis the mean hospital stay was 13.7 11.5 days . longer hospital stay was correlated with previous steroid use , ocular and systemic diseases , longer interval between symptom presentation and admission , previous ocular surgery , large ulcer size , fungal infection , ntm infection , poor visual acuity at presentation , old age , and surgery during admission ( all p < 0.05 by simple linear regression ) . in multiple linear stepwise regression analysis , 4 factors including previous ocular surgery , large ulcer size , ntm infection , and surgery during admission were associated with longer hospital stays ( table 6 ) . during the 10-year study period , 558 patients were included in this study , of which 285 ( 51.1% ) and 273 ( 48.9% ) were male and female , respectively ( table 1 ) . the mean age of patients was 50.3 22.7 years ( range , 2100 yr ) . the right eye was involved in 287 patients and the left eye was involved in 271 patients . the corneal ulcer was small in 192 eyes ( 37.7% ) , medium in 267 eyes ( 52.5% ) , and large in 50 eyes ( 9.8% ) . the location of the corneal ulcer was central in 238 eyes ( 47.1% ) , paracentral in 206 eyes ( 40.8% ) , and peripheral in 61 eyes ( 12.1% ) . the presence of hypopyon was noted in 153 eyes ( 33.8% ) . except for laterality , trends for demographics and clinical features were not statistically significant the most common risk factor was contact lens wear ( 31.4% ) , followed by systemic and ocular diseases ( 26.3% ) , trauma ( 23.5% ) , and previous ocular surgery ( 12.7% ) ( table 2 ) . a total of 54 patients ( 12.7% ) had at least 2 predisposing factors for corneal ulcers . materials causing ocular trauma included plant ( n = 24 ) , iron ( n = 16 ) , mud ( n = 5 ) , chemical ( n = 4 ) , wood ( n = 3 ) , and unidentified sources ( n = 48 ) . lagophthalmos ( n = 16 ) and dry eye ( n = 14 ) were the most common ocular surface diseases . the trend test showed that during the 10-year study period , the proportion of patients wearing contact lenses and with trauma increased ( p = 0.011 and p = 0.035 , respectively ) , and the rate of previous ocular surgery decreased ( p = 0.027 ) . two hundred thirty - eight ( 42.7% ) patients were administered topical antibiotics before referral , and 88 patients had negative culture results . regarding isolates , 210 bacterial isolates ( 59.9% ) , 62 fungal isolates ( 17.6% ) , 8 nontuberculous mycobacteria ( ntm ) isolates ( 2.3% ) , and 2 acanthamoeba isolates ( 0.6% ) were identified ( table 3 ) . seventy - one patients had polymicrobial infections ( 20.1% ) . among bacterial isolates , gram - negative bacteria ( 37.4% ) were more common than gram - positive bacteria ( 22.1% ) . the most commonly isolated bacterium was pseudomonas aeruginosa ( 28% ) , followed by coagulase - negative staphylococcus ( cns , 5.4% ) and staphylococcus aureus ( 4.5% ) . the percentage of serratia marcescens decreased significantly ( p = 0.033 ) ; the identified organisms did not change over 10 years . isolated organisms in microbial keratitis table 4 lists isolated organisms from patients with different risk factors for microbial keratitis . gram - negative bacteria , particularly p. aeruginosa , mainly accounted for contact lens - related keratitis ( 52.9% ) . keratitis associated with ocular and systemic diseases was mainly caused by bacteria ( 33.8% and 29.4% for gram - positive and gram - negative bacteria , respectively ) . all patients were initially treated with empiric antimicrobials , which were adjusted on the basis of the clinical response and the results of the drug susceptibility test . medical treatment was successful for patients with gram - positive bacterial infections ( 62.8% ) , gram - negative bacterial infections ( 78% ) , ntm infections ( 75% ) , acanthamoeba infections ( 100% ) , and polymicrobial infections ( 74.6% ) ( table 5 ) . surgical procedures included amniotic membrane transplantation , patch graft , lamellar keratectomy , penetrating keratoplasty , and evisceration . one patient with fungal keratitis , 2 patients with pseudomonal keratitis , and 3 patients with polymicrobial infections underwent evisceration to eradicate the infections . treatment for microbial keratitis the mean hospital stay was 13.7 11.5 days . longer hospital stay was correlated with previous steroid use , ocular and systemic diseases , longer interval between symptom presentation and admission , previous ocular surgery , large ulcer size , fungal infection , ntm infection , poor visual acuity at presentation , old age , and surgery during admission ( all p < 0.05 by simple linear regression ) . in multiple linear stepwise regression analysis , 4 factors including previous ocular surgery , large ulcer size , ntm infection , and surgery during admission were associated with longer hospital stays ( table 6 ) . severe infectious keratitis is a leading cause of corneal blindness . optimal clinical practice for the prevention and treatment of microbial keratitis should account for patient - specific risk factors and possible causative organisms in different regions . in this study , we focused on patients admitted with microbial keratitis ; first , we ensured that only patients with severe infection leading to admission were included in this study , and second , we used the same ( or as similar as possible ) criteria as used in a previous report conducted at another university hospital in northern taiwan between 1992 and 2001 to provide updated regional epidemiological information . our findings showed that contact lens wear remained the leading risk factor for inpatient microbial keratitis , and the trend increased significantly over the 10-year study period ; p. aeruginosa was the most common causative organism . in the current study , risk factors for microbial keratitis from 2003 to 2012 were contact lens wear , ocular and systemic diseases , trauma , and previous ocular surgery , in descending order , which was similar to the results of a previous taiwanese report . in taiwan , the leading risk factor for microbial keratitis was still contact lens wear , which was also reported in the united states , western europe australia , and hong kong . rattanatam et al found that the number of patients with contact lens - related microbial keratitis decreased in their hospital , suggesting that a higher number of such patients were treated in the community after the introduction of fluoroquinolones . by contrast , our study demonstrated an increasing trend in the rate of contact lens - related microbial keratitis in our hospital over the 10-year study period ( p = 0.011 ) . in a prospective , population - based study of contact lens - related microbial keratitis in australia , risk factors for infections included overnight use , poor storage case hygiene , smoking , internet purchase of contact lenses , less than 6 months wear experience , and higher socioeconomic class ; however , new lens types did not reduce the incidence of infection . because of the retrospective design of this study , it was difficult to correlate contact lens wearing modalities , hygiene , and other factors with infections ; however , approximately 35.8% of patients had overnight use in our study . wearing contact lenses is popular in taiwan , which is a country with high prevalence of refractive errors , and contact lens - related microbial keratitis has become a public health concern . in 2012 , corneal health for care network to advocate the 3 c 's for contact lens wearers : consulting a physician on how to fit contact lenses correctly ( correct ) , cleaning and maintaining contact lens ( care ) , and receiving regular health check - ups ( check ) . this campaign is anticipated to facilitate decreases in the rate of contact lens - related microbial keratitis in taiwan . in this study , the most common causative organisms were bacteria , followed by fungi ; gram - negative bacteria were more common than gram - positive bacteria , and p. aeruginosa was the most commonly identified isolate . the spectrum of microorganisms accounting for microbial keratitis differ depending on geographic location , climate , and etiology . for example , gram - positive bacteria are predominant in temperate climate regions , whereas gram - negative bacteria and fungi are prevalent in tropical regions;pseudomonas species are associated with contact lens - related infections , whereas fungi are related to trauma caused by plants . the predominance of p. aeruginosa infection in taiwanese studies may reflect both the geographic prevalence of the microorganism and contact lens - related keratitis . fungi are also crucial causative organisms of microbial keratitis in taiwan ; similarly , this might be due to the geography and climate , and injury caused by plant materials . notably , 4 of 5 previous studies in europe and taiwan investigating microbiological findings of hospitalized patients with microbial keratitis have reported that gram - negative bacteria are the most common causative organism . this finding might suggest that microbial keratitis caused by gram - negative bacteria , particularly pseudomonas species , tends to be severe and progresses rapidly , thus requiring admission . in the current study , medical treatment was successful in 69.5% of patients ; more than half of the patients with fungal keratitis required additional surgical interventions to control infections . medical treatment of fungal keratitis , particularly deep - seated infections , is often unsatisfactory because of delayed diagnosis , inadequate drug penetration , and slow response to therapy . surgical intervention to remove infectious elements and necrotic tissue may increase drug penetration and shorten the clinical course . however , therapeutic corneal transplantation and even destructive surgery may be indicated for severe keratitis with a poor response to medical therapy or when severe complications supervene . long hospital stay also has effects on financial resources , staffing , and turnover rate of beds in the public health service . in this study , the mean hospital stay was 13.7 days , and longer hospital stays were associated with previous ocular surgery , large ulcer size , ntm infection , and surgery during admission . a study of hospitalized patients with infectious keratitis in new zealand reported that longer hospital stay was associated with the presence of hypopyon , larger ulcers , previous ocular surgery , and poor visual acuity . as expected , large ulcers or surgery during admission prolonged hospital stay . older age and longer duration from symptom onset to diagnosis were noted in patients with previous ocular surgery ( data not shown ) , which might explain the relationship between longer hospital stay and previous ocular surgery . ntm has a relatively slow growth rate , and the infection can mimic that caused by other pathogens . thus , the delay in diagnosis and treatment of ntm infections might prolong hospital stay . morbidities caused by microbial keratitis can be assessed on the basis of surgical intervention , hospital stay , and visual loss . in this study , we did not analyze the predictors for poor visual outcome in microbial keratitis because of visual assessments with variable follow - up intervals . however , the study in new zealand reported that longer hospital stays were associated with poor visual acuity both at presentation and final assessment . we included cases with both positive and negative culture results , and we could not exclude the possibility that cultured isolates were contaminated . because our patients were admitted to a tertiary referral hospital in taiwan , the results can not be generalized . in conclusion , p. aeruginosa was the most common causative organism , and contact lens wear was the most common risk factor for microbial keratitis ; there was a significant increase in the percentage of contact lens - related keratitis during the 10-year study period in taiwan . the majority of patients with microbial keratitis were cured through medical treatment , but a high proportion of patients with fungal keratitis required surgical interventions . microbial keratitis has the potential to cause devastating visual impairment and major costs to the public health system ; our findings provide updated information and facilitate future prevention and treatment of microbial keratitis in taiwan .

abstractwe conducted a retrospective , cross - sectional study to analyze predisposing factors , clinical features , and microbiological characteristics of patients with microbial keratitis hospitalized over 10 years.the medical records of 558 patients who were diagnosed with microbial keratitis and admitted to chang gung memorial hospital ( cgmh ) , a referral center in taiwan , from january 1 , 2003 to december 31 , 2012 were reviewed . demographics , predisposing factors , isolated organisms , treatment , and hospital stay were recorded . yearly trends were tested using a linear - by - linear association.contact lens wear was the most common predisposing factor ( 31.4% ) , followed by ocular and systemic diseases ( 26.3% ) and trauma ( 23.5% ) . contact lens - related infectious keratitis increased year by year ( p = 0.011 ) . pseudomonas aeruginosa was the most commonly isolated organism ( 28% ) , followed by fungi ( 17.6% ) and coagulase - negative staphylococcus ( 5.4% ) . except for serratia marcescens , the identified organisms did not change over 10 years . most bacterial infections were controlled using antimicrobial treatment , but more than half of patients with fungal keratitis required surgical interventions . the mean hospital stay was 13.7 11.5 days . previous ocular surgery , large ulcer size , nontuberculous myycobacteris infection , and surgery during admission were related to prolonged hospital stay.in taiwan , contact lens - related pseudomonal keratitis remained the most common cause of microbial keratitis in patients hospitalized from 2003 to 2012 .

INTRODUCTION METHODS RESULTS Demographics and Clinical Features Predisposing Factors Microbiological Analysis TREATMENT DISCUSSION

the human rights act of 1977 followed by the privacy act of 1982 were the first legislative acts to protect personal information across the canadian public sector ( peekhaus 2008 ) . in 1987 , at a time when the hiv epidemic was gaining momentum across canada , the freedom of information and protection of privacy act was the first legislation to outline principles of conscientious and cautious handling of phi that institutions were responsible to adhere to ( cavoukian 1990 ) . what followed was legislation that extended the protection of personal information in the private sector in canada , and in 2001 , the personal information protection and electronic documents act ( pipeda ) came into effect ( canadian hiv / aids legal network 2004 ; peekhaus 2008 ) . since 1997 , provinces have adopted privacy legislation that specifically applies to healthcare providers regardless of whether they are engaged in commercial activities ( peekhaus 2008 ) , and , currently , nine provinces have specific laws that protect phi and impose obligations on healthcare providers to protect that information ( canadian hiv / aids legal network 2014 ) . the personal health information protection act ( phipa ) was enacted in 2004 and governs the collection , use and disclosure of phi within the ontario health sector and aims to keep phi confidential and secure while allowing for the effective delivery of healthcare and the effective operation of the healthcare system ( beardwood and kerr 2004 , 2005 ; cavoukian 2008 ; oipc n.d . ) . phi is broadly defined under phipa as identifying information about an individual in oral or recorded form that could be used to identify a specific individual , for example , the physical and/or mental health of the individual ( including the health history of the individual 's family ) , the provision of healthcare to the individual , payments or eligibility for healthcare , and the individual 's health number ( beardwood and kerr 2004 ) ; hiv status is included under this definition ( canadian hiv / aids legal network 2004 ) . under phipa , persons and organizations that provide healthcare are collectively known as health information custodians , or hics ( oipc n.d . ; peekhaus 2008 ) as they have custody or control of personal health information in connection with performing their duties or work ( beardwood and kerr 2004 : 63 ) . whether individuals are hics or agents of a hic , their obligation to abide by phipa and ensure the security , confidentiality , accuracy and integrity of phi in their custody is the same ( fletcher 2014 ; peekhaus 2008 ) . phipa requires that hics take reasonable steps to ensure phi is protected against theft , loss and unauthorized use or disclosure regardless of the type of records being used ( beardwood and kerr 2005 ; cavoukian and rossos 2009 ) . consent , disclosure and circle of care are key constructs outlined in phipa that have important implications on the activities and decision - making of hics with regard to phi . hics may imply consent for the collection , use and disclosure of phi for the delivery of healthcare services ( cavoukian 2008 ) , for example , consent is implied if a patient accepts a referral and shows up for care ( fletcher 2014 ) . with regard to disclosure , under phipa , phi may only be disclosed by hics if the individual consents or if phipa specifically permits the disclosure without consent ( oipc n.d . ) . phipa was specifically designed so that it would not prevent a barrier to the disclosure of personal health information among healthcare providers , hics are permitted to disclose phi for the purposes of providing or assisting in providing care on the basis of implied or assumed implied consent ( canadian hiv / aids legal network 2014 ; cavoukian and rossos 2009 ) . a patient 's express consent , that is verbal or written consent , is not required to share information within the circle of care to other healthcare providers ( canadian hiv / aids legal network 2014 ) . at the same time , phipa permits disclosure of phi without implied consent in a number of vague and unspecified circumstances including providing healthcare ( as an individual healthcare provider or as a facility ) ; managing risks and error ( beardwood and kerr 2004 ) ; planning and management of the health system ; and analysis of the health system , etc . the assumption of implied consent is no longer true , however , when the hic is aware that the individual wishes to withhold or withdraw their consent . furthermore , when phi is disclosed to a non - hic outside of the circle of care , or for purposes other than delivery of healthcare , express consent is required ( beardwood and kerr 2004 ; cavoukian 2008 ) . implied consent and disclosure within the circle of care for the purposes of providing or assisting in the provision of healthcare is arguably the most significant provision in phipa yet it is buried in a sub - section of the legislation ( beardwood and kerr 2004 : 65 ) . permitting disclosure of phi in a variety of circumstances for the broad purposes of providing healthcare vis - - vis a focus on implied consent means that hics need to use little or no effort to comply with the requirements of phipa ( beardwood and kerr 2004 : 67 ) . therefore , while it is essential that people who use health services trust that their privacy will be protected , at the same time , the delivery of high quality healthcare depends on the availability of accurate and complete health information phipa attempts to strike a balance between protecting privacy and facilitating care delivery ( fletcher 2014 ) . although identifying information in oral form falls under the category of phi ( beardwood and kerr 2004 ) , it appears that sharing information verbally amongst hics and to non - hics is not the central focus of phipa nor is it explicitly mentioned in the legislation compared to other forms of information sharing practices . instead , phipa focuses on health records , in particular paper and electronic records ( cavoukian and rossos 2009 ) . furthermore , phipa does not lend itself to how information is currently exchanged in healthcare institutions with the advent of new technologies and increasing use of e - mail and digital interfaces to facilitate communication between and amongst hics ( fletcher 2014 ) . although healthcare providers may mention hiv status in the process of delivering care to the patient , it may result in disclosure of phi to a non - hic or someone outside of the circle of care , for example , hospital visitors . healthcare providers may use hiv status as a label , reminder , and/or communication tool , both for themselves and within the healthcare team , to prevent risks and error in the process of delivering care ( beardwood and kerr 2004 ) . employing hiv in this way may help those providing care to ensure appropriate checks and balances within a woman 's care plan ( gagnon 2014 , 2015 ) . healthcare providers may also assume hospital visitors are aware of one 's hiv status , especially if the patient does not explicitly discuss with them the importance of keeping it private . regardless of whether a patient articulates if it is safe to disclose their hiv status is not the point ; when hiv status is disclosed without a person 's express consent , the attempt to strike a balance between maintaining privacy while delivering high quality healthcare is not achieved . what mechanisms are in place for service users to take action in the event that their privacy is breached ? how would management of the institution in which care is provided respond to this situation and take reasonable steps to right this wrong ? there are many consequences when privacy is not respected including reputational consequences and changes in therapeutic relationships with healthcare providers ( fletcher 2014 ) . most privacy breaches are avoidable , even if conducted by well - intentioned healthcare providers ; regardless of the intention behind the behaviour , the effect and consequences are the same ( fletcher 2014 ) and hics who willfully collect , use or disclose phi in contravention of phipa can be found liable ( beardwood and kerr 2005 ) . at a provincial level , enforcement of phipa individuals have up to one year to file a complaint concerning a breach of privacy under phipa ( canadian hiv / aids legal network 2012 ) . enforcement involves an adversarial system whereby the commissioner , viewed as an impartial adjudicator , has discretion to determine the course of action including initiating a review or ordering a hic to modify , cease or implement a particular information practice ( beardwood and kerr 2005 ) . patients have the choice to withdraw their consent for the use and disclosure of phi to other healthcare providers who deliver care , but the details of how this would be achieved , as well as how the hic would monitor patient consent , are unclear . how policies and practices that align with phipa are disseminated , interpreted and enforced by the hic and its agents , especially within a fast - paced and overburdened healthcare system , presents a whole different set of challenges . the bottom line is that healthcare institutions implicated under phipa do not do an optimal job to keep phi private . patients are encouraged to stand up for and assert their rights to privacy ( canadian hiv / aids legal network 2014 ; fletcher 2014 ) , but is this a realistic expectation and outcome ? although disclosure of phi often occurs by well - intentioned healthcare providers , wlwh believe there will be serious repercussions if they call on healthcare providers who have disclosed their hiv status to account for their inappropriate behaviour ( ion et al . , many wlwh who experience disclosure of phi will not report the actions of their healthcare providers . is there something unique about having hiv that positions wlwh in a particular way while in hospital ? early in the hiv epidemic , there was virtually no privacy protection for those living with hiv because of fear , ignorance and aids hysteria within the healthcare system . despite significant advances in the clinical management of hiv and its evolution into a complex chronic illness ( scandlyn 2000 ; thompson et al . 2010 ) , wlwh around the world continue to face a number of health , social and legal challenges including access to hiv treatment , hiv - related stigma , discrimination and the criminalization of hiv ( debruyn 2004 ; greene et al . wlwh continue to report stigmatizing interactions with healthcare providers , for example , being treated differently in the labour and delivery unit because of their hiv status and the societal perception that wlwh should not be having children ( greene et al . the current experiences of wlwh may reflect a long and enduring history of fear and ignorance of hiv within the healthcare system . women 's experiences may also reflect an enduring lack of knowledge and awareness about hiv amongst healthcare providers who do not work in settings that specialize in hiv care . it is critical to consider the legislative frameworks pertaining to privacy through the lens of hiv because hiv has been classified as phi that is particularly sensitive ( cavoukian 1990 ; gostin 1995 ) . it is also apparent that healthcare practices within non - hiv - specific services have not kept pace with the evolution of hiv as a chronic condition . it is clear why wlwh continue to perceive hiv - related stigma when they access care , in particular , during pregnancy and early postpartum , as well as why a climate of fear , ignorance and stigma continues to surround the hiv epidemic across canada ( canadian hiv / aids legal network 2004 ) . although patients have every right to hold hics accountable , expecting health service users to stand up for their rights is not always optimal or possible , especially when they are ill , in hospital , are not comfortable speaking up for themselves , or feel powerless to do so ( fletcher 2014 ; greene et al . 2015 ; ion and elston 2015 ; ion et al . moreover , healthcare providers and trainees may not truly understand their accountability and duty of confidentiality requirements ( canadian hiv / aids legal network 2004 ) . power dynamics may be at play between wlwh and their healthcare providers within the healthcare system . the choice of wlwh not to respond to or hold healthcare providers accountable for disclosing their hiv status sheds some light on how power dynamics may flourish within healthcare systems and may not always position the patient at the centre of care . furthermore , expecting patients to express their privacy complaints to individual healthcare providers , then channel these complaints to the upper echelons of a healthcare corporation , as well as a provincial body like the office of the information and privacy commissioner , is a tall order . although hics may not do an optimal job to protect the privacy of patients ( fletcher 2014 ) , expecting patients to advocate for themselves when privacy concerns arise and submit complaints to an adversarial system after disclosure has occurred may not be a perfect solution . could the healthcare system be organized differently to decrease the chance of a privacy breach and to facilitate the appropriate collection , use and disclosure of phi , including hiv status ? a number of steps can be taken to optimize information practices to ensure the phi of wlwh is protected and remains confidential . these steps are relevant not only to wlwh , but all people living with hiv ( plwh ) and other patients who experience challenges related to privacy and confidentiality when navigating the healthcare system . any system changes must first be grounded in the perspective that , as a matter of public policy , the right to privacy is a fundamental human right for all plwh ( canadian hiv / aids legal network 2004 ) . privacy is essential to freedom and revolves around personal control and freedom of choice ( cavoukian 2014 ) . it must also be recognized that not only do plwh have a right to privacy regarding their phi , hics owe a duty to plwh to keep their phi confidential ( canadian hiv / aids legal network 2004 ) . confidentiality of phi is fundamental to the preservation of the ethical values of autonomy , dignity and respect for the individual ; patient confidentiality is not only an essential pre - condition to successful treatment it 's an issue of human dignity and respect the concept of privacy by design ( pbd ) offers a framework for ensuring that privacy is embedded directly into the design specifications of information technologies , business practices and operational processes ( cavoukian 2014 ) . pbd emphasizes service user privacy and the need to embed privacy as a default condition by transforming service user privacy issues from a pure policy or compliance issue into a business imperative ( cavoukian 2014 : 13 ) . pbd is focused on process rather than singular , technical outcomes and recognizes the need to introduce privacy principles during architecture planning , system design including networked infrastructure , and the development of operational procedures including work processes and management structures ( cavoukian 2014 ) . scholars have suggested a number of recommendations at the macro- , meso- and microlevels of policy and practice regarding how information practices could be optimized to ensure the appropriate collection , use and disclosure of phi . at the macro level of legislation , the canadian hiv / aids legal network has taken issue with the discretionary disclosure clauses inherent in health privacy legislation noting that these clauses fail to provide the level of privacy protection accorded to health information under the canadian charter of rights and freedoms ( canadian hiv / aids legal network 2004 ) . as such , the canadian hiv / aids legal network ( 2004 ) recommends that only in exceptional and circumscribed situations should a hic be permitted to disclose health information without the express and informed consent of plwh , rather than the vague and unspecified circumstances that are currently permitted . the legal network also recommends that hics be prohibited from disclosing any information that may reasonably reveal a person 's health information to family and friends without the person 's consent ( canadian hiv / aids legal network 2004 ) . at the meso level of systems , buffet and kosa ( 2006 ) have investigated how hics ensure that patient preferences regarding disclosure of phi are acted upon . the authors note that heath information network providers , for example , digital interfaces used to facilitate communication between hics and its agents , bear some responsibility for tracking and monitoring patient consent ( buffet and kosa 2006 ) . the authors propose a systematic consent management program , which they believe will minimize , if not eliminate , risk for the hic and health information network provider ( buffet and kosa 2006 ) . the consent management system relies on utilities and assigns a valuation to patient attitudes with regard to the handling of phi . the utilities are applied as part of a risk - based consent management framework and could be updated and reviewed each time a patient 's records are accessed . at any time a hic performs an action that involves a patient 's phi , an analysis would be conducted to help the hic determine whether to proceed based on the patient 's valuations and the likelihood that the patient 's preferences would be violated in the process ( buffet and kosa 2006 ) . the system for consent and risk management would assist hics to meet their legal obligations under phipa by managing a patient 's consent for releasing phi . scholars have also highlighted how training and professional development initiatives for healthcare providers and learners are important domains where the pbd framework could be enacted . for example , privacy may only be a small portion of orientation modules trainees are expected to complete ; these modules may present privacy in very theoretical terms and lack practical and applied elements ( fletcher 2014 ) . training programs could be redesigned to better prepare and orient healthcare providers and learners to privacy protection and duty of confidentiality . concrete examples of privacy breaches could be included as a way to apply knowledge to practice including the nuances of privacy in the context of hiv and other chronic and/or stigmatizing health conditions ( fletcher 2014 ) . health professional licensing bodies also need to educate their members about legal and ethical obligations regarding privacy and confidentiality . for example , just because discretionary disclosure clauses permit disclosure of phi under privacy legislation does not mean that the disclosure is necessary and valuable for the provision of care ( canadian hiv / aids legal network 2004 ) . regulatory bodies could make the protection of phi a performance metric for meeting licensing requirements . enhancing education , training and resources for healthcare providers within regulatory licensing bodies , as well as healthcare corporations , could result in important meso- and macro - level changes . the canadian hiv / aids legal network ( 2004 ) has offered recommendations regarding how the healthcare system , and its legislative and regulatory structures , could be better organized to protect the privacy of plwh . the legal network believes that the meso - level remedies currently in place for plwh whose privacy rights have been violated could be improved . the current adversarial system in ontario , for example , which investigates complaints brought forward by service users , could be made more accessible vis - - vis increasing the modes through which people could file a complaint such as audio or videotape in addition to written form , and eliminating fees associated with filing a complaint ( canadian hiv / aids legal network 2004 ) . accessibility could also be enhanced by increasing the public 's awareness of the system and improving transparency ; for example , the privacy commission of ontario could develop education programs to inform the public about the existence of and rights under privacy legislation including information about the complaint process and remedies ( canadian hiv / aids legal network 2004 ) . the legal network ( 2004 ) believes that remedies currently available to plwh whose privacy rights have been violated should also be strengthened ; for example , a system of deterrents should be implemented if hics improperly use and disclose phi including increased enforcement and compensation to patients . at the micro level of patient and provider , the legal network ( 2014a ) reminds us that healthcare providers should only ask questions that are relevant to providing care , for example , there is no need to ask about hiv status if that information is not required to examine or treat someone . plwh have often remarked about being asked how they acquired hiv during a routine clinical encounter ; when telling these stories they are always outraged and annoyed because healthcare providers , who they often have never met before , are driven more by their curiosity than delivering high quality , patient - centred care . asking relevant questions during a healthcare encounter is incredibly important for plwh and highlights one of the nuanced ways that the sensitivity of hiv can be considered in practice because it prioritizes the person 's privacy . healthcare providers should have a confidential , one - on - one conversation with their patients in advance so that a plan to maintain privacy and respect confidentiality is co - created and agreed to . how will we get there , especially considering that policies in the current political climate are considered within the micro - space of marginal incremental objectives , continually building out from the current situation ( lindblom 1959 ) ? at the same time , healthcare decisions are also influenced by economic , social , environmental and political forces ; policy makers and governments make decisions based on public opinion , electoral considerations , personal preferences and crisis management ( fafard 2008 ) . luckily , pbd has received global acceptance and endorsement by public and private sector privacy regulators around the world ( cavoukian 2014 ) . also , a number of scholars have spoken out to highlight public opinion regarding how governments handle phi ( peekhaus 2008 ) ; why hiv should be framed as health information that is particularly sensitive ( canadian hiv / aids legal network 2004 , 2012 , 2014 ) ; and how issues of privacy and hiv - related stigma complicate access to care and result in negative care experiences for plwh ( carter et al . forthcoming ; mccoy 2005 ; ohtn 2010 ; wong - wylie and jevne 1997 ) . as a result , community champions , social workers , researchers , activists and legal experts working in the hiv sector , as well as those concerned with privacy and its protection more broadly , are positioned with sufficient ammunition to enable a paradigm shift . the time is now to ensure that policy and practice decisions that affect privacy at the micro- , meso- and macro - levels are on the government 's agenda , and at the very least , are informed by evidence ( fafard 2008 ) , and grounded in the lived experiences and current realities of service users .

in the process of receiving perinatal care , women living with hiv ( wlwh ) in canada have experienced disclosure of their hiv status without their express consent . this disclosure often occurs by well - intentioned healthcare providers ; however , from the perspective of wlwh , it is a breach of confidentiality and leaves wlwh to manage the consequences . this paper is a critical review of the regulatory and legislative infrastructure that exists to protect the personal health information of wlwh in ontario and canada ; the recourse that wlwh have in the event that their confidentiality is breached ; and potential approaches that could be applied to organize the system differently to decrease the chance of a privacy breach and to facilitate appropriate collection , use and disclosure of personal health information .

Regulatory Frameworks and Privacy: What Legislative Infrastructure Exists in Canada? The Personal Health Information Protection Act in Ontario Enforcing PHIPA Recommendations for System Redesign: Optimizing Information Practices

in 2005 , cardiovascular disease ( cvd ) was the underlying cause of death in 864,480 of the approximately 2.5 million total deaths in the united states [ 1 , 2 ] . cvd is the leading cause of death and the most costly disease in america and is expected to increase in costs to $ 1.48 trillion by 2030 . although the focus has been to reduce the modifiable risk factors for cardiovascular disease ( such as lipid levels , diabetes , and sedentary lifestyle ) , the unmodifiable risk factor , aging , is a major risk factor for coronary disease , hypertension , congestive heart failure , and stroke . the number of the aged in the united states is projected to increase by more than 20% by the year 2030 . this growth in the elderly population is expected to significantly test our already overloaded health care system . although years of research have been conducted in regard to aging , we are still a long way from understanding the intricacies of age - related changes in human physiology , in particular the cardiovascular system . whether aging in animal models mimics many of the cardiovascular changes seen in humans is not well understood . in addition , the process of delineating the effect gender has on aging provides yet another variable to be considered . aging is believed by some to be a progressive disorder that decreases an organism 's ability to maintain reproductive capacity and normostasis . indeed a strong correlation exists between aging and higher incidence of several diseases including dementia , parkinson 's disease , diabetes , cancer , and alzheimer 's disease . in the cardiovascular system , aging is positively correlated with an increasing risk of cardiac problems including arrhythmias and is a major independent risk factor for cardiovascular - related morbidity and mortality . indeed , more than 70% of males and females over 75 years of age present some clinically evident cardiovascular disease ( cvd ) . cvd is the number one killer of women in western nations . among women , more than 200,000 of the 454,613 total cvd deaths occurred in those above 85 years of age . although cardiovascular risk increases with age in both sexes , the increase in age - associated risk appears to be sharper in women . why cvd risk may differ between men and women is not well understood but may be related to differences in age - associated cardiovascular function . whereas congestive heart failure in men is oftentimes due to systolic insufficiencies , congestive heart failure in women is often related to diastolic dysfunction [ 10 , 11 ] . the incidences of ischemia , cardiac failure , and cardiac rupture in addition to ventricular remodeling have also been shown to differ between genders with aging and often lead to worse outcomes in women [ 1215 ] . recent data suggests that premenopausal women have a decreased risk of cvd compared to men of comparable age [ 16 , 17 ] . this cardioprotective benefit appears to be lost over time as the risk of developing cardiovascular disease in postmenopausal women increases to a rate that is similar to that observed in men . the reason(s ) for this increase in cvd risk following menopause is currently unclear ; however , it is well known that early menopause is associated with an increased risk for coronary heart disease and cardiovascular disease death [ 1921 ] . this finding is thought to be related , at least in part , to diminished estrogen exposure . consistent with this notion , each year of increasing age at natural menopause has been found to be associated with a 2% increase in total cardiovascular mortality [ 22 , 23 ] . whether differences in estrogen alone or changes in estrogen along with other factors can fully explain the sex related differences in age - related cardiovascular function is unknown and requires further investigation . human aging research is limited due to cost , differences in lifestyle / history , and more importantly the time required for data collection as well as analysis of that particular system . importantly , there are a number of different animal models that can be used to acquire information on how aging affects the female cardiovascular system . primates are the closest in regard to female human aging due to the fact that they are the only species to undergo menstrual sloughing of the endometrial lining . nonetheless , it is important to note that female primates do not experience menopause . rats are widely used in aging research as they exhibit a relatively short lifespan and are genetically quite homogenous . among the various strains of rats , the wistar , f344 , and f344/brown norway are although rats do not experience menses , they do experience estrus cycling and ovarian aging . table 1 presents the stages of ovarian aging in female rats . reproductive maturity is reached at five months when there is an estrous cycle that lasts four to five days . aging rats exhibit periods of persistent estrous cycle which consists of elevated and constant levels of estradiol , low levels of progesterone , and lack of luteinizing hormone ( lh ) surges , in addition to ovulation [ 24 , 31 , 32 ] . ovarian decline occurs between six and eighteen months depending on the rodent strain and is characterized by low levels of estradiol and progesterone , with little or no developing follicles and increased prolactin secretion [ 24 , 31 , 32 ] . comparisons between aging research in female rats models and humans can be complicated due to the differences in the mechanisms of ovarian / hormone aging in human females and its potential impact on cardiovascular disease . though not fully understood , it is thought that the loss of the hormones estrogen and progesterone in aging human females is due to the decrease in the ovarian follicular reserve . conversely , aging female rats experience a persistent estrous cycle due to chronic anovulation which consist of pseudopregnant / disestrus estrogen levels as well as high progesterone levels from increased ovulation and the corpora lutea . therefore , reproductive senescence in female rats consists of alterations in the hypothalamic - pituitary axis while reproductive senescence in human females is classified as ovarian follicle depletion . due to these reasons and others , although estrogen secretion by the ovary promotes hypothalamic changes , the chronic administration of estrogen can damage neurons in the arcuate nucleus and medial basal hypothalamus . therefore , it can be important to remember that the surgical removal of the ovaries can lead to neurological changes that may affect other organs during the aging process . the pharmacological acceleration of ovarian aging using 4-vinylcyclohexene diepoxide ( vcd ) ( accelerated ovarian failure ( aof ) model ) or ovariectomy ( ovx ) procedures are often performed due to the lack of natural menopause in rat research models because they are believed to better mimic the hormone milieu seen in aging human females . vcd has been shown to target the plasma membrane of the primordial and primary follicles through direct inhibition of autophosphorylation of the oocyte - associated receptor , kit . kit acts as an antiapoptotic factor in primordial follicular survival [ 36 , 37 ] . although an effective model of chemical ovotoxicity limited research has been performed using female rat models [ 3841 ] and to date no published data exist regarding vcd and rat cardiovascular aging . vcd in juvenile ( 1 month ) and adult ( 3 months ) sprague - dawley rats depleted follicles but had no effect on the duration or onset of persistent estrus . however , fsh levels were significantly high in vcd treated animals with no change in cyclicity or serum levels of 17-estradiol . the absence of a human based clinical menopausal hormone profile in the aof model brings into question the ability to apply data derived from this model for translational relevant treatment of cardiovascular human based menopausal pathologies . an ovariectomy is the removal of the ovaries which induces a surgical menopause characterized by the cessation of estrogen and progesterone , as well as reduced production of testosterone . surgical menopause leads to more severe and sudden symptoms compared to that observed during the natural human menopause where ovaries produce lower levels of hormones over time . like that seen in menopausal human females , ovariectomy in rats also increases cardiovascular risk [ 42 , 43 ] . studies have found that the majority of human females who undergo natural menopause exhibit different age - associated cardiovascular alterations than those who underwent ovariectomy - induced menopause . elective bilateral removal of the ovaries at a young age is associated with an increased risk for cardiovascular disease and premature death . in addition to declines in well - being and sexual function , it is thought that elective oophorectomies are also associated with elevated risk of cognitive impairment , dementia , and parkinsonism . it is not known whether the increased risk to cardiovascular disease is due to alterations in hormones or the hypothalamic - pituitary axis . as expected , bilateral oophorectomy is associated with different hormonal alterations including changes in estrogen production and reduced levels of progesterone and testosterone , as well as increases in gonadotropins ( lh and follicle stimulating hormone ( fsh ) ) , compared to those that occur in human females who experience natural menopause [ 45 , 46 ] . in rats , ovariectomy causes alterations to heart structure and function which include increases in cardiac interstitial space , cardiac fibrosis , heart weight , and left ventricular weight . ovariectomized rats appear to exhibit increased evidence of oxidative stress and cardiac apoptosis [ 4751 ] . in rats , cytokine expression ( tumor necrosis factor - alpha ( tnf- ) and interleukin-1 beta ( il-1 ) ) and angiotensin converting enzyme ( ace ) and angiotensin ii type 1 receptor gene expression also appear to be increased following ovariectomy [ 48 , 50 ] . when estradiol treatment was given to ovariectomized rats , it prevented the reduction of cardiac contractility as well as the increase in apoptosis and cytokine expression [ 48 , 51 ] . research in other rats , namely , aging female dahl salt - sensitive rats , has shown that female rats are more likely to develop hypertension after ovariectomy . the researchers postulated that estrogen protects against the increased activity of the renin angiotensin system . however , in human females , research has shown that postmenopausal hypertension occurs approximately five to 10 years after menopause , not immediately after starting menopause . therefore , other age - related causes must occur independently of estrogen loss that promotes cvd in postmenopausal human females , as is shown by the fact that estrogen replacement therapy is not cardioprotective after menopause [ 53 , 54 ] . interestingly , both natural and surgically induced reproductive senescence rats exhibit increased fsh as well as decreased levels of estradiol and inhibin ( a / b ) . surgically induced reproductive senescence in rats has also been shown to alter dopamine receptor affinity in the heart . alterations in cardiac structure and function are not the only alterations found after ovariectomy . compared to aged controls , human females that underwent prophylactic bilateral salpingo - oophorectomy exhibit increased total and low - density lipoprotein cholesterol levels . in light of the fact that within human females natural menopause and ovariectomy results altered hormonal profiles and cardiovascular risk , the data suggest that age - associated cardiovascular disease in human females may be associated with both time dependent hormone deprivation and age - associated changes in structure , function , and protein signaling . of note is the fact that most studies have used ovariectomized female rats at young ages ( 612 weeks old ) to examine the effects of hormone deprivation on the cardiovascular system which can lead to unreliable results given that the cardiovascular system has not yet aged . the increased risk of cvd in human females may be due to differences in the type or magnitude of age - associated alterations in cardiac structure and function . olivetti and colleagues have demonstrated a small decrease in human heart weight with aging in males but not females . additionally , no significant change was noted with myocardium , left ventricular , or right ventricular free wall weight in the aging human female heart with no underlying cardiovascular pathology nor were age associated changes detected in proportion , shapes , size , or number of mononucleated and binucleated cardiac myocytes . given this information , it appears that natural aging in human females is not associated with cardiac remodeling during or after menopause in the absence of preexisting pathologies or loading abnormalities . although a limited number of studies have looked at how cardiac structure and function change with age in female rats , very few have directly investigated how sex may affect cardiac structure and function . go demonstrated that cardiovascular aging in male and female rats demonstrated significant gender differences in lv size and function . boluyt and colleagues investigated how aging may affect cardiac structure and function in female f344 rats using echocardiography . aging female rat cardiac structural changes included a dilatation of the left ventricle between 13 and 22 months of age . this dilatation was characterized by increases in posterior and septal wall thickness during diastole at 22 and 30 months of age . aging in the female f344 was also associated with increases in collagen content and collagen cross - linking . in addition , boluyt and colleagues demonstrated mild systolic dysfunction ( decline in left ventricle ejection fraction , fractional shortening , and velocity of circumferential fiber shortening ) in female 22-month - old animals when compared to young adults and that these changes preceded the development of mild diastolic dysfunction [ 61 , 63 ] . these authors suggested that this modest decline in systolic function was due , at least in part , to a shift in the amount of alpha and beta myosin heavy chains [ 61 , 62 ] , similar to what is seen in the human failing heart . additional work , perhaps using other rat models , is needed in order to truly distinguish if these alterations are due to increasing age or if they are simply specific to the f344 strain . fannin and colleagues found that increased age in the female f344xbn was associated with increases in oxidative stress and damage . age - related changes in cardiac structure were also found , such as increase in heart to body weight ratio , cardiomyocyte cross - sectional area , posterior wall thickening , and left ventricle chamber dilatation . further , increased age was also associated with diastolic dysfunction , alterations in heart rhythm intervals , and alternations in connexin 43 expression . the presence of age - associated cardiac structural changes in the f344 and f344xbn animal models calls into question the use of these strains for translational data , in light of the absence of age - associated change in human females reported by olivetti et al . . additionally , the menstrual cycle induced hormonal changes have been shown to cause cardiac functional fluctuations . this fact could indicate that the time at which functional measurements are taken may impact the results and must be a consideration in comparing all female derived cardiac functional data . in addition , aging is also associated with an increase in the percentage of incidence of atrial fibrillation [ 60 , 69 ] . in male rats , age - associated increases in cardiac fibrosis as well as the changes in gap junction morphology have been hypothesized to cause changes in cardiac conduction in addition to the incidence of arrhythmias and death [ 68 , 70 ] . as humans age , there is an increase in the ekg abnormalities which are associated with increased mortality . men tend to experience more atrial fibrillation , early repolarizations , brugada syndromes , and sudden cardiac death than women , while women are more likely to be at risk to develop long qt syndrome - based arrhythmias as well as bradycardia - induced torsades de pointes . although sex related differences in cardiac electrocardiogram parameters have been observed , electrocardiogram abnormalities seem to vary between studies . in men , there is an increase in qrs duration and sinus cycle length while women tended to a have a higher maximum heart rate [ 73 , 74 ] . electrocardiogram analysis in elderly men and women has demonstrated large or intermediate q waves , left axis deviation , negative t - waves , and complete right bundle branch block as well as atrial fibrillation / flutter . in aging men , the qrs complex has been found to be narrowed , while aged men and women have been shown to undergo a leftward shift of the qrs axis . another work has suggested that puberty in men is associated with a shortened qt interval which is not present in women and that aging in men demonstrates an increase in pr intervals and a prolongation of the qt interval . conversely , another research found that aging did not appear to alter ekg tracings in either men or women . similar alterations in electrocardiogram parameters have also been found in aging rats . in male wistar rats , the pr and corrected qt intervals alterations in aging electrocardiogram parameters have been shown to be rat strain dependent and could only be detected subsequently to heart failure as in male shhf / mcc - cp rat strain at 19 months of age . though not fully understood , these differences in gender associated gonadectomy induced arrhythmia frequency have been attributed to higher heart rates in male animals and longer qt intervals , as well as gender differences in ion channel expression . fannin and colleagues found that the arrhythmias were not significantly higher in older female f344xbn rats when compared with younger rats ; however , the study did find that valvular dysfunction was increased in the older rats when compared to the younger ones . the exact mechanisms of the gender associated reduction of arrhythmias in humans and the role estrogen may play are not fully understood but two potential indirect mechanisms have been proposed . one potential indirect mechanism may be related to the ability of estradiol to reduce the incidence of cardiac ischemia . other works have suggested that high levels of estradiol in female and male rats may directly reduce arrhythmias , possibly by causing slowing of the inward calcium current [ 80 , 81 ] . although multiple studies have investigated ekg changes in aging rats , these parameters have not been determined in the nia approved aging f344xbn rat model . however , according to the ich - s7b , the use of rats for the safety testing of drugs is not considered appropriate due to differences in the ionic mechanisms of repolarization . arrhythmia mechanisms , resting heart rate , ca homeostasis , and action potential configuration are all important differences that limit the translational relevance of rat generated cardiovascular data for human application . aging in the male rat heart is associated with the accumulation of oxidative damage to lipids and proteins as well as decline in mitochondrial enzymes . in addition , an increasing number of mutations in mitochondrial dna ( mtdna ) has been gradually observed during aging . the level of 8-ohdg mtdna adducts and deletions increase exponentially with age . in human muscle , liver , and brain tissue , complex iv and mitochondrial oxidative phosphorylation this decline in function is correlated with the accumulation of mtdna mutations , including deletions , and base substitutions . similarly , aged male rat hearts exhibit an increase in superoxide , 4-hydroxynonenal ( 4-hne ) , and nitrosative stress levels which appear to be highly correlated to increases in left ventricular thickness . the exact role that increased levels of oxidative stress may play in the development and progression of age - associated cardiovascular disease remains to be determined . compared to that observed in females , the hearts of aging male rats exhibited increases in protein carbonylation , advanced oxidation protein products , nitrotyrosine , nonprotein thiol , reduced glutathione , and iron levels . although aging is associated with increases in oxidative stress in both male and female rat hearts , female rat hearts exhibited lower mitochondrial hydrogen peroxide production , oxidative damage , and a greater mitochondria differentiation compared to that seen in male animals . it is thought that higher mitochondrial differentiation is a metabolic adaptation to increase energy efficiency as it is associated with their lower mitochondrial free radical production and oxidative damage . in addition to differences with sex , it is also likely that reactive oxygen species ( ros ) levels vary with animal strain as female wistar and f344 rats exhibit lower ros production and indices of oxidative damage than that seen in female sprague - dawley rats which may help to explain their greater mean lifespan [ 91 , 92 ] . the mitogen - activated protein kinase ( mapk ) cascades are evolutionary conserved serine / threonine protein kinases that regulate several important cellular functions including proliferation , differentiation , development , cell cycle , and cell death . the major mapk signaling pathways are the extracellular signal - regulated protein kinase cascade ( p44/42 ) , c - jun amino - terminal kinase / stress - activated protein kinase cascade ( jnk / sapk ) , and the p38 cascade . stimuli that include stress or injury typically activate the jnk and p38 mapk kinases , while the p44/42 mapks are stimulated by mitogenic and growth factors [ 94 , 95 ] . mapk signaling is involved in many of the age - associated physiological changes ( hypertrophy , oxidative stress , and apoptosis ) as well as other cardiac pathologies . works investigating mapk signaling in aging human hearts have demonstrated reduced p38 mapk activity / signaling in heart failure and impaired activation of mapk target genes following increase in oxidative stress [ 9698 ] . it is well recognized that increases in cellular stress cause the upregulation of the heat shock protein ( hsp ) . hsps range in size from 10 to 150 kda and function to prevent protein aggregation and assist in ensuring proper protein folding , as well as helping to mediate the translocation of damaged proteins . hsps are also involved in cardiac hypertrophy , in response to vascular wall injury , ischemic preconditioning , and aging . during aging , there is an accumulation of damaged or misfolded proteins which may cause a burden on maintaining proteostasis [ 102104 ] . an important function of heat shock proteins is to protect against age - related protein misfolding [ 103105 ] . in aged male f344 rats ( 24 months ) , it appears that aging decreases hsp70 upregulation following chronic exercise and heat stress [ 106108 ] . similar to the mapk proteins , the nuclear factor-b ( nf-b ) pathway is thought to play a role in cardiac remodeling , apoptosis , acute ischemia and reperfusion , and unstable angina , as well as heart failure in both humans and rodents [ 109112 ] . the nf-b family consists of rela ( p65 ) , c - rel ( rel ) , relb , nf-b1 ( p50 ) , and nf-b 2 ( p52 ) . figure 1 presents the nf-b signaling pathway . in the cytoplasm , the p50 and p65 are found as an inactive heterodimer due to the binding of the ib kinase inhibitory proteins ( ikk and ikk ) . cellular stimuli known to activate the nf-b pathway include ros , tnf- , il-1 , and bacterial lipopolysaccharides . it has been shown that the ibs must first become phosphorylated and degraded before p50/p65 activation can occur . the p50/p65 when phosphorylated ( activated ) then translocates into the nucleus to induce the transcription of chemokines ( il-8 , mcp-1 ) , cytokines ( tnf- , il-1 , il-2 , il-6 ) , adhesion molecules ( icam-1 , vcam-1 , e - selectin ) , acute phase proteins , antimicrobial peptides , secondary inflammatory enzymes ( cox-2 , inos , pla2 , mnsod ) , and antiapoptotic factors . upon termination of the nf-b stimuli , p50/p65 binds to new ibs and the complex it is thought that the mapk pathway works in concert with the nf-b pathway to increase transcription of inflammatory genes . extensively review the role of sex hormones in intracellular signaling mechanisms within the myocardial inflammation . although the effects of estrogen on mapk signaling have been studied in several pathological processes , for example , breast cancer , migraines , and polycystic ovarian syndrome , little is known regarding how this molecule affects mapk signaling in the heart . it is thought that differences in mapk activation between male and females may be regulated , at least in part , by the influence of estrogen [ 66 , 115 , 116 ] . estrogen has been shown to stimulate the activation of p44/42 mapk and jnk mapk in different model systems including cardiomyocytes , mammary cancer cells , pituitary tumor cells , tissue slices of the hippocampus , and endometriotic stromal cells [ 117119 ] . in males as well as ovariectomized females mapk signaling is decreased after acute ischemia [ 115 , 116 ] . in a model of cardiac pressure overload , estradiol treatment appeared to exhibit antihypertrophic effects by increasing the expression of atrial natriuretic peptide ( anp ) and inhibiting p38-mapk activation . in addition to the regulation of mapk proteins , there is evidence to suggest that gender also has an effect on the hsp response . in hearts that were not exposed to increased cellular stress , male hearts have half as much hsp72 expression compared to that observed in the female heart . estradiol treatment has also been found to increase hsp27 , hsp70 , hsp72 , and hsp90 expression in the heart [ 122124 ] . to our knowledge , no studies have looked at hsp27 , hsp70 , and hsp90 in the aging female heart . aging female and male hearts have shown increased apoptosis and inflammation , as well as age - associated gender differences in nf-b signaling . to our knowledge , only a few studies have investigated the changes in nf-b expression and activity in the aging female rat heart . apoptosis or programmed cell death is a highly conserved and regulated cell response to inhibit the abnormal proliferation of cells . cardiomyocytes are not capable of self - regeneration and are terminally differentiated but can undergo apoptosis , necrosis , or autophagy when unduly stressed . apoptosis is increased in many cardiovascular diseases such as dilated and ischemic cardiomyopathy , hypertrophic heart disease , and arrhythmias [ 126 , 128 , 129 ] . in rats , increased age has been shown to cause apoptotic susceptibility in the heart following oxidative injury [ 130 , 131 ] . aging in the male f344xbn heart is characterized by increases in the amount of cytochrome c , aif , bax , rate of permeability transition pore opening , and fragmented dna . the age - associated increase in apoptosis appears to be due not only to the activation of proapoptotic molecules but also to decreases in antiapoptotic nf-b , bcl - xl , and grx1 signaling . another work has demonstrated that the aging male f344xbn rat heart is characterized by increases in tdt - mediated dutp nick end labeling- ( tunel- ) positive nuclei , caspase-3 activation , caspase - dependent cleavage of alpha - fodrin , and diminished phosphorylation of protein kinase b / akt ( thr308 ) . the increase of apoptosis in the aging male f344xbn was highly correlated to age - associated increases in oxidative - nitrosative stress . though not demonstrating cause and effect , these results suggest that increased cellular ros and cardiomyocyte apoptosis may play a role in age - related cardiac remodeling . the incidence of cells undergoing apoptosis in the heart has been shown to differ with sex in humans and rats . in humans , a higher number of tunel positive myocytes are seen in young males compared to females , aging appears to increase cardiac apoptosis in male but not female ; however , there was no gender difference in apoptosis in male and female f344 rat [ 92 , 135 ] . structural changes found in the aging vasculature include an enlarged lumen , media - intimal thickening , irregularly shaped endothelial cells , migration and proliferation of vascular smooth muscle cells , increased deposition of extracellular matrix , increased expression of adhesion molecules , and alterations in the expression of metalloproteinases as well as cytokines [ 4 , 136138 ] . functional changes with age in the vasculature consist of impaired distensibility , increased stiffness , increased endothelial permeability , attenuation of 2-adrenoceptor vasodilator response to agonists , and diminished response to adrenergic receptor stimulation [ 4 , 139 ] . like humans , rats also appear to exhibit several age - associated changes in vascular structure and function including changes in intimal thickening , elevations in inflammation - associated molecule expression , and increased evidence of oxidative stress [ 4 , 140 ] . similar to that seen in other parts of the cardiovascular system , differences in aortic structure and function between genders have also been observed in the aging human and animals . in males , aortic wall and intimal - medial thickness are greater during aging than that observed in women ; however , stiffness was not different between men and women . similarly , there were no differences in distensibility in the aging aorta between men and women . although the human aging aorta did not show increases in aortic stiffness , it has been documented that the increased vascular stiffness with aging is more prominent in male than female animal models [ 143 , 144 ] . this may be due in part , to the lower total peripheral resistance and higher cardiac output seen in women compared to men of similar arterial pressure and age . abnormal proliferation and migration of vascular smooth muscle cells ( vsmcs ) play important roles in the pathophysiology of atherosclerotic diseases . previous studies have shown that vsmcs isolated from old animals replicate more actively than those obtained from young animals [ 145 , 146 ] . similarly , aging has been shown to be associated with an increased proliferative response of vsmcs after balloon angioplasty . endothelial dysfunction is considered to be a common and early feature of vascular disease and impaired endothelium - dependent relaxation has been demonstrated in several rat models of hypertension , experimental diabetes , atherosclerosis , and high salt diet , as well as aging . the factors regulating endothelial dysfunction are likely complex and may vary between models and with aging . nitric oxide ( no ) is a vasodilator that is produced by endothelial nitric oxide synthase ( enos ) that plays a crucial role in blood pressure regulation . no production is stimulated by shear stress , cyclic strain , acetylcholine , vascular endothelial growth factor , bradykinin , estrogen , sphingosine-1 phosphate ( s-1p ) , hydrogen peroxide , and angiotensin ii . it is thought that enos activity is regulated by enos phosphorylation at serine 1177 , serine 635 , and serine 617 by phosphatidylinositol 3-kinase ( pi3k)/akt , adenosine monophosphate - activated protein kinase ( ampk ) , and cyclic amp - dependent protein kinase signaling [ 149 , 150 ] . given its important role in regulating vascular function , it is not surprising that abnormalities in vascular no production are thought to contribute to the pathogenesis of atherosclerosis and hypertension [ 151 , 152 ] . advanced age has been found to be associated with impaired endothelial no synthesis and endothelial dysfunction [ 153 , 154 ] . the mechanisms responsible for age - associated alterations in no synthesis are not fully known but may include changes in activity or expression of enos , increased breakdown of no due to oxidative stress / oxidative injury , changes in antioxidant defense systems , and decreased availability of enos substrate , l - arginine , although the activity of enos is generally thought to be diminished with aging in the aorta . in addition to changes in enos activity , decreased no availability during aging can also be caused by oxidative stress and alterations in enos structure [ 155 , 156 ] . although estrogen ( 17- estradiol ) has been shown to activate enos , estrogen studies in rats have shown no effect or increased enos expression following estrogen treatment . conversely , in humans , estrogen replacement appears to be largely ineffective as a means of decreasing cvd risk [ 159 , 160 ] . in women , the incidence of vascular dysfunction is thought to be related , at least in part , to the cessation of ovarian hormone production . in vascular smooth muscle cells from young female wistar kyoto rats , estrogen inhibited vsmc proliferation following stimulation with fetal calf serum similar decreases in vsmc proliferation with estrogen treatment following cell stimulation by growth factors or mechanical stress have also been noted [ 161 , 163 ] . like estrogen , progesterone is also thought to inhibit the proliferation of aortic vsmc , most likely via its ability to inhibit dna synthesis . nonetheless , it should be noted that the effects of estrogen in vivo are likely more complex than those seen in cell culture . for example , in aortas from diabetic female rats , estrogen failed to reverse the impaired basal release of nitric oxide and the abnormal relaxation to histamine . the mapk signaling pathways function to regulate many processes in the aorta including vsmc proliferation , contraction , migration , differentiation , and cell survival [ 166168 ] . aging has also been shown to increase the activation ( phosphorylation ) of p44/42 and jnk mapks in the aorta while another work has shown that aging affects the ability of the aged aorta to activate p38 and jnk mapk signaling following mechanical loading . in aged animals the activation of p44/42 mapk in vascular smooth muscle was increased compared to young animals [ 145 , 171 ] . similar to that observed for the heart , age - associated changes in the expression / activity of nf-b were found in the vascular smooth muscle cells of the aorta . aging has been found to increase the sensitivity of nf-b to glucose in aortic vscm cells . another research has shown that vscm proliferation and nf-b activation stimulated by interleukin-1 were increased more in aged female rats than young female rats . in premenopausal women , receiving hormone treatment appeared to reduce nf-b activation suggesting that estradiol reduces nf-b activation . how estrogen may inhibit nf-b activity is currently unclear but may be related to increased ib levels or decreased levels of circulating tnf- . in summary , the loss of ovarian function during aging may influence cardiovascular structure and function . however , the fact that human females do not display cardiac hypertrophy or myocyte loss in the absence of loading abnormalities with aging may suggest the presence of estrogen prior to menopause acts to attenuate the cardiac response to preexisting underlying pathologies . due to the complexities of the aging process , more studies are needed to distinguish whether observed differences are due to aging alone , hormone deprivation , or a combination of both . in order to overcome the difficulties found in aging human research ( ethics , cost , and long lifespan ) , rat models have been used to provide great insight in investigating age - related changes in cardiovascular structure and function . however , additional research examining how aging may affect cardiac structure and function in rats is needed to determine whether the alterations seen in rats mimic the changes seen in humans . because of the systemic nature of reproductive senescence , the use of surgical practices to induce pseudomenopause or ovariotoxicity may be incomplete in the overall approach to female aging and cardiovascular changes . the global changes in the body with age may play a cumulative role in combination with the hormonal changes seen in reproductive senescence . aging - associated increases in oxidative stress can lead to alterations in the cardiac cell signaling which may lead to deleterious changes in the heart . because cardiac structure , function , signaling , and hormonal response appear to differ between male and female rats and humans with aging , care must be taken in evaluating current literature with regard to specific finding and gender . studies investigating the details of cardiac signaling with gender and aging are likely to lead to a better understanding of the mechanisms responsible for cardiovascular diseases in both male and females . these findings may prove critical in the application of current prophylactic practices and pharmaceutical interventions in patient outcomes .

cardiovascular disease is the leading cause of death in women in the united states . aging is a primary risk factor for the development of cardiovascular disease as well as cardiovascular - related morbidity and mortality . aging is a universal process that all humans undergo ; however , research in aging is limited by cost and time constraints . therefore , most research in aging has been done in primates and rodents ; however it is unknown how well the effects of aging in rat models translate into humans . to compound the complication of aging gender has also been indicated as a risk factor for various cardiovascular diseases . this review addresses the systemic pathophysiology of the cardiovascular system associated with aging and gender for aging research with regard to the applicability of rat derived data for translational application to human aging .

1. Introduction 2. Discussion 3. Conclusion

the mycotoxic nephropathy ( mn ) is a renal disorder caused by alimentary ingestion of secondary fungal metabolites possessing nephrotoxic properties and encountered in feeds / foods / forages made mainly from cereals or fibrous plants , and kept in storehouse conditions and increased humidity . since its discovering the disease has been described using various names : nephrosis provoked by moulds , chronic interstitial fibrosis of kidneys , chronic interstitial nephritis , etc . recently , the terms ochratoxicosis and mycotoxic nephropathy are the most frequently used ones for describing this nephropathy . during the last years this kidney disease has gained high publicity in some countries with well developed stock - breeding ( especially pig - breeding ) as denmark , sweden , bulgaria , etc . in order to prevent human exposure to various nephrotoxic mycotoxins , mainly ochratoxin a ( ota ) , via consuming the meat of animals with nephropathy , the timely diagnosis of disease during the meat inspection at slaughterhouses is very important . in such a way the exposure of humans to this very hazardous and relatively heat stable toxin from chicken / pigs meat can be prevented . there are some variances in the manifestation of the disease , especially in the clinicomorphological picture , which in a lot of the cases is influenced by the secondary bacterial infections as a result of the pronounced immunosuppression in the affected animals [ 2 , 3 , 4 , 5 , 6 ] . therefore , this review paper could contribute to timely diagnostics of mycotoxic nephropathy in farm animals ( mnfa ) as well as could help the organization of the prophylactic measures against this disease . in addition , some information is given how to make an evaluation of human exposure to ota on the base of the found concentrations of ota in human serum via calculating the daily ota intake of humans in endemic areas . although many nephrotoxic fungal compounds such as fumonisins are known , only ota and partly citrinin can be firmly associated with spontaneous cases of mn . the first and the most toxic compound discovered is ota , which was isolated from aspergillus ochraceus and chemiclly defined in 1965 by several south african scientists [ 811 ] . ota consists of a dihydroisocoumarin moiety linked through its 7-carboxyl group by an amide bond to one molecule of l- phenylalanine ( 7-carboxy-5-chloro-8-hydroxy-3 - 4-dihydro-3r - methylisocoumarin ) . citrinin was discovered in 1931 by hetherington and raistrick , who isolated the compound from several strains of penicillium citrinum . fungi that produce ota are widespread in nature and various foods or drinks [ 13 , 14 , 15 ] , but commonly contaminate stored grain . penicillium viridicatum , predominantly a storage fungus , is the mould that usually develops on grains at low temperatures and also is the major producer of ota in the colder areas of the world ( northern europe and canada ) . on the other hand aspergillus ochraceus probably is the most impotant producer of ota in tropical and semitropical areas [ 16 , 17 ] . however , the growth of a. ochraceus on natural substrates has not been associated with a natural occurrence of ota . not all isolates of p. viridicatum and a. ochraceus are ota producers , but some of them produce other toxic compounds , e.g. , citrinin , penicillic acid ( pa ) , hydroxyaspergillic acid and secalonic acid . a positive correlation has been observed between the frequency of spontaneous porcine nephropathy in bulgaria and the rate of ota in corresponding feed samples . however , the analysis for ota of various feed samples from farms with high frequency of spontaneous mycotoxic porcine nephropathy ( mpn ) revealed that the values were substantially low and ranged from 38 to 552 ( mean 207.1 65.14 ) ng / g for 1993 and from 42 to 427 ( mean 114.06 35.79 ) ng / g for 1994 respectively . sometimes the problem seemed to come from certain feed plants whose grains , collected during moist and rainly days , had not been properly dried . all farms supplied by these plants subsequently produced some pigs with nephropathy and growth depression , but after changing the certain suspected feeds the problems with poor growth of pigs disappeared . as a whole , the frequency and duration of the observed nephropathy in different batches of slaughtered pigs varied significantly ( from 12% up to 8090% frequency ) and have depended on the duration of feeding of various suspected feeds stored for a long time in poor conditions and at high humidity [ 1821 ] . it is important to mention that kidney damages in spontaneous mpn in bulgaria ( figure 1 ) are more similar to those in balkan endemic nephropathy ( ben ) in humans , than in danish mpn . for example , the fibrotic changes and contraction of kidneys in the end - stages of mpn in bulgaria are very comparable to those in ben in humans , whereas such changes are not seen in the classical description of mpn as made in denmark . in addition to the contraction of kidneys in later stages of bulgarian mpn and ben , there are various other similarities between ben in humans and bulgarian mpn as the low contamination levels of ota in feeds or foods and serum , neoplastic changes in kidneys ( pigs figure 2 ) or urinary tract ( humans ) , retention cyst formation in proximal tubules of kidneys , vascular lesions in kidneys , electron dense formations and myelin - like figures in mitochondria of epithelial cells ( figure 3 ) . however , these pathological changes have not been observed in classical mpn as described in denmark or elsewhere . this circumstance , in addition to low feed / food levels of ota in bulgarian mpn and ben , suggest a possible interaction between ota and other mycotoxins , which needs to be proved ( figure 3 ) . recently , it was found that pathomorphological changes , including electron - dense formation and myelin - like figures , induced in pig 's kidneys by combined exposure to such low contamination levels of ota together with pa , are more similar to those in spontaneous mpn in bulgaria than to classical danish mpn . all feed samples originating from farms with high frequency of spontaneous avian nephropathy in bulgaria were also positive for ota and similarly to pig farms , the levels of ota were substantially low ( the values ranged from 90.8 to 310 ng / g ( mean 196.2 ng / g 45.9 ) . the nephropathy in chickens was usually observed during the spring and summer , similarly to porcine nephropathy and in the same regions as well . the occurrence of nephropathy in different batches of slaughtered chickens varied from 12 per cent up to 90100 per cent . the continuance of this nephropathy , also differed widely from one month to about 56 months or even throughout a year . it is known that experimental pigs exposed to ota contaminated feed at levels of about 200 ppb develop only microscopic lesions after a period of 34 months [ 24 , 25 ] . the average contamination level of ota in bulgarian feeds for pigs in 1993 only exceeded 200 ppb ota . on the other hand , the low contamination levels of ota in pigs / chicks feeds suggest a possible synergism between ota and other compounds produced by the same ochratoxinogenic fungi . it is very likely that feeds contain other nephrotoxic mycotoxins or compounds which enhance the toxicity or produce a synergistic interaction with ota [ such as pa , fumonisin b1 ( fb1 ) , citrinin , viomelein , xanthomegnin etc ] . several years ago , it has been found that the toxicity of various strains of the same aspergillus ochraceus group is very different , independently of the capacity of ota production [ 22 , 26 , 27 ] . a potent synergistic effect was found between ota and pa , mycotoxins produced by the same ochratoxinogenic fungi , when the same mycotoxins were given simultaneously to pigs and chickens [ 22 , 2630 ] . the production of multiple toxins by a single organism , such as aspergillus ochraceus ( which produces ota and pa simultaneously ) , or by mixture of fungi presents a problem that has not been sufficiently investigated . pa was suspected to be carcinogenic and was found to have dna - attacking ability in the rec assay as well as to induce chromosome aberations . some data suggest that feed contamination levels of pa itself up to 4000 ppb have little toxicity ( mostly hepatotoxicity ) and do not adversely affect body or organ weights or biochemical parameters in chickens . only dna breaks in mammalian cell lines [ 36 , 37 ] and an inhibition of rat liver glutathione s - transferase activity in crude extracts in vitro as well as pa - induced hepatobiliary excretory dysfunction and liver hemorrhages have been found so far . the higher toxicity of ota in all above mentioned studies might be due to synergistic toxic effects between ota and pa , as has been reported in mice [ 41 , 42 ] and in poultry . previous studies have suggested an important role of the pancreatic enzyme carboxypeptidase a in the detoxification of ota in the small intestine [ 43 , 44 ] . parker and colleagues showed that pa inhibits carboxypeptidase activity in vitro and in vivo and such inhibition may significantly impair the primary detoxification of ota in the intestinal tract and so be partly responsible for the enhanced toxicity of ota observed in combination with pa [ 22 , 2630 ] . the pa - induced hepatobiliary excretory dysfunction may also result in decreasing of hepatobiliary excretion of ota . such synergism between ota and pa or other mycotoxins under field conditions may be responsible for the spontaneous mycotoxic nephropathy in bulgaria , which can be caused by relatively low contamination levels of ota in feed [ 18 , 21 , 23 ] . this could explain why the low levels of ota in bulgarian feeds for pigs [ 18 , 21 ] , chickens or humans have such high toxic effect on kidneys , when ingested with spontaneous contaminated feeds . the concentration of ota in feeding forages is probably more important than that of pa , because the higher contamination levels of pa ( 60000 ppb pa in combine with 1000 ppb ota ) have not been able to produce more significant toxic effects . this findings clearly suggest that the increase in ota - toxicity is probably due to impaired detoxification of ota via pa - inhibited carboxypeptidase activity in the intestinal tract . several years ago , stoev and collaborators induced macroscopic kidney damages in pigs fed on a mouldy diet containing low levels of ota ( 180 ppb ) in combination with pa for a 3 months feeding period , whereas microscopic lesions in kidneys in the same experiment were observed in pigs fed on a diet containing only 90 ppb ota in combination with pa . the observed changes were similar to those caused in pigs at the end of the 4-month period of exposure to higher levels ( markedly above 200 ppb ) of pure ota in feed . it is therefore important to investigate the effect of combined administration of ota and other mycotoxins , produced by the same ochratoxinogenic fungi as occur in the field . in combined administration of ota and pa much lower concentrations of ota ( about 100 ppb ) are enough for a significant toxic effect in chickens , expressed by degenerative changes in internal organs , by a depletion of cells in lymphoid tissue and decrease of lymphoid organ 's weight as well by known changes in various biochemical parameters . moreover , degenerative and weight changes in kidneys , liver and lymphoid organs as well as immunosuppression were seen recently in chickens at only 0.2 or 0.3 ppm ota in combination with pa [ 26 , 27 , 29 , 30 , 47 ] , whereas such changes had been seen in chickens exposed to significantly higher levels of pure ota ( about 4 ppm ) in feed [ 4851 ] . according to other authors a similar but less pronounced synergistic toxic effect has been observed at higher contamination levels of ota ( 1000 ppb ) . the mentioned above low experimental levels of ota correspond very well to the feed levels of ota found in spontaneous cases of mycotoxic avian nephropathy ( man ) in bulgaria ( 0.090.31 ppm ) , which suggests a possible multicausal nature of man in bulgaria . furthermore , to ascribe the source of ota to the a. ochraceus group of fungi is unwise because ochratoxinogenic forms are isolated so rarely [ 18 , 52 ] . therefore , it is already of great importance to carry out more mycological investigations in order to isolate the ochratoxinogenic fungi in bulgarian feeds . in addition , the overall concentration of ota in feed samples ( from 0.38 to 0.552 ppm ) were substantially lower than the 1 to 2 ppm required to reproduce mpn / man of severity similar to that observed in spontaneous cases [ 18 , 21 , 23 , 53 ] . it seems , therefore , that the mpn / man in bulgaria may have a multitoxin or multifactor etiology , because it can not be explained by the concentration of ota alone . the multimycotoxin etiology of mpn / man in bulgaria is recently confirmed as high contamination levels of pa ( 838,6 223,9 g / kg - 88% positives ) and fb1 ( 5564,1 584 g / kg - 96% positives ) in bulgarian feed samples from farms with mycotoxic porcine nephropathy were found , whereas the levels of ota ( 188,8 27,3 g / kg - 100% positives ) in the same feeds were consecutively lower ( unpublished personal data ) . a similar multimycotoxin etiology was also found for south african mpn as the same mixture of mycotoxins ( 67,8 39,2 g / kg ota - 83,3% positives ; 149,2 64,1 g / kg pa - 41,7% positives , 5046,2 1301 g / kg fb1 - 80% positives ) was found in south african feed samples from pig farms with nephropathy problems ( unpublished personal data ) . some mycological investigations of ota - contaminated feeds in bulgaria revealed the common presence of p. aurantiogriseum complex , which is a potent producer of pa . pa is produced by numerous species of penicillium ( especially p. aurantiogriseum ) and aspergillus at temperatures ranging from 4c to 30c , with the maximum rate of production occurring at about 25c . the production of pa decreased sharply at low oxygen concentrations , while fungal growth was not noticeably influenced . the rate of toxin coupling with -sh radicals increases with ph as well as in high temperature . the resulting complexes are much less toxic than the uncoupled molecules , resulting in actual detoxification . as a result , this toxin usually accumulates at relatively low temperatures during the winter at which detoxification is more reduced than toxin production . that could explain why bulgarian nephropathy in pigs / chickens was usually observed during the spring and summer it has recently been found that administration of p. polonicum extract ( not containing ota ) to rats can provoke profound and persistent histopathological damages such as apoptic and karyomegalic or mitotic changes in the nuclei of tubular epithelium in kidneys of rats , including dna - adducts formation . the same p. polonicum strain , which is a common food / feed spoilage mould in warm temperate latitudes , has been found as a frequent contaminant in bulgarian feeds , suspected of causing spontaneous porcine nephropathy [ 18 , 52 ] . it is known that the p. aurantiogriseum group ( including p. polonicum strain ) is a potent producer of pa , which can also provoke dna breaks in mammalian cell lines as has been reported . in such a way it could appear that the source of pa in bulgarian feeds can be different from the source of ota . also , the same changes ( apoptosis and karyomegaly in tubular epithelium ) , provoked by p. polonicum extract , could be induced by another unknown mycotoxin ; this would be of interest , because the same mycotoxin could be partly responsible for the nephrotoxic damages described in bulgarian nephropathy . moreover , the quiet apoptosis , induced by p. polonicum nephrotoxin , could be a possible model for the cryptic and clinically - silent onset of renal atrophy in the idiopathic balkan endemic nephropathy in humans . on the other hand , it has been reported that ota has a high potential to induce apoptosis and dna - adducts in vitro [ 61 , 62 ] and in rodent in vivo . also , rahimtula et al . showed that lipid peroxidation in kidney microsomes was enhanced by ota and produced malondialdehyde . in such a way , the extent to which ota may interact with other components of commercial chicken / pig rations or human food compounded from agricultural produce may also influence the significance of the relatively lower doses of ota that commercial chickens [ 23 , 27 ] , pigs [ 18 , 21 ] or humans may encounter in some feed / food . various different strategies can be employed to reduce human / animal exposure to ota or to reduce its toxic effects when it is fed to animals . the same include the use of management practices to prevent the production of ota by the storage fungi , the feeding of contaminated grain to animal species that are less susceptible to the toxic effects of ota , such as ruminants , modification of the diet to promote enhanced hydrolysis of ota in the gastrointestinal tract or reduced absorption , the use of a feeding regimen that can counteract the metabolic effects of ota , the use of various procedures that can destroy ota , such as physical and chemical methods for decontamination of commodities as well as the use of various feed additives , which can protect against the toxic effects of ota etc . . gastrointestinal microorganisms have a large effect on the disposition of ota because they promote the hydrolysis of ota to its nontoxic form ot. this is particularly important in ruminants and therefore they are less susceptible to ota - toxicity . in addition , the type of diet also affects the disposition of ota in the rumen [ 67 , 68 ] . ruminal fluid obtained from hay - fed animals ( ph 7.0 ) was able to hydrolyze ota to ochratoxin ( ot ) in vitro five times faster than ruminal fluid obtained from grain - fed animals ( ph 5.5 ) . disappearance of ota from the rumen and the corresponding formation of ot was also much faster for hay - fed than for grain - fed sheep . the bioavailability of ota in sheep fed grain was 4.3 times greater than that in sheep fed hay . thus , the rumen of the sheep has an important role in the detoxification of ota and the type of diet affects the rate and extent of this process . as a result , the bioavailability of ota , and probably the toxicity to ruminants , is decreased . in addition , the intestinal microflora of nonruminant animals , also affect the bioavailability of ota , because the microorganisms in the intestines and particularly in the caecum and large intestine are also responsible for the hydrolysis of ota to its nontoxic form ot . another way to prevent the production of ota is prevention of the growth of the storage fungi . the most important factors in safeguarding stored feedstuffs / foodstuffs from ota prodution by moulds are their moisture content ( water activity ) and temperature . large quantities of ota can be produced at intermediate and high ambient temperatures and high moisture contents by species belonging to the penicillium and aspergillus , whereas at lower ambient temperature the toxin can be produced by penicillium spp . only . in addition , the moulds in the corn when stored at 21% moisture produced a large quantity of ota ( about 3.6 ppm ) , whereas none of this toxin was found in corn stored at 16% moisture . these and some other studies indicate that fungi in cereal grains stored at moisture contents lower than 15% generally do not produce ota , which suggests that the moisture content in the cereal grains have to be decreased under 15% via various drying procedures before storage . storage at higher moisture levels requires that grain be maintained under anaerobic conditions , which prevent growth of fungi . otherwise , a combination of mould inhibitors and sterilization techniques , possibly coupled with the inoculation of nontoxigenic competitive microflora , have to be applied to prevent the growth of ota - producing fungi . chelack and collaborators [ 72 , 73 ] have used another way to control the growth of ota - producing fungi . they demonstrated that gamma or electron beam irradiation is a highly effective means of destroying spores from some ota - producing fungi as a. alutaceus [ 72 , 73 ] . various other types of radiation ( x - rays , ultraviolet light , microwaves ) in addition to -irradiation are also used as a means for detoxification of mycotoxins as well as to control the growth of fungi [ 74 , 75 ] . leitao and collaborators demonstrated that phosphine ( ph3 ) was effective at inhibiting both fungal growth and sterigmatocystin production by a. versicolor , and this could be considered as another specific way to control the growth of ota - producing fungi . for foods with ph values from 5 to 6 , such as the cereals or sorghum , the antimicrobial agent ( food additive ) that is able to prevent the growth and ota - production by aspergillus and penicillium species , is methyl paraben or potassium sorbate . small concentrations of these compounds are able completely to inhibite the growth by both genera of fungi and their ota - production . at ph 4.5 , as occurs in silage , fungal growth and ota - production was completely inhibited by 0.02% potassium sorbate , 0.7% methyl paraben , and 0.2% sodium propionate . the use of various procedures that can destroy ota , such as physical and chemical methods for decontamination of commodities can be considered as another important way for safely utilizing of feedstuffs . madsen and collaborators reported that treatment of ota - contaminated barley with 5% nh3 for 96 h at 70c or warming of grain to 105c in the presence of 0.5% naoh as well as autoclaving at 132c for 0.5 h can destroy the main part of ota in the barley , but such treatments are not practical and are not able effectively reduce the concentration of ota in grain . chelkowski and collaborators reported that treatment of ota - contaminated grain with ammonia reduced ota concentrations to undetectable levels . moreover , weight gains of chickens fed the ota - contaminated grain were markedly decreased , whereas there was no reduction in weight gains when the chickens were fed the ota - contaminated grain that had been ammoniated . they concluded that ammoniation of grain not only detoxifies several mycotoxins , including ota , but also inhibits mould growth . feeding studies have shown no toxic effects related to the ammoniation process , but there are some changes in the nutritional quality of the feed , such as a decrease in lysine and sulfur containing amino acids . in addition , adequate aeration after ammoniation is necessary for acceptance of the feed by animals . however , other than ammoniation , many of the techniques proposed to remove mycotoxins are currently perceived as impractical , ineffective , and/or potentially unsafe for largescale use . rotter and collaborators demonstrated that inoculation of barley with a lactobacillus species followed by ensiling ( decreasing of ph ) reduced the concentration of ota by approximately 50% . also , scott reported that ota is completely destroyed during malting of barley . according to laciakova and collaborators formic acid of 0.25% concentration degraded ota after 3 h exposure , propionic and sorbic acids in 1% concentration after 24 h exposure and benzoic acid in 0.5% concentration after 24 h exposure . several different approaches have been used to reduce ota absorption , including the use of hydrated sodium calcium aluminosilicate ( hscas ) , bentonite , charcoal , and cholestyramine . the addition of hscas ( 1% ) and bentonite ( 1% and 10% ) to a diet containing ota had no effect on ota concentration in swine blood , serum , tissues , and bile . the addition of 1% activated charcoal to the diet , in contrast , caused a slight decrease in the concentration of ota in swine blood , whereas 10% charcoal decreased the concentration of ota in blood , liver , kidney , spleen , and heart by 50% to 80% . however , the supplementation of ota - contaminated diet with activated charcoal was considered as an impractical method of reducing ota toxicity in chicks or pigs that were continuously consuming ota . cholestyramine , in contrast to the nonspecific absorbent discussed above , seems to be an effective absorbent of ota in the gastrointestinal tract of nonruminant animals . cholestyramine is a commercial anion exchange resin that has been shown to reduce blood ota concentrations by 50% , when it was included in 0.5% in a rat diet containing 1 ppm ota . in addition , the faecal excretion of ota was significantly increased in rats given a single oral dose of ota , which were fed on diet containing cholestyramine . another way to reduce ota - toxicity supposed a good understanding of the mechanisms of ota - toxicity : inhibition of phenylalanine metabolizing enzymes ( having in mind a phenylalanine moiety of ota ) , enhancing of lipid perooxidation , and inhibition of mitochondrial atp production . it is well known that the phenylalanine moiety of ota competitively inhibits at least two enzymes : phenylalanyl - trna synthetase and phenylalanine hydroxylase , resulting in reduced protein synthesis and altered rates of tyrosine production from phenylalanine . the addition of phenylalanine to cell cultures containing ota in vitro or coadministration of phenylalanine with ota [ 88 , 89 ] reduced ota - induced inhibition of protein synthesis . in vivo experiments show that injection of phenylalanine also prevented ota - induced immunosuppression in mice [ 90 , 91 , 92 ] and partially reduced teratogenesis in rats . the phenylalanine has to be used in 5:1 molar ratio towards ota , because it had been found that phenylalanine given in higher doses as 10:1 molar ratio , provides only a slight protection against ota , because of increasing the absorption of ota from the stomach and intestine . moreover , the higher supplementation of pure phenylalanine in chick diets contaminated with ota also tended to create an amino acid imbalance , which reduced b. w. gain and feed conversion efficiencies [ 95 , 96 ] . these studies suggest that little or no benefit is obtained by supplementing ota - contaminated diets with phenylalanine . the concentration of protein in the diet of growing chicks was also shown to ameliorate the toxicity of ota [ 97 , 98 ] . the consumption of high - protein diets ( 26% ) by growing chicks over a three - week period compared to a diet containing a lower concentration of protein ( 14% ) decreased the toxicity of 5 ppm ota in diet as indicated by rate of growth , mortality and relative organ weights of chicks [ 97 , 98 ] . this treatment , however , may not be practical because a large change in the protein concentration in diet may be costly and produces relatively small benefits the ascorbic acid supplementation ( 300 mg / kg ) of laying hen diet that contained 3 ppm ota has been reported to have a good protective effect against ota - action and can partially ameliorate ota - toxicity ( including the negative effect of ota on the eggs production and on the weight of the eggs ) [ 99 , 100 ] . the mechanism of the protective effect is not clear but it is supposed to affect the production of lipid peroxides by ota and to improve the membrane integrity of cell organelles , which is always affected by ota - action . the use of various feed additives can also protect against the toxic effects of ota and can reduce farm losses from a decrease of weight gain in stock chicks avoiding the rejecttion or condemnation of ota - contaminated feed [ 27 , 30 , 94 ] . sesame seed ( rich in proteins - about 20% and relatively rich in l- phenylalanine - about 4.3% ) , given in 8% to the feed , offered a cheaper agronomic additive to supply animals with phenylalanine and thereby to protect against toxicity of ota . it might also avoid the increase of ota - absorption from the stomach and intestine provoked by the pure phenylalanine in feeds . simultaneously , the increase of urinary excretion of ota in presence of phenylalanine could be preserved in a later stage . moreover , the sesame seed ( 8% to the feed ) increases the energy metabolism in animals , which usually is disturbed by ota . ( hofmann la roche , grenzach - wyhlen , austria ) is a polyenzyme complement produced by fungi trichoderma , which contains : cellulase , xylanase , endo - beta 1,3 - 1,4 glucanase , pectinase and amylase . roxazyme - g , given in concentration 0.2g / kg feed , was reported to improve digestive dissimilation of polysaccharides to easily assimilated substances , that could improve utilization of feed by increasing digestible energy production by 813% , thereby counteracting ota impairment of energy production in chicks . artichoke - extract is a complex containing compounds which might protect against ota intoxication [ 26 , 27 , 30 , 94 ] . the 5% total water - extract of artichoke ( cynara scolymus l ) , prepared as a steam infusion from dried leaves of artichoke and given to chicks in concentration 5 ml / kg.b.w . via the feed or water , usually contains various biologically active compounds : cynarine , flavonoids , cynaropicrin , dehydrocynaropicrin , grosheimin as well as a high content of calcium and ascorbic acid . it has been recommended as a diuretic agent in cardiac and renal insufficiency , and might accelerate the urinary route of excretion of ota [ 26 , 27 , 30 ] . moreover , the cynarine content in such extract stimulates metabolism of cholesterol , decreases serum urea and lipids , improves diuresis and increases biliary secretion , which probably augment the hepatobiliary route of excretion of ota ( ota is mainly eliminated via bile and urine ) and thereby has been found to protect against ota nephrotoxicity and hepatotoxicity [ 26 , 27 , 29 , 30 , 94 ] as well as against the immunosuppressive effect of ota [ 27 , 94 , 104 ] or against ota - induced anaemia . cynarine and flavonoids as well as some medical preparations prepared from artichoke - extract ( chophytol and chophytamine ) have a potent hepatoprotective effect against hepatotoxic damage [ 105107 ] . also , antipermeability and vasoconstrictive effects of water extract of artichoke could decrease oedema in various internal organs provoked by ota [ 18 , 27 , 29 , 30 , 94 ] . the high content of calcium and vitamin c in the steam extract probably has also a protective effect against ota - toxicity via improving membrane integrity of cell organelles and skeleton - strength of the chicks or via some other ways [ 27 , 29 , 94 ] , since some authors reported that ascorbic acid supplementation ( 300 mg / kg ) of laying hen diet that contained ota ( 3.0 mg / kg ) partially reduced ota - toxicity . rosallsat , which is another plant extract ( a polyextract of bulbus alii sativi and seminum rosai canina ) , was seen also to have a protective effect against ota - toxicity , when it was given per os in 0.6 ml per kg body mass daily as a supplement to the feed . the rosallsat is a new natural galenic phytosubstance of balanced polyvitamine , phytoncide and steroid - saponine composition - described completely in patent no 98915 bg / 1994 , which contains biological active compound allicin ( 12.08 mg / g ) as well as large quantities of vitamins a ( 0.0332 mg / g ) , f ( 0.8112 mg / g ) , e ( 0.1548 mg / g ) and furostanic sapogenine of c57h96o30 [ 26 , 29 , 30 ] . the allicin in that study was formed in advance and included in a natural oil extract in commercial lots stabilized with natural antioxidants . this is an advantage when compared with fresh or dried garlic cloves since thiosulphinate is hardly formed in the ph conditions of the stomach and intestine after eating fresh or dried garlic . the precise mechanism of protection of rosallsat against ota - toxicity has not been investigated in depth , but it is supposed that the biological active compound allicin and the high quantities of vitamins a , f and e in this plant extract probably are of great importance [ 26 , 29 , 30 ] . the rosallsat is found to inhibit lipid peroxidation ( unpublished personal data ) and in such a way prevents one of the mechanism of ota toxicity . it was found that the intensity of macroscopical and histopathological changes , the deviations in relative organs weight and body weight ( table 1 ) , the decrease of antibody titer ( table 2 ) , as well as the intensity of changes in various haematological and biochemical parameters ( table 3 ) were slighter in chicks treated with some antidotes ( 5% artichoke - extract , 0.02% roxazyme - g , 8% sesame seed , 0.0025% phenylalanine ) in addition to 5 ppm ota than in chicks only treated with 5 ppm ota . in chicks of groups treated with sesame seed or artichoke - extract the described changes , especially the antibody titer against newcastle disease , were more similar to those ones observed in chicks exposed to an approximately 5 times lower contamination levels of ota ( 1 ppm ) ( table 2 ) . moreover , it appeared that all of the mentioned above antidotes , used as supplements to the feeds , especially sesame seed and artichoke - extract , had a potent protective effect against ota - induced toxicity and could be used as a practical approach for safely utilizing of ota - contaminated feed . in such a way the rejection or condemnation of such feed will be avoided as well as there will be no need to eliminate ota from the feed , if its contamination levels are similar to these encountered in the practice ( normally upto 12 ppm ota ) . however , in the pigs which are more sensitive to ota , the levels of 1 ppm ota can be more dangerous [ 2 , 22 , 108 ] and this requires additional experimentation to clarify the antidote effect of the same additives in these animals . a protective effect against ota - induced decrease of total serum protein was found in chicks of all antidote - treated groups mentioned above , but a protection against the increase of serum glucose was only seen in groups treated with roxazyme - g and artichoke - extract . also , a protective effect against ota - indiced increase of serum creatinine and urea was seen in all antidote - treated groups especially on day 70 . in addition , the protective effect against the increase of serum values of uric acid and creatinine was strongest in the group treated with sesame seed ( table 3 ) . however , it appeared that the protective effect of phenylalanine was slighter than expected one in contrast to some of the other studied antidotes . the main reason for that could be the circumstance that phenylalanine was found to increase the absorption of ota from the stomach and intestine as well as the gastrointestinal transit of ota . this resulted in eightfold and fourfold higher levels of ota in serum and liver of mice , respectively , during the first 12 h , which could also increase its elimination . therefore , phenylalanine has to be given after changing the feed source and stopping ota - exposure of animals . the strong protection of roxazyme - g against ota - induced increase of serum glucose could be due to the improved energy metabolism , whereas the protection of artichoke - extract against that increase could be due to an improvement of diuresis . the protective effect of sesame seed and phenylalanine against ota - provoked immunosuppression in humoral immune response and changes in differential wbc count might be due to an improvement of protein synthesis , which usually is disturbed by ota and subsequently , an improvement of ota - induced delay of the division of the cells of the immune system . it is known that ota induces an increase of the weight of the organs taking part in its detoxification or elimination ( kidneys and liver ) as well as a decrease of the lymphoid organs weight and body weight , whereas some of the studied feed additives ( 5% artichoke - extract , 0.02% roxazyme - g , 8% sesame seed , 0.0025% phenylalanine ) significantly protects against these weight changes as the protective effect of sesame seed and roxazyme - g against these weight changes was stronger than the protective effect of artichoke - extract and phenylalanine . in other experiments [ 26 , 30 , 110 ] it was also confirmed that artichoke - extract has a significant protective effect against ota - toxicity . in the same studies , another plant extract rosallsat was also seen to protect against ota - toxicity , expressed by statistically significant protection against the ota - induced changes in serum levels of total protein , uric acid and cholesterol ( table 4 ) . the serum levels of uric acid , which were most significantly influenced of the quantity of ota in fed forages , usually give an exact assessment of the impairment of kidney function in chicks . the absence of a confident increase in that parameters in artichoke - extract and rosallsat treated groups suggests a slight impairment in kidney function in these groups and confirms the protective effects of these feed additives against ota - toxicity . on the other hand , the protective effect of artichoke - extract and rosallsat on the liver function may contribute to the higher cholesterol levels in the groups of chicks treated with the same antidotes . in addition , the toxicological investigations in the same experiments revealed that the contamination levels of ota in kidneys are lower in chicks treated with artichoke or rosallsat in addition to ota than in chicks only treated with the same feed level of ota . by 1990 , at least nine countries have regulations for ota levels or had proposed and enforced official limits for ota . regulatory control of nephrotoxic mycotoxins in animal feeds in european countries has been summarized by van egmond and later by boutrif and canet . on the other hand , european union decided on an official limit for ota in cereals designed for direct consuming of about 5 g / kg . the official limits for ota in cereals in some countries can be seen in various fao reports and commonly ranged between 2 g / kg ( switzerland ) and 20 g / kg ( czech republic ) , and rarely reached up to 50 g / kg ( uruguay ) . in countries with wide spreading of mpn and man residues of ota were often found in animal tissues samples during the meat inspection [ 18 , 23 , 114 , 115 , 116 ] . the contamination levels of ota in liver , muscles and fat tissues of pigs can be easily found using the following experimental calculations : by 1990 , at least nine countries have regulations for ota levels or had proposed and enforced official limits for ota . regulatory control of nephrotoxic mycotoxins in animal feeds in european countries has been summarized by van egmond . because of the absence of correlation between macroscopic changes and concentration of ota in pig kidneys it is very difficult to prevent the exposure of humans to the toxin from pork by toxicological investigations of pale or mottled kidneys as it is occurred in denmark . the practice in denmark of monitoring kidney tissues in only those pigs that have detectable kidney lesions could not provide a good basis for condemnation of carcases , because the intake of ota may have not been of sufficient duration to produce detectable lesions . therefore , the regulations in denmark , according to which all enlarged and mottled kidneys are investigated for residues of ota at slaughter time and all carcasses , whose kidneys contained ota levels above 10 g / kg are condemned are not very safe and clever , because macrscopic changes in kidneys can be found only after 13 months ota exposure via the feeds [ 22 , 118 ] . some investigations made by various authors revealed that at least 12 months are needed to develop characteristic macroscopical renal lesions in pigs exposed to natural or experimental ota contaminated barley [ 24 , 119 ] . unfortunately , characteristic for mpn macroscopic changes in kidneys can not be found after a shorter period of ota exposure via the feeds even with such high levels as 1000 ppb ota and animal products containing dangerous ota contamination levels could easily pass meat inspection at slaughterhouses . on the other hand the renal lesions in kidneys ( mottled appearance ) induced at an early age do not disappear when the pigs are fed on ota - free diet , which also indicates the lack of efficiency of such control system and the implementation of some unnecessary toxicological investigations , which could be avoided [ 21 , 22 ] . in spite of the toxicological investigations of such kidneys ota - contaminated pork may enter the human food chain and thus represents a potential public health hazard . since the toxin is relatively heat stable , a limited occurrence of ota might anticipated in prepared food from such animals . because of the assumption that mycotoxins and especially ota are involved in etiology of balkan endemic nephropathy [ 1 , 122124 ] the exposure of humans to this very hazardous toxin from pork or chicken meat ( by the way feed pork / chicken - food ) need to be prevented . a much better procedure for preventing the exposure of humans to the toxin from meat would be a toxicological analysis of a few blood samples of pigs / chicken from farms , suspected of having mn , several weeks ( in pigs ) or several days ( in chicken ) before slaughter and a change in the feed source for a week ( pigs ) or for 23 days ( chicken ) , if it is necessary . also , the period of feed deprivation of pigs / chicken before slaughter could be prolonged [ 21 , 23 ] . because of the short half - life of ota in pigs ( 72 120 hours ) and especially in chickens ( 4 hours ) its concentration in blood and various tissues quickly decreases after changing the feed source or after prolonging the period of feed deprivation before slaughtering . thus , the loss of condemnation of pig / chicken production would be prevented and a better procedure ( than toxicological invesigations of enlarged mottled kidneys accepted in denmark ) would be realized for preventing the exposure of humans to ota from meat [ 2123 , 114 ] . the preventive measures in already slaughtered chicks could include condemnation and removing of the kidneys and liver , where ota is accumulated . however , having in mind that a combined administration of ota and other mycotoxins such a pa , often occurrs in the practice and synergistic effects between ota and the same mycotoxins are reported , the lower contamination levels of ota in food / feed would be also very dangerous for human and animal health [ 22 , 30 ] . balkan endemic nephropathy ( ben ) in humans is another renal disorder having a similar pathological characteristics as mpn . it is most commonly observed in farmers at ages between 30 and 50 years , has oligosymptomatic clinical picture , silent onset and poor prognosis , progresses very slowly and manifests itself when the function of majority of nephrons have become impaired . at this time there is agreement that the disease represents an unusual type of chronic interstitial nephropathy of unknown etiology , although there is an assumption that mycotoxins are involved . the disease has a strict endemic character and is encountered in the rural populations in bulgaria , romania and ex - yugoslavia . the high humidity and the poor food storage conditions in endemic villages , the low living and hygienic standard as well as the consumption of home prepared food from own crops - bread , bean and others [ 123 , 127 ] correspond to the fact that the moulds producing mycotoxins contaminate mostly home produced grain feeds stored in bad conditions and high humidity , while the situation in the towns is markedly different - foodstuff supply is centralized and quality controlled ( that explain the family character of ben only in rural inhabitants ) . also , the focality and zonality ( characteristic for ben ) are typical for mycotoxicosis ( i. e. endemic dissemination ) and are determined mainly by the ecological conditions of the regions which are optimal for the development of the corresponding fungi and by the altitude . the seasonal and yearly variations in incidence and prevalence of the disease and ota - content in the human food as well as the positive correlation between the excess rainfall and the number of patients suffering from ben , who died during the following two years , probably because of additional intake of moulded food ( provoking new degenerative damages in kidneys and impairing the disease state ) , give additional support to the mycotoxin hypothesis of ben . however , the etiology of the disease is still unknown in spite of various etiological investigations including the search for heavy metals , trace elements , infectious agents and genetic factors as well as various bacteriological , toxicological , and hydrological investigations of water . nephrotoxic mycotoxin ota has been suggested as one of possible disease determinants of ben based on a comparison of morphological and functional kidney impairment between ota - induced mpn and ben . in endemic areas , ota has been found in human blood as well as in human food [ 123 , 126 , 130 , 131 , 132 ] and in animal feed in higher concentrations than in nonendemic areas or the countries without ben . it is important to mention that the feed levels of ota in animal feeds from endemic for mpn regions ( 38552 ppb ) are approximately the same as in the foods from endemic for ben regions ( 10890 ppb ) , which additionally suggest a possible common etiology . the mean concentration of ota in human blood in bulgarian - endemic areas ranges respectively from 20 g / l for 1986 to 27.2 g / l for 1989 [ 130 , 132 ] . these high concentrations suggest for a possible role of ota in a disease causation of ben . however , ota was detected in a concentration range of 5100 g / l only in 0.4 to 2.5% of a total 13,797 analysed blood samples from 14 endemic areas in slavonski brod ( croatia ) during 9-year ( 19811989 ) period and in a concentration range 250 g / l in 0.2 to 4.5% of a total 6,910 analysed serum samples from endemic village of kaniza over the 10-year ( 19851994 ) period . these concentrations of ota in human blood are significantly lower towards those ones in bulgaria , but correspond quite well to the prevalence of the disease ( ranged between 1% and 4.5% during the last 15 years ) and the incidennce of newly recorded ben - suffering patients ( ranged between 1.0 and 2.5 per thousand ) in the same endemic areas . the disease is also closely associated with a high frequency of carcinoma in the renal pelvis , ureter and urinary bladder [ 135 , 136 ] , which could be due to the carcenogenicity of nephrotoxic mycotoxin ota [ 125 , 137 , 138 ] , but the working group that evaluated the carcinogenicity of ota to humans could not draw any conclusion . interestingly , some tumors in kidneys and bladders from patients living in areas associated with balkan endemic nephropathy , have been found to contain dna adducts similar to those obtained from the kidneys of mice exposed to ota , which provides new evidence of the possible role of ota in the development of tumors of the urinary tract . moreover , 11 of 31 patients with urinary tract tumors in france exhibited a ota - specific dna adduct pattern similar to those found in bulgarians suffering from ben , ota - treated rodent and bulgarian pig suffering from nephropathy . it was found that changes in debrisoquine 4-hydroxylation in vivo can occur in the rat in response to mouldy food and patients suffering from ben and/or upper urothelial tumors were more frequently extensive metabolizers of debrisoquine ( db ) . a recent study in egypt revealed a positive correlation between ota and the end stage renal disease ( esrd ) or urothelial tumors in humans . patients with esrd as well as patients with nephrotic syndrom or urothelial tumors have significantly higher mean concentrations of ota in blood ( between 0.52 and 2.19 ng / ml ) and urine ( between 0.36 and 3.09 ng / ml ) than patients from control groups ( between 0 and 0.08 ng / ml in blood and between 0.01 and 0.26 ng / ml in urine ) . it is established that ota decreases natural killer cell activity by the specific inhibition of endogenous interferon levels . as natural killer cells are involved in the distribution of tumor cells , the ability of ota to modulate the activity of these cells might contribute to its capacity to induce renal and hepatic carcinomas . recently , it has been found that ota has a significantly longer plasma half - life in humans ( 33.55 days ) than in animals . a slow excretion of ota suggests a tendency to an increase of toxin levels in the serum as well as assumes a very constant rate of ota in human blood for a long period of time , even after repeated exposure to very low concentrations . in such way the humans are more influenced by the dose and less influenced by the frequency , duration and quantity ( if the quantity of the toxin ingested is comparatively low ) of ota exposure than other species , because in the humans ota has a very constant and continuous effect on the proximal tubules even in little amounts , because of the very low unbound ( free ) fraction of ota in blood . it can be supposed that only the free ota in the macroorganism has a toxic effect on the tissues as well that continuous persistence of ota in human blood and its slow excretion via kidneys may be influenced on the slow progression of ben . thus , consumption of food containing very low concentrations of ota over a long period of time may become toxicologically significant . a method is also presented for calculating the daily consumption of ota through various food products on the basis of the measured content in plasma , even though the toxicokinetic constants are unknown . the renal filtration rate of ota in humans , calculated from the glomerular filtration rate of inulin and the free fraction of ota is 0.67 ml / kg body weight per day . the kidney filtration has been found to correspond to the total plasma clearance of ota ( almost 100% ) in monkeys , but only to 10% in mice , rats and pigs . the equation : k0 = clp.cp/a shows the relationship between continuous intake ( k0 , ng / kg b. w. per day ) , the plasma clearance ( clp , ml / kg b. w. per day ) , the plasma concentration of ota ( cp , ng / ml ) and the bioavailability ( a , proportion of toxin absorbed - about 50% ) . . the same equation was used to calculate the daily intake of ota from the concentration of the toxin in plasma of humans in endemic regions in bulgaria . the daily intake of humans , calculated on the basis of the average plasma levels of ota in endemic area in bulgaria was found to be between 26.8 and 36.4 ng / kg b. w. as can be seen from table 5 [ 1 , 124 ] . it is important to mention , that this calculation should give an underestimate of the intake , since the value used for plasma clearance only involves glomerulal filtration . this underestimate could be as much as ten times , if the ota clearance in man is more similar to those in rat , mouse and pig than that in monkey . because of the increase of scientific reports on ota contamination in beverages and many kinds of food , the who / fao joint expert committee on food additives ( jecfa ) assessed the available information and proposed 112 ng / kg b.w . having in mind the strong carcinogenic effect of ota its ptwi was later decreased to 100 ng / kg b.w . , which corresponds to about 14 ng / kg body weight per day . it can be concluded that the average daily intakes in the endemic areas in bulgaria from 26.8 ng / kg b. w. for 1988 [ 1 , 124 ] , 36.4 for 1989 and 34.2 for 1990 respectively , exceeds strongly ptwi ( 100 ng / kg b. w. or 14 ng / kg b. w. per day ) , proposed by the jecfa . jecfa bases its calculation of the tolerable intake mainly on the nephrotoxicity of ota and does not address the question of the toxin 's carcenogenic effect . kuiper - goodman and scott , however , regard the carcinogenic effect as the most important effect and base their analysis on this . tolerable daily intakes ( tdis ) , depending on the method used and calculated on the base of carcinogenic effect of ota , range from 0.2 to 4,2 ng / kg b. w. it can be concluded that the maximum tolerable daily intake of ota is 5 ng / kg b. w. . it can be seen that the average daily intake of the endemic and in some extent nonendemic areas exceeds strongly the tdi calculated on the base of cancerogenic effect of ota . the performed calculations on tdis show that ota contamination of food and feed constitutes a public health problem of unknown dimensions . therefore , it is quite relevant to investigate to what extent ota is hazardous to humans . because of the high thermal stability of this mycotoxin , ordinary cooking does not eliminate it . recently , however , more and more evidences appeared , that exposure to more than one mycotoxin may be an important factor in etiology of ben and mpn . moreover , the synergism between ota and various other mycotoxins such a pa , citrinin or fb1 in field conditions may be responsible for an enhanced toxicity of ota [ 22 , 27 , 30 ] . due to the potent toxic and synergistic effects of ota and pa or citrinin [ 22 , 150 ] as well as between ota and fb1 [ 151 , 152 ] , simultaneous exposure to those mycotoxins might be an important factor for development of chronic renal diseases in animals and humans , especially after long - term exposure . the same mycotoxins were recently found in high contamination levels ( especially fb1 and pa ) in most of the feeds originated from farms with mycotoxic porcine or avian nephropathy in bulgaria ( unpublished personal data ) . because of that it is of great importance to investigate the combined effect between ota and other mycotoxins produced by the same species as it is occurred in field conditions .

various etiological factors contributing to the development of mycotoxic nephropathy in farm animals and humans are reviewed . the possible synergistic effect between ochratoxin a ( ota ) and other mycotoxins , as penicillic acid ( pa ) and fumonisin b1 ( fb1 ) , contributing to this nephropathy is also considered and discussed . the most convenient ways of prophylaxis and various preventive measures against ota contamination of feeds or foods are reviewed . a reference is made concerning the most successful methods of veterinary hygiene control in the slaughterhouses in order to prevent the entering of ota in commercial channels with a view to human health . the economic efficacy of these prophylactic procedures is also considered . an evaluation of human exposure to ota is made .

Introduction 1. Complex Etiology of MN 2. Methods of Prophylaxis and Various Preventive Measures in Regard to the Main Etiological Agent of MN - OTA 3. A Possible Hygiene Control in Regard to OTA 4. A Possible Involvement of Mycotoxins in Etiology of Balkan Endemic Nephropahy and Evaluation of Human Exposure to OTA

distal anterior cerebral artery aneurysms , also known as pericallosal artery ( pa ) aneurysms , are located on the anterior cerebral artery distal to the anterior communicating artery . these aneurysms represent approximately 6% of all intracranial aneurysms and 4% of those that rupture49 ) . furthermore , they were similarly represented in the international subarachnoid aneurysm trial ( isat ) study population , in which they accounted for 95 of the 2143 patients randomized ( 4.4%)14 ) . pa aneurysms present several problems for direct surgical clipping , that is , difficult exposure via an interhemispheric approach , especially in a swollen brain after subarachnoid hemorrhage ( sah ) , sacrifice of a bridging vein for adequate surgical exposure ( thus increasing the risk of postoperative morbidity ) , difficult controlling the parent artery , and the unfavorable orientation of the aneurysm fundus . as a consequence , surgical morbidity has been reported to be relatively high , with incidences ranging from 0 to 25%141221 ) . advances in endovascular techniques and devices have led to the rapid evolution of coil embolization of ruptured and unruptured intracranial aneurysms , and it has now become an efficient treatment technique that is comparable to surgical clipping . however , because of their distal location and the small diameter of the parent vessel , pa aneurysms pose a technical challenge for endovascular therapy and , as is the case for most bifurcation cerebral aneurysms , have a potential high recurrence rate18 ) . a search of the literature revealed relatively few studies have been performed on the endovascular treatment of ruptured pa aneurysms , and that the studies performed involved relatively small numbers of patient . accordingly , it is evident that the effectiveness and safety of the endovascular treatment of ruptured pa aneurysms have not been well - defined . here , we present 30 patients that were treated endovascularly for a ruptured pa aneurysm and describe their clinical and radiologic outcomes . we retrospectively analyzed the angiographic and clinical records of all patients that underwent endovascular treatment for a ruptured pa aneurysm at our institution from september 2003 to december 2013 . the treatment strategy for ruptured pa aneurysm at our institution was to attempt endovascular treatment as the first - line treatment . this strategy was applied to those accompanied by intraparenchymal hematoma ( ich ) , and surgical evacuation , if necessary , was performed in the immediately following coiling . however , microsurgery was used in the early years of this study because the technology for coiling of distally located aneurysms was in its infancy . therefore , surgical clipping was first attempt in 6 cases during the same period . there was no technical failure in both clipping and coiling groups . due to the retrospective nature , the present study is exempt in accord with the institutional review board standards of our institution . thirty ruptured pa aneurysms in 30 patients were treated endovascularly , and there were 10 males and 20 females of mean age 55.9 years ( range , 24 to 88 years ) . dissecting or fusiform aneurysms , aneurysms associated with a brain arteriovenous malformation , and traumatic or mycotic aneurysms were excluded . size of aneurysms and parent arteries were measured automatically and manually on two - dimen sional digital subtraction angiography working views . aneurysm size was defined as the maximum distance of the dome from the neck plane . aneurysm and neck sizes ranged from 2.5 to 10.4 mm ( mean 5.0 mm ) and from 0.9 to 6.1 mm ( mean 2.2 mm ) , respectively . 21 of the 30 saccular aneurysms were defined as wide - neck aneurysms ( a large neck size of 4 mm and/or a dome - to - neck ratio of 2 ) . of the 30 pa aneurysms , 23 ( 76.7% ) were located at the pericallosal - callosomarginal junction and 7 ( 23.3% ) at the pericallosal - frontopolar junction . fifteen pa aneurysms were on the left side , 13 on the right , and 2 at midline ( azygos artery ) . all patients were clinically assessed at admission using hunt and hess grades ; 12 patients ( 40.0% ) grade ii , 9 ( 30.0% ) grade iii , 6 ( 20.0% ) grade v , and 3 ( 10.0% ) grade iv . fifteen patients ( 50.0% ) had a concomitant ich that was managed conservatively in all but two cases that required surgical evacuation . in general , endovascular treatment was performed as soon as possible after sah , regardless of clinical condition . coiling of aneurysms was performed under local anesthesia , and intravenous bolus injection of standard heparin was not performed during the procedure . however , drip infusion of heparin was carried out employing catheter pressure infusion systems with continuous infusion of 1000 u of heparin per 1000 ml of saline . the aim of the coiling was to obtain a packing of the aneurysm as attenuated as possible . the remodeling techniques using a balloon or stent or multiple catheters were unavailable for the aneurysms treated during the early part of our series . however , we have recently favored these remodeling techniques to achieve higher postoperative occlusion rates . when an aneurysm could not be reached using standard procedures because of a distal location and instability of the microcatheter , the guiding catheter was placed more distal into the internal carotid artery . care was taken not to interfere with the flow of the carotid or parent artery . by using these means , all aneurysms were reached with a microcatheter . in the case of intraprocedural thromboembolic complication , various strategies for thrombolysis were applied , such as mechanical thrombolysis and intra - arterial or intravenous abciximab , or heparin infusion during or after the procedure . immediately after the procedure furthermore , all patients underwent a non - enhanced brain computed tomography ( ct ) scan to evaluate possible hemorrhagic complications . unless the thromboembolic complication occurred or a stent was used , further antiplatelet or anticoagulant medications were not administered . the indications for long - term antiplatelet therapy of acetylsalicylic acid and/or clopidogrel were ; the formation of a thrombus at the end of the coil or thromboembolic events , stent - assisted coiling , and coil protrusion into the parent artery . occlusion was classified as ; complete when no contrast filling of the aneurysmal sac and neck was observed , near - complete when contrast filling of the neck of the aneurysm was slow or partial when any degree of contrast filling was observed within the aneurysm sac . clinical results were assessed upon discharge from hospital or at last visit using the modified rankin scale ( mrs ) , as follows : 0 , no symptoms at all ; 1 , no significant disability ; 2 , slight disability ; 3 , moderate disability ; 4 , moderately severe disability ; 5 , severe disability ; and 6 , death . complications were defined as all adverse events related to the procedure , and these were studied retrospectively using medical and operative reports . early rebleeding was defined as expanding hemorrhage with worsening of the patient 's condition within 30 days after coiling , and diagnostic confirmation was made by ct when it showed an increased amount of hemorrhage compared with immediate postprocedural ct . univariate and multivariate logistic regression analyses were used to identify risk factors associated with periprocedural rebleeding or a poor clinical outcome . results are presented as odds ratios ( ors ) and 95% confidence intervals ( cis ) . variables showing a univariate association with a dependent risk factor ( p<0.10 ) were included in the multivariate logistic analysis . chicago , il , usa ) . statistical significance was accepted for p values of < 0.05 . we retrospectively analyzed the angiographic and clinical records of all patients that underwent endovascular treatment for a ruptured pa aneurysm at our institution from september 2003 to december 2013 . the treatment strategy for ruptured pa aneurysm at our institution was to attempt endovascular treatment as the first - line treatment . this strategy was applied to those accompanied by intraparenchymal hematoma ( ich ) , and surgical evacuation , if necessary , was performed in the immediately following coiling . however , microsurgery was used in the early years of this study because the technology for coiling of distally located aneurysms was in its infancy . therefore , surgical clipping was first attempt in 6 cases during the same period . there was no technical failure in both clipping and coiling groups . due to the retrospective nature , the present study is exempt in accord with the institutional review board standards of our institution . thirty ruptured pa aneurysms in 30 patients were treated endovascularly , and there were 10 males and 20 females of mean age 55.9 years ( range , 24 to 88 years ) . dissecting or fusiform aneurysms , aneurysms associated with a brain arteriovenous malformation , and traumatic or mycotic aneurysms were excluded . size of aneurysms and parent arteries were measured automatically and manually on two - dimen sional digital subtraction angiography working views . aneurysm size was defined as the maximum distance of the dome from the neck plane . aneurysm and neck sizes ranged from 2.5 to 10.4 mm ( mean 5.0 mm ) and from 0.9 to 6.1 mm ( mean 2.2 mm ) , respectively . 21 of the 30 saccular aneurysms were defined as wide - neck aneurysms ( a large neck size of 4 mm and/or a dome - to - neck ratio of 2 ) . of the 30 pa aneurysms , 23 ( 76.7% ) were located at the pericallosal - callosomarginal junction and 7 ( 23.3% ) at the pericallosal - frontopolar junction . fifteen pa aneurysms were on the left side , 13 on the right , and 2 at midline ( azygos artery ) . all patients were clinically assessed at admission using hunt and hess grades ; 12 patients ( 40.0% ) grade ii , 9 ( 30.0% ) grade iii , 6 ( 20.0% ) grade v , and 3 ( 10.0% ) grade iv . fifteen patients ( 50.0% ) had a concomitant ich that was managed conservatively in all but two cases that required surgical evacuation . in general , endovascular treatment was performed as soon as possible after sah , regardless of clinical condition . coiling of aneurysms was performed under local anesthesia , and intravenous bolus injection of standard heparin was not performed during the procedure . however , drip infusion of heparin was carried out employing catheter pressure infusion systems with continuous infusion of 1000 u of heparin per 1000 ml of saline . the aim of the coiling was to obtain a packing of the aneurysm as attenuated as possible . the remodeling techniques using a balloon or stent or multiple catheters were unavailable for the aneurysms treated during the early part of our series . however , we have recently favored these remodeling techniques to achieve higher postoperative occlusion rates . when an aneurysm could not be reached using standard procedures because of a distal location and instability of the microcatheter , the guiding catheter was placed more distal into the internal carotid artery . care was taken not to interfere with the flow of the carotid or parent artery . by using these means , all aneurysms were reached with a microcatheter . in the case of intraprocedural thromboembolic complication , various strategies for thrombolysis were applied , such as mechanical thrombolysis and intra - arterial or intravenous abciximab , or heparin infusion during or after the procedure . immediately after the procedure furthermore , all patients underwent a non - enhanced brain computed tomography ( ct ) scan to evaluate possible hemorrhagic complications . unless the thromboembolic complication occurred or a stent was used , further antiplatelet or anticoagulant medications were not administered . the indications for long - term antiplatelet therapy of acetylsalicylic acid and/or clopidogrel were ; the formation of a thrombus at the end of the coil or thromboembolic events , stent - assisted coiling , and coil protrusion into the parent artery . occlusion was classified as ; complete when no contrast filling of the aneurysmal sac and neck was observed , near - complete when contrast filling of the neck of the aneurysm was slow or partial when any degree of contrast filling was observed within the aneurysm sac . clinical results were assessed upon discharge from hospital or at last visit using the modified rankin scale ( mrs ) , as follows : 0 , no symptoms at all ; 1 , no significant disability ; 2 , slight disability ; 3 , moderate disability ; 4 , moderately severe disability ; 5 , severe disability ; and 6 , death . complications were defined as all adverse events related to the procedure , and these were studied retrospectively using medical and operative reports . early rebleeding was defined as expanding hemorrhage with worsening of the patient 's condition within 30 days after coiling , and diagnostic confirmation was made by ct when it showed an increased amount of hemorrhage compared with immediate postprocedural ct . univariate and multivariate logistic regression analyses were used to identify risk factors associated with periprocedural rebleeding or a poor clinical outcome . results are presented as odds ratios ( ors ) and 95% confidence intervals ( cis ) . variables showing a univariate association with a dependent risk factor ( p<0.10 ) were included in the multivariate logistic analysis . chicago , il , usa ) . statistical significance was accepted for p values of < 0.05 . the microcatheter was navigated into the lumen of the pa aneurysm and coils were delivered in all 30 patient . the treatment of 19 aneurysms was straightforward , but the other 11 required an adjunctive technique , that is , double catheter , balloon - assisted , or a stent - assisted technique . immediate post - procedural angiograms showed complete aneurysm occlusion in 21 ( 21/30 , 70.0% ) and near - complete occlusion in 9 ( 9/30 , 30.0% ) . procedure - related thromboembolic complications developed in two patients ( 2/30 , 6.7% ) . in one patient , occlusion of a callosomarginal artery occurred due to thrombus formation at the artery origin , which resulted in right leg monoparesis . the other thromboembolism occurred in the inferior m2 segment of the middle cerebral artery during the procedure , a relevant infarction was observed 2 days after coiling . diagnosis of intraprocedural rupture was made based on the angiographic visualization of contrast material extravasation in two cases . the coil was observed partially outside the aneurysm with a sudden rise in arterial blood pressure but without angiographic demonstration of extravasation of contrast medium in one case . the mean delay between coiling and early rebleeding was 31 hours ( range , 2 - 83 hours ) . in these three patients , intravenous bolus of 3000 iu of heparin was injected during the procedure only in one case needed a stent . besides drip infusion of heparin during the procedure , further antiplatelet or anticoagulant medications were not administered in the other two cases . of these six patients with periprocedural rebleeding , five had small ( 4 mm ) aneurysms , but one had large ( 9.9 mm ) and multi - lobulated aneurysm . furthermore , the five patients ( 5/6 , 83.3% ) concerned had an adjacent hematoma on initial ct scans , and three ( 3/6 , 50.0% ) showed contrast retention and delayed washout by the aneurysm by preprocedural digital subtraction angiography . in these six patients with periprocedural rebleeding , no new neurologic deficit developed in three patients ( 3/6 , 50.0% ) , but deterioration of consciousness , resulting in death , occurred in two patients ( 2/6 , 33.3% ) . it could be questioned as to whether the early rebleeding had worsened clinical status in one patient , because the patient had initial severe sah and died within 5 days after sah onset . thus , the periprocedural mortality in our cohort was 6.7% ( 2/30 ) and morbidity was 3.3% ( 1/30 ) . table 3 provides a summary of the risk factors found to be associated with periprocedural rebleeding . the associations of all variables with periprocedural rebleeding were examined by univariate logistic regression analysis , and contrast retention was found to be associated with an increased risk of periprocedural rebleeding ( or : 23.000 ; 95% ci : 1.773 to 298.446 ; p=0.016 ) . furthermore , multivariate logistic - regression analysis adjusted for adjacent hematoma confirmed the validity of this relationship ( or : 21.352 ; 95% ci : 1.310 to 347.917 ; p=0.032 ) . only one patient ( 1/30 , 3.3% ) suffered from symptomatic vasospasm and , fortunately , no negative long - term effects were observed . clinical results were assessed upon discharge from hospital or at last follow - up visit using the mrs . at the end of the observational period , 18 patients were independent with a mrs score of 0 - 2 ( 18/30 , 60.0% ) , and the other 12 ( 12/30 , 40% ) were dependent ( mrs score , 3 - 6 ) . seven of the 12 had a mrs score of 6 ( due to a poor preoperative status in five and procedure - related rebleeding in two ) , three had a mrs score of 5 ( due to poor preoperative status or old age ) , one had a mrs score of 4 ( due to old age ) , and one had a mrs score of 3 ( due to a large ich ) . the 23 surviving patients were followed for 6 - 68 months ( mean , 32.7 months ) . no neurologic deterioration or hemorrhagic complication occurred during follow - up period in any of these patients . univariate logistic - regression analysis showed that age ( or : 1.080 ; 95% ci : 1.003 to 1.163 ; p=0.043 ) , adjacent hematoma ( or : 13.000 ; 95% ci : 2.074 to 81.479 ; p=0.006 ) , and hunt and hess grade 3 - 5 ( or : 13.750 ; 95% ci : 1.452 to 130.239 ; p=0.022 ) were associated with a poor clinical outcome . however , multivariate logistic - regression analysis adjusted for age and hunt and hess grade 3 - 5 showed that only adjacent hematoma was associated with a poor clinical outcome ( or : 47.734 ; 95% ci : 1.596 to 1427.240 ; p=0.026 ) . seventeen of the 23 surviving patients underwent follow - up conventional angiography between 1 and 65 months postoperatively ( mean , 16.5 months ) . according to the follow - up images obtained , 9 of the 10 initially occluded aneurysms remained completely occluded and the remaining one showed partial recanalization due to coil compaction . repeat follow - up angiography at 20 months showed in this patient that the condition was stable , and the patient concerned declined additional therapy . five of the 7 aneurysms initially showing near complete occlusion remained stable at follow - up . recoiling was undertaken in the patient showing major recanalization at 13 months postoperatively , and complete occlusion was achieved . thus , the overall stable occlusion rate was 82.4% ( 14/17 ) and the recanalization rate was 17.6% ( 3/17 ) . parent arteries of the aneurysm treated by y - stent - assisted coiling remained patent with no evidence of intimal hyperplasia or in - stent stenosis . the microcatheter was navigated into the lumen of the pa aneurysm and coils were delivered in all 30 patient . the treatment of 19 aneurysms was straightforward , but the other 11 required an adjunctive technique , that is , double catheter , balloon - assisted , or a stent - assisted technique . immediate post - procedural angiograms showed complete aneurysm occlusion in 21 ( 21/30 , 70.0% ) and near - complete occlusion in 9 ( 9/30 , 30.0% ) . procedure - related thromboembolic complications developed in two patients ( 2/30 , 6.7% ) . in one patient , occlusion of a callosomarginal artery occurred due to thrombus formation at the artery origin , which resulted in right leg monoparesis . the other thromboembolism occurred in the inferior m2 segment of the middle cerebral artery during the procedure , a relevant infarction was observed 2 days after coiling . however , the patient concerned did not experience any neurological symptom . diagnosis of intraprocedural rupture was made based on the angiographic visualization of contrast material extravasation in two cases . the coil was observed partially outside the aneurysm with a sudden rise in arterial blood pressure but without angiographic demonstration of extravasation of contrast medium in one case . the mean delay between coiling and early rebleeding was 31 hours ( range , 2 - 83 hours ) . in these three patients , intravenous bolus of 3000 iu of heparin was injected during the procedure only in one case needed a stent . besides drip infusion of heparin during the procedure , further antiplatelet or anticoagulant medications were not administered in the other two cases . of these six patients with periprocedural rebleeding , five had small ( 4 mm ) aneurysms , but one had large ( 9.9 mm ) and multi - lobulated aneurysm . furthermore , the five patients ( 5/6 , 83.3% ) concerned had an adjacent hematoma on initial ct scans , and three ( 3/6 , 50.0% ) showed contrast retention and delayed washout by the aneurysm by preprocedural digital subtraction angiography . in these six patients with periprocedural rebleeding , no new neurologic deficit developed in three patients ( 3/6 , 50.0% ) , but deterioration of consciousness , resulting in death , occurred in two patients ( 2/6 , 33.3% ) . it could be questioned as to whether the early rebleeding had worsened clinical status in one patient , because the patient had initial severe sah and died within 5 days after sah onset . thus , the periprocedural mortality in our cohort was 6.7% ( 2/30 ) and morbidity was 3.3% ( 1/30 ) . table 3 provides a summary of the risk factors found to be associated with periprocedural rebleeding . the associations of all variables with periprocedural rebleeding were examined by univariate logistic regression analysis , and contrast retention was found to be associated with an increased risk of periprocedural rebleeding ( or : 23.000 ; 95% ci : 1.773 to 298.446 ; p=0.016 ) . furthermore , multivariate logistic - regression analysis adjusted for adjacent hematoma confirmed the validity of this relationship ( or : 21.352 ; 95% ci : 1.310 to 347.917 ; p=0.032 ) . only one patient ( 1/30 , 3.3% ) suffered from symptomatic vasospasm and , fortunately , no negative long - term effects were observed . clinical results were assessed upon discharge from hospital or at last follow - up visit using the mrs . at the end of the observational period , 18 patients were independent with a mrs score of 0 - 2 ( 18/30 , 60.0% ) , and the other 12 ( 12/30 , 40% ) were dependent ( mrs score , 3 - 6 ) . seven of the 12 had a mrs score of 6 ( due to a poor preoperative status in five and procedure - related rebleeding in two ) , three had a mrs score of 5 ( due to poor preoperative status or old age ) , one had a mrs score of 4 ( due to old age ) , and one had a mrs score of 3 ( due to a large ich ) . the 23 surviving patients were followed for 6 - 68 months ( mean , 32.7 months ) . no neurologic deterioration or hemorrhagic complication occurred during follow - up period in any of these patients . univariate logistic - regression analysis showed that age ( or : 1.080 ; 95% ci : 1.003 to 1.163 ; p=0.043 ) , adjacent hematoma ( or : 13.000 ; 95% ci : 2.074 to 81.479 ; p=0.006 ) , and hunt and hess grade 3 - 5 ( or : 13.750 ; 95% ci : 1.452 to 130.239 ; p=0.022 ) were associated with a poor clinical outcome . however , multivariate logistic - regression analysis adjusted for age and hunt and hess grade 3 - 5 showed that only adjacent hematoma was associated with a poor clinical outcome ( or : 47.734 ; 95% ci : 1.596 to 1427.240 ; p=0.026 ) . seventeen of the 23 surviving patients underwent follow - up conventional angiography between 1 and 65 months postoperatively ( mean , 16.5 months ) . according to the follow - up images obtained , 9 of the 10 initially occluded aneurysms remained completely occluded and the remaining one showed partial recanalization due to coil compaction . repeat follow - up angiography at 20 months showed in this patient that the condition was stable , and the patient concerned declined additional therapy . five of the 7 aneurysms initially showing near complete occlusion remained stable at follow - up . recoiling was undertaken in the patient showing major recanalization at 13 months postoperatively , and complete occlusion was achieved . thus , the overall stable occlusion rate was 82.4% ( 14/17 ) and the recanalization rate was 17.6% ( 3/17 ) . parent arteries of the aneurysm treated by y - stent - assisted coiling remained patent with no evidence of intimal hyperplasia or in - stent stenosis . ruptured pa aneurysms may be technically difficult to clip because of the narrow exposure afforded by an interhemispheric approach , difficulty control of the parent artery , and sacrifice of a bridging vein to achieve adequate surgical exposure . surgical morbidity has been reported to be relatively high , with incidences ranging from 0 to 25%141221 ) . endovascular treatment of ruptured pa aneurysms has been reported to be technically difficult , primarily because of the long access route , which complicates microcatheter manipulation , and the small aneurysmal lumen . the introduction of new microcatheter and microguidewire with improved trackability , pushability , and torque and novel hydrophilic coatings have facilitated navigation into distant cerebral arteries , such as , the pa . furthermore , the availabilities of various soft coils may have contributed to the higher success rate of small aneurysm coiling without intraoperative rupture . we found that the pa could be reached and coiling of the aneurysm accomplished in all 30 patients . in fact , even a 2.5 mm sized aneurysm located at a distal junction was occluded successfully . in our series , an independent daily living activity level ( mrs score , 0 - 2 ) was achieved by 60% of our patients ( 18/30 ) . the poor clinical outcomes ( mrs score , 3 - 6 ) of the other 12 patients resulted from poor preoperative status , periprocedural rebleeding , old age , and large ich . waldenberger et al.25 ) reported that the most important negative contributor to the outcome of pa aneurysm coiling was the presence of ich , and this was followed by thromboembolic complications and poor preoperative status . ruptured pa aneurysms cause ich , in addition to sah , in more than one - half of cases , and are associated with poorer outcomes after rupture than aneurysms in other locations311 ) . in the present study , 15 of the 30 patients ( 50.0% ) had a concomitant ich that was visualized on initial ct scans , and 10 of the patients ( 10/15 , 66.7% ) had a poor clinical outcome ( mrs score , 3 - 6 ) . furthermore , multivariate logistic - regression analysis showed that adjacent hematoma was the only independent predictor of a poor clinical outcome ( or : 47.734 ; 95% ci : 1.596 to 1427.240 ; p=0.026 ) . other variables , such as , age and hunt and hess grade were of borderline significance , possibly due to the small number of patients enrolled ( table 4 ) . we have summarized the reported studies in which more than 10 patients with ruptured pericallosal artery aneurysms were treated with endovascular treatment in table 5 . in a recently reported meta - analysis of endovascular treatment of pa aneurysms including 16 studies , the authors concluded that endovascular treatment of pa aneurysms is associated with high angiographic occlusion rates , but the complication rates are higher compared with other aneurysms in the circle of willis . procedure - related ischemia and iatrogenic rupture occurred in 5% and 7% of patients , respectively . procedure - related permanent morbidity rate was 8%23 ) . in the present study , the overall procedure - related complication rate was 26.7% ( 8/30 cases ) . two patients experienced thromboembolic events and acute infarctions and developed corresponding neurologic deficits , though fortunately showed no neurological deficit at follow - up . intraprocedural rupture occurred from three aneurysms ( 10.0% ) , which was markedly greater than anticipated . a review of the literature revealed pa aneurysms have a higher procedure - related rupture rate than non - pa aneurysms . keston et al.9 ) reported an intraprocedural rupture rate of 18% ( 3/17 patients ) for pa aneurysms , which compared with an overall departmental five year ( 1999 - 2003 inclusive ) intraprocedural rupture rate of only 1.1% ( 7/640 patients ) for non - pa aneurysms . nguyen et al.15 ) reported a 12% ( 3/25 patients ) procedure - related perforation rate in their endovascular series of pa aneurysms . based on these reports and the fact that previous reports included unruptured cases , whereas we did not , we believe that our intraprocedural rupture rate is not excessive . it is well known pa aneurysms are smaller than non - pa aneurysms tend and that they tend to rupture at a smaller size101327 ) . in a study of a large number of aneurysm cases , the average diameter of ruptured aneurysms was reported to be 8.2 mm8 ) , whereas the average aneurysm diameter in our patients was 5.0 mm . we believe this difference could explain the higher intraprocedural rupture rate in the endovascular treatment of ruptured pa aneurysms . in addition , to aneurysm size , the risk of procedure - induced perforation may also be related to anatomical peculiarities . a more distal location leads to catheter positioning via a longer segment in a small - caliber vessel , which reduces the degree of catheter deflection when a coil encounters resistance15 ) . prior studies on early rebleeding after coiling have reported rupture rates of 1.4% to 2.6%6722 ) . incomplete embolization , adjacent hematoma , small aneurysms , and post - procedural anticoagulation have been reported to be associated with early rebleeding after coiling . the cerebral aneurysm rerupture after treatment study reported that the risk of posttreatment rebleeding was closely associated with the degree of aneurysm occlusion7 ) . however , we experienced three cases with early rebleeding after coiling ( 10.0% ) , despite all three aneurysms being complete embolized during the procedure . the presence of adjacent hematoma is a strong , independent risk factor of early rebleeding and a thrombosed pseudoaneurysm and hematoma could cause early reopening and rehemorrhage22 ) . in our series , all three cases with early rebleeding accompanied by adjacent hematoma . at coiling , a pseudoaneurysm may or may not be visible by angiography . if a pseudoaneurysm is apparent by angiography , coiling of the true aneurysm and the pseudoaneurysm can be considered , but the fragile wall of the pseudoaneurysm is prone to perforation , and this has been prompted to advise against catheterization into false lumen16 ) . on the other hand , cho et al.2 ) , who reported on early recurrent hemorrhage after coil embolization in ruptured intracranial aneurysms , hypothesized another possibility that rebleeding with ich may be caused by delayed hemorrhage or propagation of the initial ich due to vulnerability of the parenchyma adjacent to the ich , rather than by rebleeding of the coiled aneurysm . early rebleeding after coiling of a ruptured aneurysm and intra - procedural rupture are major concerns , because associated mortality rates are high . when a ruptured pa aneurysm accompanied by adjacent hematoma , decisions regarding endovascular treatment should be taken only after carefully considering the possibility of periprocedural rebleeding . therefore , it may be better to consider the surgical clipping of a ruptured pa aneurysm with adjacent hematoma as a first option to prevent periprocedural rebleeding . with regard to treatment durability , recurrence requiring recoiling due to the risk of re - rupture , developed in one of our 17 cases , who developed major recanalization . in the present study , the overall recanalization rate was 17.6% ( 3/17 ) but the major recanalization rate was only 5.9% ( 1/17 ) . recurrence after coiling has been reported to be associated primarily with aneurysm size7162226 ) . in one study , follow - up angiographic studies after complete occlusion showed recurrence rates of 14% for aneurysms of < 5 mm , 27% for aneurysms of 5 - 10 mm , and 57% for aneurysms of 10 mm23 ) . therefore , the lower rate of recurrence in our series could have been due to the lower proportion of cases with aneurysms 5 mm ( 9/30 , 30.0% ) . however , in a recent series of 16 cases of pa aneurysm coiling , there was a comparatively high recurrence rate ( one major and five minor recurrences , 37.5% ) , attributed to the loose coil packing rate19 ) . this study is limited by its retrospective design , patient - selection bias , small cohort , relatively short angiographic follow - up , by the fact that it was conducted at a single institution . nonetheless , our findings suggest that the endovascular treatment of ruptured pa aneurysms appears to be a valid treatment strategy given suitable levels of institutional and operator expertise . our experiences show that the endovascular treatment of ruptured pa aneurysms is both feasible and effective . however , they show periprocedural rebleeding occur far more frequently ( 20.0% ) than is generally considered and that these rebleeding are associated with preprocedural contrast retention . accordingly , our findings caution that decisions regarding endovascular treatment in cases showing preprocedural contrast retention should approached by carefully considering the possibility of periprocedural rebleeding . furthermore , an existing adjacent hematoma was found to have a severe negative influence on clinical outcome .

objectiveaneurysms arising from the pericallosal artery ( pa ) are uncommon and challenging to treat . the aim of this study was to report our experiences of the endovascular treatment of ruptured pa aneurysms.methodsfrom september 2003 to december 2013 , 30 ruptured pa aneurysms in 30 patients were treated at our institution via an endovascular approach . procedural data , clinical and angiographic results were retrospectively reviewed.resultsregarding immediate angiographic control , complete occlusion was achieved in 21 ( 70.0% ) patients and near - complete occlusion in 9 ( 30.0% ) . eight procedure - related complications occurred , including intraprocedural rupture and early rebleeding in three each , and thromboembolic event in two . at last follow - up , 18 patients were independent with a modified rankin scale ( mrs ) score of 0 - 2 , and the other 12 were either dependent or had expired ( mrs score , 3 - 6 ) . adjacent hematoma was found to be associated with an increased risk of poor clinical outcome . seventeen of 23 surviving patients underwent follow - up conventional angiography ( mean , 16.5 months ) . results showed stable occlusion in 14 ( 82.4% ) , minor recanalization in two ( 11.8% ) , and major recanalization , which required recoiling , in one ( 5.9%).conclusionour experiences demonstrate that endovascular treatment for a ruptured pa aneurysms is both feasible and effective . however , periprocedural rebleedings were found to occur far more often ( 20.0% ) than is generally suspected and to be associated with preoperative contrast retention . analysis showed existing adjacent hematoma is predictive of a poor clinical outcome .

INTRODUCTION MATERIALS AND METHODS Patient population and aneurysm morphologies Endovascular procedure Clinical and angiographic follow-up Statistical analysis RESULTS Immediate angiographic results and complications Clinical status at discharge or during follow-up Follow-up angiographic results DISCUSSION CONCLUSION

cardiac implantable electronic device ( cied ) implantation continues to grow with permanent pacemaker implantation in the usa increasing by 56% between 1993 and 2009 . in europe , there has been a 75 and 115% increase in implantable cardiac defibrillator ( icd ) and cardiac resynchronization therapy pacemaker / defibrillator ( crt - p / crt - d ) implants between 2004 and 2008 . as a result of increasing indications for complex pacing , there is an expanding need for patients with pre - existing devices to undergo system revision due to lead failure or upgrade to allow icd and/or crt implant . in a recent european survey , 28% of crt implants were performed in patients with pre - existing devices , and recent lead advisories have necessitated an increase in lead extraction cases . the combination of such factors means that the need for system revision and upgrade is likely to increase in the future . a major obstacle to device revision and/or upgrade is the presence of asymptomatic ipsilateral central venous obstruction . older reports suggested obstruction occurred in up to 50% of cases with symptoms affecting only 13% , but more contemporary reports have demonstrated a lower incidence closer to 30% . this perhaps reflects improvements in lead design over time , but it remains clear that asymptomatic venous obstruction is not infrequently encountered . various strategies to overcome venous occlusion exist including contralateral lead or device implantation , venoplasty , and surgical epicardial lead implantation . the addition of extra pacemaker and/or icd leads is not without its drawbacks . in a recent prospective us registry of pacemaker / icd generator replacements , the need for an additional lead increased the rate of major complications from 4.0 to 15.3% . lead extraction may be an alternative option to overcome this problem , but it is not without risk and this should be weighed against the benefits of removing any leads and also the likelihood of symptom recurrence in cases where there are symptoms of venous occlusion . the practice of laser lead extraction to re - canalize venous obstruction has previously been described in a limited number of patients and the suitability of this technique on a larger scale has not been reported . we describe our experience using laser lead extraction to overcome venous occlusion to enable ipsilateral device revision and/or upgrade . guy 's and st thomas ' hospital is a quaternary referral centre for cied extraction . all patients undergoing device extractions are prospectively entered into a computer database recording patient demographics , comorbidities , device and lead type , reason for extraction , procedural success , and complications . complications are classified according to those recommended in the heart rhythm society consensus report on transvenous lead extraction . deaths were adjudicated by senior cardiologists within our department , none of whom had any input in the current study . patients from this database were included in the present study if the indication for lead extraction was to upgrade or revise an existing device in the presence of ipsilateral venous obstruction . in all cases , venous obstruction was identified on the basis of venography performed ( either in the radiology department at least 1 day prior to the procedure or in the cardiac catheter laboratory on the day of the procedure ) prior to each procedure to ascertain the patency of the venous system ( figure 1 ) . all patients provided written informed consent and all procedures were performed in our cardiac catheter laboratory under general anaesthesia . in patients with ipsilateral venous occlusion , or stenoses severe enough such that a hydrophilic guide wire would not cross the obstruction , laser lead extraction with retention of the outer sheath in the vascular tree was performed . in cases of device upgrade , any non - functional leads were removed ; if there was no non - functional lead ( for example , upgrade from dual - chamber pacing to crt - p ) , then the atrial lead was extracted with an attempt to preserve the existing right ventricular ( rv ) pacing lead . any redundant leads were also extracted . in cases of device upgrade , where there was also a failed lead in need of extraction , the failed lead was extracted and two hydrophilic wires passed through the outer sheath of the laser . after opening the existing generator pocket and disconnecting the leads from the generator , the suture sleeve of the lead being extracted was released . the proximal end of the lead was cut and a locking stylet ( liberator beacon , cook medical inc . or lld ez lead locking device ; spectranetics ) was advanced as distally as possible and locked in place . a silk suture was then tied to the lead to aid traction and this was fed through the laser sheath ( sls ii excimer laser sheaths ; spectranetics ) with the outer sheath also in position . both sheaths were advanced over the lead and the inner sheath advanced until resistance was met at which point laser energy was applied in short pulses to free the lead body from the surrounding vessel wall or cardiac musculature ( cvx-300 excimer laser system ; spectranetics ) . lasing was performed as necessary , up to the final 1 cm proximal to the distal electrode , and the lead was freed with counter - traction using the outer sheath . the lead and inner sheath were removed in their entirety leaving the outer sheath in place thereby maintaining vascular access . a venogram was performed to ensure that the sheath remained in the vascular / cardiac space and a hydrophilic wire ( terumo ) passed through the outer sheath when intravascular position was confirmed . a long haemostatic sheath(s ) was then placed to allow lead implantation in a standard fashion ( see figure 2 ) . ( a ) suggestive of collateral formation ( white arrow ) , which is confirmed with more medial panning during the venogram ( white arrows ; b ) . figure 2sequential images of the steps taken to successfully extract an atrial pace / sense ( patient in atrial fibrillation and in need of upgrade to a biventricular pacemaker ) , with maintenance of venous access across the level of occlusion . ( a ) the inner sheath is advanced with the outer sheath trailing . as demonstrated in this case , the laser is often required to overcome fibrosis at the clavicular level before the outer sheath can be advanced ( b ) . ( c ) further lasing upto and beyond the point of occlusion with passage of the outer sheath beyond the occlusion aided by rotational torque ( d ) . ( e ) the atrial lead is successfully extracted in its entirety using a combination of forward pressure on the outer sheath and manual traction on a locking stylet . ( f ) the inner sheath and lead are removed leaving the outer sheath in the vascular space just beyond the level of occlusion . ( g ) a hydrophilic wire is then passed through the outer sheath allowing passage of an introducer sheath and subsequent lv lead placement ( h ) . ( a ) suggestive of collateral formation ( white arrow ) , which is confirmed with more medial panning during the venogram ( white arrows ; b ) . sequential images of the steps taken to successfully extract an atrial pace / sense ( patient in atrial fibrillation and in need of upgrade to a biventricular pacemaker ) , with maintenance of venous access across the level of occlusion . ( a ) the inner sheath is advanced with the outer sheath trailing . as demonstrated in this case , the laser is often required to overcome fibrosis at the clavicular level before the outer sheath can be advanced ( b ) . ( c ) further lasing upto and beyond the point of occlusion with passage of the outer sheath beyond the occlusion aided by rotational torque ( d ) . ( e ) the atrial lead is successfully extracted in its entirety using a combination of forward pressure on the outer sheath and manual traction on a locking stylet . ( f ) the inner sheath and lead are removed leaving the outer sheath in the vascular space just beyond the level of occlusion . ( g ) a hydrophilic wire is then passed through the outer sheath allowing passage of an introducer sheath and subsequent lv lead placement ( h ) . between july 2004 and april 2012 , 242 upgrade / revision procedures were performed and of these 71 ( 29% ) were performed in patients with occluded or severely stenosed venous anatomy . complete ipsilateral occlusion was present in 52 of 71 patients ( 73% ) in this series . the remainder had severe stensoses that did not allow passage of hydrophilic guide wires and/or introducer sheaths and this was taken as indicating functional obstruction . the vast majority of obstructions were identified in the subclavian vein ( 67 of 71 ) with the remainder being at the junction of the subclavian vein with the superior vena cava ( svc ) ( see figure 1 ) . twenty - nine ( 41% ) patients had a history of ischaemic heart disease and 19 ( 27% ) had prior cardiac surgery ( coronary artery bypass surgery or valve surgery ) . the mean left ventricular ( lv ) ejection fraction was 38% ( derived from 2d echocardiography using simpson 's biplane method ) . three patients ( 4% ) had symptoms of venous occlusion ( arm swelling and pain on the side of device implant ) . table 1patient characteristicscharacteristicage ( years)62 15gender , n ( % ) male 55 ( 77)female 16 ( 23)ejection fraction ( % ) 38 15comorbiditiesihd , n ( % ) 29 ( 41)cardiac surgery , n ( % ) 19 ( 27)diabetes , n ( % ) 5 ( 7)hypertension , n ( % ) 16 ( 23)pvd , n ( % ) 3 ( 4)stroke , n ( % ) 5 ( 7)copd , n ( % ) 14 ( 20)ckd , n ( % ) 12 ( 17)ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . patient characteristics ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . most extractions were performed in patients with existing dual - chamber pacemakers , icds , or crt - ds ( 24 , 37 , and 27% , respectively ) . in total , 129 leads were extracted from 71 patients ( see table 2 ) . of these , 40 ( 31% ) were passive fixation right atrial ( ra ) or rv pacemaker leads , 33 ( 26% ) were active fixation ra or rv pacing leads , 41 ( 31% ) were single- or dual - coil defibrillator leads , and 15 ( 12% ) were coronary sinus lv pacing leads , which were extracted due to sub - optimal function ( phrenic nerve capture , or sub - optimal lead positioning ) . the commonest indications for extraction were lead malfunction ( 56% ) and need for device upgrade ( 40% ) . table 2device characteristicscharacteristicdevice , n ( % ) vvi2 ( 3 ) ddd17 ( 24 ) icd26 ( 37 ) crt - p7 ( 9 ) crt - d19 ( 27)indication for extraction , n ( % ) lead failure40 ( 56 ) upgrade28 ( 40 ) symptoms3 ( 4)nature of upgrade , n ( % ) ppm to icd8 ( 29 ) ppm to crt - p9 ( 32 ) icd to crt - d11 ( 39)number of leads extracted , n ( % ) total129 passive a / v40 ( 31 ) active a / v33 ( 26 ) icd41 ( 31 ) cs15 ( 12)mean duration of lead implant ( months)80 62vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . device characteristics vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . it was necessary to snare leads from the femoral venous approach in two cases following unsuccessful laser extraction when the lead fragmented despite the obstruction being crossed . new leads were successfully re - implanted via the laser sheath across the venous obstruction / stenosis in 67 ( 94% ) cases . in four cases , the laser sheath was unable to pass the obstruction due to intense fibrosis / calcification , and in three of these cases a subclavian vein puncture medial to the venous occlusion was performed to obtain venous access and successfully place the lead . in one case , a transvenous lead could not be placed and an epicardial lv pacing lead was surgically implanted during a later procedure . mean fluoroscopic screening time was 16 13 min and mean radiation dose was 837 1269 cgycm . there were two major complications ( 3% ) that were both cases of infection of a previously sterile site , and four ( 6% ) minor complications ( ipsilateral pneumothorax , phrenic nerve palsy , acute renal failure , and pocket haematoma ) . table 3procedural characteristics and outcomecharacteristicobstruction crossed , n ( % ) 67 ( 94)lead successfully extracted via laser sheath , n ( % ) 69 ( 97)transvenous lead successfully sited70 ( 99)procedural time ( min)116 32fluoroscopy time ( min)16 13radiation dose ( cgycm)837 1269complications major , n ( % ) 2 ( 3 ) minor , n ( % ) 4 ( 5 ) 30-day mortality , n ( % ) 2 ( 3 ) procedural characteristics and outcome medium- to long - term device follow - up was available in 65 of 71 ( 92% ) cases with a mean follow - up of 26 19 months . one patient developed non - procedure - related sepsis ( urinary sepsis following urethral instrumentation for urinary retention ) while recuperating from their procedure . the second death occurred in a patient with severe heart failure ( new york heart association iv ) with a pre - existing crt - d that had a non - functioning lv pacing lead . the patient had refractory hypotension and hyponatraemia limiting the use of medical therapy and a decision was made to attempt revision of the crt - d device as a procedure of last resort . the patient died in hospital from end - stage heart failure despite successful lead extraction . both deaths were adjudicated by senior physicians within our department , who were blinded to the interventions in the current study . it was necessary to further revise cied implants following the index procedure in four ( 6% ) cases . two cases required re - intervention for defibrillator leads with diminished sensing , one patient developed phrenic nerve capture from their lv pacing lead , and one patient deteriorated with a sub - optimal lv lead position that required revision . twenty - nine ( 41% ) patients had a history of ischaemic heart disease and 19 ( 27% ) had prior cardiac surgery ( coronary artery bypass surgery or valve surgery ) . the mean left ventricular ( lv ) ejection fraction was 38% ( derived from 2d echocardiography using simpson 's biplane method ) . three patients ( 4% ) had symptoms of venous occlusion ( arm swelling and pain on the side of device implant ) . table 1patient characteristicscharacteristicage ( years)62 15gender , n ( % ) male 55 ( 77)female 16 ( 23)ejection fraction ( % ) 38 15comorbiditiesihd , n ( % ) 29 ( 41)cardiac surgery , n ( % ) 19 ( 27)diabetes , n ( % ) 5 ( 7)hypertension , n ( % ) 16 ( 23)pvd , n ( % ) 3 ( 4)stroke , n ( % ) 5 ( 7)copd , n ( % ) 14 ( 20)ckd , n ( % ) 12 ( 17)ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . patient characteristics ihd , ischaemic heart disease ; pvd , peripheral vascular disease ; copd , chronic obstructive pulmonary disease ; ckd , chronic kidney disease . most extractions were performed in patients with existing dual - chamber pacemakers , icds , or crt - ds ( 24 , 37 , and 27% , respectively ) . in total , 129 leads were extracted from 71 patients ( see table 2 ) . of these , 40 ( 31% ) were passive fixation right atrial ( ra ) or rv pacemaker leads , 33 ( 26% ) were active fixation ra or rv pacing leads , 41 ( 31% ) were single- or dual - coil defibrillator leads , and 15 ( 12% ) were coronary sinus lv pacing leads , which were extracted due to sub - optimal function ( phrenic nerve capture , or sub - optimal lead positioning ) . the commonest indications for extraction were lead malfunction ( 56% ) and need for device upgrade ( 40% ) . table 2device characteristicscharacteristicdevice , n ( % ) vvi2 ( 3 ) ddd17 ( 24 ) icd26 ( 37 ) crt - p7 ( 9 ) crt - d19 ( 27)indication for extraction , n ( % ) lead failure40 ( 56 ) upgrade28 ( 40 ) symptoms3 ( 4)nature of upgrade , n ( % ) ppm to icd8 ( 29 ) ppm to crt - p9 ( 32 ) icd to crt - d11 ( 39)number of leads extracted , n ( % ) total129 passive a / v40 ( 31 ) active a / v33 ( 26 ) icd41 ( 31 ) cs15 ( 12)mean duration of lead implant ( months)80 62vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . device characteristics vvi , single - chamber pacemaker ( lead in rv ) ; ddd , dual - chamber pacemaker ; icd , implantable cardioverter defibrillator ; crt - p , cardiac resynchronization therapy - pacemaker ; crt - d , cardiac resynchronization therapy - defibrillator ; ppm , permanent pacemaker ; passive a / v , passive fixation atrial / ventricular leads ; active a / v , active fixation atrial / ventricular leads ; and cs , coronary sinus . all 129 leads were successfully extracted in their entirety . it was necessary to snare leads from the femoral venous approach in two cases following unsuccessful laser extraction when the lead fragmented despite the obstruction being crossed . new leads were successfully re - implanted via the laser sheath across the venous obstruction / stenosis in 67 ( 94% ) cases . in four cases , the laser sheath was unable to pass the obstruction due to intense fibrosis / calcification , and in three of these cases a subclavian vein puncture medial to the venous occlusion was performed to obtain venous access and successfully place the lead . in one case , a transvenous lead could not be placed and an epicardial lv pacing lead was surgically implanted during a later procedure . mean fluoroscopic screening time was 16 13 min and mean radiation dose was 837 1269 cgycm . there were two major complications ( 3% ) that were both cases of infection of a previously sterile site , and four ( 6% ) minor complications ( ipsilateral pneumothorax , phrenic nerve palsy , acute renal failure , and pocket haematoma ) . table 3procedural characteristics and outcomecharacteristicobstruction crossed , n ( % ) 67 ( 94)lead successfully extracted via laser sheath , n ( % ) 69 ( 97)transvenous lead successfully sited70 ( 99)procedural time ( min)116 32fluoroscopy time ( min)16 13radiation dose ( cgycm)837 1269complications major , n ( % ) 2 ( 3 ) minor , n ( % ) 4 ( 5 ) 30-day mortality , n ( % ) 2 ( 3 ) procedural characteristics and outcome medium- to long - term device follow - up was available in 65 of 71 ( 92% ) cases with a mean follow - up of 26 19 months . one patient developed non - procedure - related sepsis ( urinary sepsis following urethral instrumentation for urinary retention ) while recuperating from their procedure . the second death occurred in a patient with severe heart failure ( new york heart association iv ) with a pre - existing crt - d that had a non - functioning lv pacing lead . the patient had refractory hypotension and hyponatraemia limiting the use of medical therapy and a decision was made to attempt revision of the crt - d device as a procedure of last resort . the patient died in hospital from end - stage heart failure despite successful lead extraction . both deaths were adjudicated by senior physicians within our department , who were blinded to the interventions in the current study . it was necessary to further revise cied implants following the index procedure in four ( 6% ) cases . two cases required re - intervention for defibrillator leads with diminished sensing , one patient developed phrenic nerve capture from their lv pacing lead , and one patient deteriorated with a sub - optimal lv lead position that required revision . we have described our experience using laser lead extraction to overcome ipsilateral venous obstruction in patients undergoing device revision and/or upgrade . to date , this is the largest series of cases , where laser lead extraction has been used to overcome venous obstruction thereby allowing ipsilateral lead revision or device upgrade . we have shown that the technique is feasible in the vast majority of cases . in 100% of cases , the targeted lead(s ) were completely extracted , and in 94% re - canalization of the obstructed vein allowed successful lead implantation via the laser sheath . in the remaining patients transvenous lead implantation was successful on the ipsilateral side with a medial puncture and in only one case did the patient need a further procedure ( surgical lv lead implant ) to achieve implant success . our complication rate is low even in a cohort of patients with reduced lv systolic function and significant comorbidities . the heart rhythm society ( hrs ) consensus statement on lead extraction in relation to venous obstruction states that lead removal is a class i recommendation in patients with ipsilateral venous occlusion preventing additional lead placement when there is a contraindication for using the contralateral side ( e.g. contralateral atrioventricular fistula , shunt or vascular access port , and mastectomy ) . lead removal in patients with ipsilateral venous occlusion and no contraindication to using the contralateral side is a class iia indication . obstruction and thrombosis of the access vein after implantation of permanent pacing and defibrillator leads are well described . the reported incidence of asymptomatic cases is up to 50% in older series and up to 30% in more contemporary series . symptomatic occlusion occurs in 13% , thus highlighting the importance of developing strategies for overcoming such obstacles at the time of device upgrade or lead revision . in our study , the finding of venous obstruction precluding device revision or upgrade was 29% in keeping with recent reports . in such cases , it may be possible to obtain venous access with a de novo puncture , but it is often not possible to advance introducer sheaths across a very tight stenosis and there is the added risk of increased lead lead interaction . these issues are avoided by using the technique described in this report and we have demonstrated that the procedure can be performed safely . laser technology is increasingly being used to facilitate lead extraction . in the plexes trial , use of the laser sheath resulted in complete removal of 94% of leads compared with only 64% where non - laser tools were used ( predominantly telescoping sheaths ) . more recently , bordachar et al . showed that laser extraction results in shorter procedures with lower radiation exposure to operators when compared with femoral snare techniques . the lexicon study was an observational retrospective study of 1449 consecutive laser lead extractions in north america and confirmed high success rates and low complication rates , particularly in high volume centres . the use of the laser sheath to overcome venous obstruction was first described by bracke et al . in three patients , where the laser was only used up to the point of obstruction and the lead left in situ . the largest previous series by gula et al . included 18 patients , where laser lead extraction was performed to facilitate system upgrade in the presence of central venous occlusion . in both earlier reports , our current study provides an expanded assessment of the technique in a larger number of patients . procedures were performed by three experienced operators in a single centre , each with extensive experience in laser lead extraction . our patients tended to have a longer duration of lead implant ( 80 62 vs. 70.8 43.5 months in the series of gula et al . ) . in addition , our cohort of patients is typical of those in whom these procedures are performed , namely depressed ejection fraction and attendant comorbidities . no outer sheath was used . in a minority of cases , tissue build - up at the tip of the laser sheath prevented the withdrawal of the lead tip through the laser sheath . this necessitated extraction of a functional atrial lead to ensure maintenance of venous patency . also , our study included patients requiring non - functional lead extraction rather than just device upgrade . in such cases , extraction may be preferable to adding extra leads . in our study , all leads were removed in their entirety , but it was necessary to snare two leads from the femoral venous route . therefore , 98% of leads were removed using the laser sheath alone and in total , venous access was maintained with the outer sheath in 94% of cases . no leads were unintentionally damaged and it was not necessary to extract any extra leads . this is important as it may have implications in reducing the risk of the procedure . there are inherent risks in extracting leads , particularly from the thin - walled ra , and so if extraneous extraction can be avoided this is preferable . in the current study , leads were successfully extracted in all cases after the laser sheath had successfully overcome fibrosis at the subclavicular level . there were two major procedural complications , both of which were cases of infection of a previously sterile site and this possibly reflects the added risk in performing upgrade or revision procedures . of the four minor complications , none were specifically related to the use of the laser sheath to overcome the obstruction . the alternative options available if ipsilateral venous occlusion is present include : insertion of new leads via the contralateral subclavian vein with either tunnelling of the leads across the sternum or abandonment of the pre - existing leads;venoplasty of the occluded vessels;alternative venous access;surgical epicardial lead implant.each of the techniques listed have their own specific drawbacks . the practice of adding leads is not without risk ; in the replace registry , there was a 15.3% major complication rate and a 1.1% 6-month mortality rate in patients undergoing generator change with a planned lead addition or revision . the hrs lead extraction consensus statement states that lead removal is reasonable in patients if cied implantation would require more than four leads on one side or more than five leads through the svc ( class iia recommendation ) . the presence of redundant leads is also associated with increased risks of infection and erosion . tunnelling across the sternum to the contralateral position of the existing generator potentially avoids the risk of svc syndrome , but there is a heightened risk of lead erosion , patient discomfort , and bilateral occlusion . venoplasty has been previously described , where a wire is passed through the occlusion , and balloon venoplasty performed to open a patent channel . the results of this procedure are encouraging with lead implantation possible in 96% of patients in a recent report . the technique may not be ideal in cases , where there is an indication for lead revision ( such as in defibrillator lead failure ) , where extraction of the malfunctioning lead may be the preferred option . lead malfunction was the primary indication for revision in 56% of our cases and the use of the laser sheath to overcome obstruction and remove the malfunctioning lead represents an attractive option in such cases . use of other venous access sites , such as the internal jugular vein , with subsequent tunnelling of leads has been described ; however lead erosion remains an issue . surgical implantation of epicardial leads negates the need for a transvenous approach but requires a thoracotomy and lead failure is not uncommon . insertion of new leads via the contralateral subclavian vein with either tunnelling of the leads across the sternum or abandonment of the pre - existing leads ; venoplasty of the occluded vessels ; alternative venous access ; surgical epicardial lead implant . this study is a single - centre experience with experienced operators and therefore our results may not be widely applicable in less experienced centres . lead extraction is not without risk and should only be performed in centres with experienced staff and necessary equipment / tools and with access to onsite emergency surgery . a prospective study of laser extraction to treat venous obstruction would be necessary to extrapolate our results . our results suggest that laser lead extraction to overcome ipsilateral venous obstruction is effective and safe , and therefore represents an attractive approach to deal with device upgrade / lead revision in patients with obstructed or severely stenosed venous anatomy . m.o.n . received research funding from st jude medical and biosense webster ; j.g . received research funding from biosense webster and st jude medical ; c.a.r . is a consultant to spectranetics and received research funding from st jude medical , boston scientific , and medtronic ; m.s .

aimsthe number of procedures involving upgrade or revision of cardiac implantable electronic devices ( cieds ) is increasing and the risks of adding additional leads are significant . central venous occlusion in patients with pre - existing devices is often asymptomatic and optimal management of such patients in need of device revision / upgrade is not clear . we sought to assess our use of laser lead extraction in overcoming venous obstruction.methods and resultspatients in need of device upgrade / revision underwent pre - procedure venography to assess venous patency . in patients with venous occlusion or stenosis severe enough to preclude passage of a hydrophilic guide wire , laser lead extraction with retention of the outer sheath in the vasculature was performed with the aim of maintaining a patent channel through which new leads could be implanted . data were recorded on a dedicated database and patient outcomes were assessed . between july 2004 and april 2012 , laser lead extractions were performed in 71 patients scheduled for device upgrade / revision who had occluded or functionally obstructed venous anatomy . new leads were successfully implanted across the obstruction in 67 ( 94% ) cases . there were two major complications ( infection ) and four minor complications with no peri - procedural mortality . device follow - up was satisfactory in 65 ( 92% ) cases with mean follow - up up to 26 19 months.conclusionlaser lead extraction is a safe and effective option when managing patients with central venous obstruction in need of cied revision or upgrade .

Introduction Methods Results Patient characteristics Device and lead characteristics Procedural characteristics Device follow-up Discussion Study limitations Conclusion Funding

the incidence of age - related diseases is rising , seriously affecting the health of millions of people around the world . according to the united nations ( un ) and the world health organization ( who ) musculoskeletal , rheumatic , and arthritic conditions are leading causes of morbidity and disability throughout the world , giving rise to enormous healthcare expenditures and loss of work ( woolf and pfleger , 2003 ; source : http://www.arthritis.org/ ) , . many types of rheumatic diseases and arthritic conditions are essentially age - related inflammatory disorders where the inflammation facilitates disease progression . the term arthritis characterizes a group of conditions involving inflammatory damage to synovial joints ( di paola and cuzzocrea , 2008 ) . it involves pain , redness , heat , swelling , and other harmful effects of inflammation within the joint . however , the most common and important form of arthritis is osteoarthritis ( oa ) , also known as osteoarthrosis or degenerative joint disease ( djd ) . oa is the most prevalent of the chronic diseases affecting the elderly ( aigner et al . , 2004 ) . the majority of the population over 65 years of age demonstrate radiographic evidence of oa in at least one joint . although oa is rare in people under 40 , it becomes much more common with age . a 2005 study in the usa estimated that oa is one of the top five causes of disability amongst non - hospitalized adults [ source : center for disease control ( cdc ) , usa ] . in 2006 it was estimated that around 35 million to 40 million europeans suffer from oa and nearly 25% of people aged 60 and above suffer from oa induced disability . it is also anticipated that by the year 2030 , 20% of adults will have developed oa in western europe and north america . therefore , oa is expected to place a heavy economic burden on healthcare systems and community services throughout the world . the risk factors for oa are well known and include age , overweight / obesity , underlying metabolic or endocrine disease , genetics , and joint trauma ( lotz and kraus , 2010 ) . with increasing life expectancy , growth in the elderly population and an alarming escalation of chronic , inflammatory , and age - related conditions ( such as oa ) , it is a mechanically unique and resilient connective tissue responsible for load - bearing and low - friction movement in the synovial joints of all vertebrates ( buckwalter et al . , cartilage is avascular and as a consequence it has a very limited capacity for intrinsic repair ( brittberg , 1999 ; tew et al . , 2001 ) . it highly prone to structural degradation making it particularly difficult to restore once it is damaged or lost . the ecm consists of three classes of molecules : collagens , aggregating proteoglycans , and non - collagenous proteins . type ii , ix , and xi collagens form a fibrillar framework of macromolecules that give the tissue form , tensile stiffness , and mechanical strength ( buckwalter and mankin , 1998b ; eyre , 2004 ) . large aggregating proteoglycans ( predominantly aggrecan ) allow cartilage to swell and resist compressive forces ( hardingham and fosang , 1992 ; kuettner , 1992 ) . small proteoglycans including decorin , biglycan , and fibromodulin , bind to other matrix macromolecules and help to stabilize the ecm . other collagenous and non - collagenous macromolecules present within the ecm perform a variety of structural and informational roles , facilitate cell cell and cell - matrix interactions , and bind growth factors ( hardingham and fosang , 1992 ; feng et al . , 2006 ) . the chondrocyte is the only cell type present in articular cartilage ( archer and francis - west , 2003 ) . during embryonic development chondrocytes synthesize a cartilaginous template for endochondral ossification and skeletal development and in postnatal life they maintain the ecm by regulating the turnover of matrix components in response to biomechanical , biochemical , and endocrine signals ( goldring and marcu , 2009 ) . chondrocytes actively synthesize new ecm components as well as the proteolytic enzymes such as matrix metalloproteinases ( mmps ) , a disintegrin , and metalloproteinase ( adams ) and a disintegrin and metalloproteinase with thrombospondin motifs ( adamtss ) are responsible for tissue remodeling during development . these enzymes are also involved in the catabolic breakdown of cartilage in oa ( aigner et al . , osteoarthritis is a degenerative disease that involves joint inflammation , bone remodeling , and catabolic destruction of the articular cartilage component ( goldring and goldring , 2007 ; samuels et al . , 2008 ) . in oa there is an imbalance between the synthesis and degradation of ecm macromolecules ( felson , 2004 ) . this can be due to increased enzymatic activity of mmps ( okada et al . , 1992 ) , and pro - inflammatory mediators such as cytokines ( goldring and goldring , 2004 ) , prostaglandins , and nitric oxide ( goldring and berenbaum , 2004 ) , coupled with the reduced anabolic capacity of chondrocytes ( aigner et al . , 1997 ) and the tissue s inherently poor reparative capacity due to its avascular nature ( archer and francis - west , 2003 ) . the degradation and release of proteins and glycoproteins from cartilage in oa can vary according to the stage of the disease process . for example , elevated serum cartilage oligomeric matrix protein ( comp ) is correlated with the presence of oa and disease severity ( clark et al . , 1999 ) . aging is a major contributor to musculoskeletal degeneration and the development of oa ( hamerman , 1998 ; lotz and carames , 2011 ) . age - related changes in articular cartilage contribute to the development and progression of oa . although the degeneration of articular cartilage is not simply the result of aging and mechanical wear , aging nevertheless modifies the articular joint including cartilage , subchondral bone , muscle , soft tissues , synovial membrane , and synovial fluid ( buckwalter and mankin , 1998a ; hamerman , 1998 ) . although older age is the greatest risk factor for oa , oa is not an inevitable consequence of growing old ( shane anderson and loeser , 2010 ) . cell stress and oxidative damage contribute to chronic inflammation that promotes age - related diseases . in oa this results in senescence - associated secretory phenotype , which has many of the characteristics of an osteoarthritic chondrocyte in terms of the cytokines , chemokines , and proteases produced ( loeser , 2011 ) . a major focus of clinical research in recent years has been the identification of new disease markers that can facilitate early diagnosis and optimize individualized treatments . such markers can also facilitate the drug discovery process by reducing the high levels of attrition in clinical trials . a biomarker is classically defined as a biochemical entity that is used to measure the progress of a disease or the effects of treatment on clinical outcome . biochemical markers can be measured in blood , serum , and urine or a variety of other body fluids and tissues . the national cancer institute ( nci ) defines a biomarker as a biological molecule found in blood , other body fluids , or tissues that is a sign of a normal or abnormal process , or of a condition or disease , and the terms molecular markers or signature molecules have also been used to describe such markers . the term biomarker is all encompassing and can include proteins , protein fragments , metabolites , carbohydrates , nucleic acids ( rna and dna ) , cellular features , and images . osteoarthritis is unambiguously diagnosed when it is detected by the best available test . thus far the best test for this purpose has been radiography , the so - called gold - standard . this process also requires clinical signs in the patient , which often occur well into the progression of the disease . however , there is often early , pre - clinical evidence of disease provided by various biomarkers , which if detected , may facilitate earlier diagnosis and treatment . such an approach is particularly pertinent in the case of oa , a disease often characterized by a prolonged pre - clinical molecular phase , a pre - radiographic phase , and a recalcitrant radiographic phase by which time there are structural changes to joints along with pain and loss of function . biomarkers have the potential to provide an early warning of joint degeneration which could prompt earlier , more targeted treatment to prevent the tissue destruction that results in the characteristic chronic disability associated with oa . in this context , biomarkers could make a significant contribution to the early diagnosis of oa , as well as informing key aspects of disease prognosis , monitoring , and therapy . the national institute of arthritis and musculoskeletal and skin diseases ( niams ) established the osteoarthritis biomarkers network to develop and validate standardized , sensitive biomarker assays in blood and urine to facilitate the diagnosis of the pre - radiologic stage of oa in humans and in animal models . such markers can help us understand the biological processes involved in disease progression and allow us to monitor the effects of surgical or pharmacological treatment , thus accelerating the pace of drug discovery . such biomarkers could also potentially be used to identify patients at increased risk of developing oa . existing biomarkers of oa have major limitations : they do not flag the pre - radiographic phase of the disease ; they are not specific for the various stages of oa , and in some cases , may not even be specific for oa . considering these challenges , the osteoarthritis research society international ( oarsi ) and the us food and drug administration ( fda ) have recently established a new oa biomarkers working group , which has proposed the division of potential markers into two major groups : the so - called soluble or biomarkers , which typically reflect a modulation in an endogenous substance in body fluids such blood , serum , plasma , urine , or synovial fluid ) ; and the dry biomarkers , which usually consist of visual analog scales , performed tasks , or images of joints ( kraus et al . , 2011 ) . therefore , the ability to detect biomarkers of cartilage degradation and/or inflammation in biological samples , such as serum , urine , or synovial fluid , may enable clinicians to diagnose sub - clinical oa as well as determining the disease stage in both human and companion animals . identifying these biomarkers will also aid drug discovery and drug safety / efficacy monitoring in patients and in animal models . using combinations of biomarkers may be more effective in achieving these goals , thus having a panel of biomarkers will help researchers and the pharmaceutical industry to monitor disease progression as well as to assess responses to treatment in experimental models of oa ( rousseau and delmas , 2007 ; williams , 2009 ) . systems biology is increasingly applied in orthopedics and rheumatology to cartilage and synovium in arthritis . these techniques include genomics , transcriptomics , proteomics , metabolomics , glycomics , and bioinformatics and can be applied to the study of cartilage , synovium , synovial fluid , and even blood ( serum ) or urine from oa patients . proteomics involves the application of specialized analytical techniques that allow the evaluation of the protein composition of tissues , cells , and culture supernatants . proteomics is being increasingly applied in basic cartilage biology ( polacek et al . , 2010 ) and oa research ( ruiz - romero et al . , 2010 ) . characterization of cell lysates from isolated chondrocytes has yielded valuable information regarding the intracellular proteins of the chondrocyte proteome , and paved the way for future studies on cartilage pathologies such as oa ( ruiz - romero et al . , 2005 ; ruiz - romero and blanco , 2010 ) . studies of soluble proteins in cartilage tissue from oa patients has increased the knowledge of the proteins contained within the ecm of diseased versus normal tissue ( wu et al . , 2007 ) . a number of papers have reported on proteins secreted from the cartilage ecm in response to pathological insults such as interleukin ( il)-1 and all - trans - retinoic acid ( wilson et al . , 2008a , b ; ruiz - romero and blanco , 2010 ) , il-1 and tnf- ( cillero - pastor et al . , 2010 ) and mechanical compression ( stevens et al . , 2008 ; identifying proteins released from cartilage has the potential to give an indication of disease biomarkers likely to be present in the synovial fluid or blood of patients in the early stages of oa . understanding healthy aging is a key research priority , along with a better understanding of the pathophysiology of aging that occurs in a number of age - related diseases , such as arthritis . by gaining a better understanding of healthy musculoskeletal aging we can provide better care and new therapies for common musculoskeletal problems . physiology and pathophysiology of musculoskeletal aging is a research topic that is intended to bring together basic researchers and clinicians working in the broad area of musculoskeletal aging . the topic includes mechanisms of healthy aging in tissues of the musculoskeletal system ( i.e. , skeletal muscle , articular cartilage , subchondral bone , tendon , and ligament ) . the discovery and validation for biomarkers of oa has accelerated significantly as our understanding of joint tissue molecules and their complex interactions have increased ( kraus , 2005 ) . one of the main drivers in this context has been the urgent need for improved oa outcome measures in clinical trials ( kraus , 2005 ; hunter et al . , in particular there is a pressing need for new biomarkers that indicate early responses of the joint cartilage to degeneration that will be useful in detecting early , pre - radiographic changes . novel markers that characterize the status and prognosis of oa , and that can be used to monitor response to therapy are also required ( mobasheri and henrotin , 2010 ) . research aims to develop an analytical toolbox which is hoped will contribute to the clinical development process ( bay - jensen et al . , 2010 ; combinations of existing biomarkers may improve their prognostic accuracy and help identify at - risk patients ( williams , 2009 ) . omics - based technologies in order to identify sensitive and reliable pre - radiographic biomarkers that can be accurately and reproducibly measured in body fluids . biomarkers that flag early stage oa will be particularly useful in curbing disease progression by identifying patients that would benefit from early therapeutic intervention . in this research topic gharbi and co - workers ( gharbi et al . , 2011 ) their aim is to improve our understanding of the physiopathology of the disease its underlying mechanisms and to discover disease - specific biomarkers and identify new therapeutic targets . this timely and focused review summarizes the currently available data regarding proteomic techniques and their applications to oa research . the authors discuss technical limitations and solutions to real and practical problems including sample preparation . although proteomics has many potential applications in this area , there are technical challenges that still remain . the author declares 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 incidence of age - related musculoskeletal impairment is steadily rising throughout the world . musculoskeletal conditions are closely linked with aging and inflammation . they are leading causes of morbidity and disability in man and beast . aging is a major contributor to musculoskeletal degeneration and the development of osteoarthritis ( oa ) . oa is a degenerative disease that involves structural changes to joint tissues including synovial inflammation , catabolic destruction of articular cartilage and alterations in subchondral bone . cartilage degradation and structural changes in subchondral bone result in the production of fragments of extracellular matrix molecules . some of these biochemical markers or biomarkers can be detected in blood , serum , synovial fluid , and urine and may be useful markers of disease progression . the ability to detect biomarkers of cartilage degradation in body fluids may enable clinicians to diagnose sub - clinical oa as well as determining the course of disease progression . new biomarkers that indicate early responses of the joint cartilage to degeneration will be useful in detecting early , pre - radiographic changes . systems biology is increasingly applied in basic cartilage biology and oa research . proteomic techniques have the potential to improve our understanding of oa physiopathology and its underlying mechanisms . proteomics can also facilitate the discovery of disease - specific biomarkers and help identify new therapeutic targets . proteomic studies of cartilage and other joint tissues may be particularly relevant in diagnostic orthopedics and therapeutic research . this perspective article discusses the relevance and potential of proteomics for studying age - related musculoskeletal diseases such as oa and reviews the contributions of key investigators in the field .

The Global Challenge of Musculoskeletal Diseases Characterized and Exacerbated by Aging Articular Cartilage Structure and Function Cartilage Degradation in Osteoarthritis Aging and Osteoarthritis Biomarkers of Osteoarthritis Systems Biology and Proteomic Approaches for the Discovery of Osteoarthritis Biomarkers Relevance of Biomarkers and Proteomic Techniques to Physiology and Pathophysiology of Musculoskeletal Aging Conflict of Interest Statement

a patient 's journey may begin with transfer from an ambulance to the emergency room and then sometimes continues to surgery and intensive care followed by a general ward . intensive care is designed and meant for the sickest patients with potential life threats and vital organs dysfunction ; it requires advanced monitoring , technique , diagnosis , and treatments . organizing and performing patient transfers in the continuum of care is part of the work of nurses and other staff of the multiprofessional healthcare team . whittaker and ball ( 2000 ) argue that it is important to perform the preparations for a transfer to the general ward accurately and correctly . if this is not done , the patient must be readmitted to the intensive care unit ( icu ) and be exposed to further stress . this depending on multiple reasons and common causes could be their present sedation or altered mental status . discharge planning in general is described as a process which should provide continuity of care for patients . in this study , icu transitional care is defined as care provided before , during , and after the transfer of an icu patient to another care unit with the aim of ensuring minimal disruption and optimal care for the patient . this care may be provided by icu nurses , acute care nurses , physicians , and other healthcare professionals . hence , in this study , care is defined as a mix of nursing care and medical care . it is important that patients ' transfers from the icu are done properly and at the right time when there is no longer a need for intensive care [ 4 , 5 ] . patients want to feel safe and secure both before and after the transfer , and they can easily become dependent on the staff . there are studies that describe how patients perceive their safety during transfer and how they feel about their recovery , but it has also been reported how perceived physical illness can affect experiences of displacement . patients sometimes also struggle with feelings of abandonment , vulnerability , helplessness , and unimportance . ambivalent feelings about the upcoming transfers are also shown to be common ; both positive and negative emotions have been reported [ 911 ] . different factors have impact on patients ' recovery from intensive care , premorbid state , social , family , psychological , physical status , and employment . the struggle for hospital bed placement is becoming more and more frequent , and nowadays hospitals often are overcrowded , which also implies that the organization of transfers is especially important for patient safety . discharge guidelines and policies are seen as important in the management of transfers [ 14 , 15 ] and are also considered an effective management tool to reduce length - of - stay in the icu and improve the utilization of icu resources [ 16 , 17 ] . however , the results of previous studies indicate that discharge planning often lacks guidelines and tends to be ad hoc and influenced by patient acuity [ 18 , 19 ] . priorities in icu may be necessary to enable admission for the most ill patients , leading to unplanned discharges even during night which are related to higher risks . a study by goldfrad and rowan ( 2000 ) found that the overall icu mortality is 25 times higher if the patient is discharged at night . in their study , the staff estimated that only 44% of these patients were fully ready for the transfer , compared with over 80% of patients who were transferred during the day . discharging patients recently discharged from the icu may be particularly at risk for adverse events and readmissions to icu . to complete a patient 's transfer from a high technological icu to a general ward , many studies demonstrate the experiences of the transition but there is no clear description of the process and how it could be organized in order to be safe [ 511 ] . based on the experiences described by those involved in the process , it can be argued that it is important to learn more about how to enhance and organize icu transitional care . therefore , the aim of this study was to describe , as experienced by intensive care and general ward staff , what strategies could be used when organizing patient 's care before , during , and after transfer from intensive care . before , during , and after transfer from the icu to a general ward , patients experience a transition process . the patients are transferred from the context of high technology to the culture of the general ward . the specific process of transition from the icu to the general ward has become a topic of interest because difficulties that arise during the process have been increasingly frequent . transitions can be initiated by such events as acute illness or injury , which also explains why the concept is a nursing concern . the process requires a beginning , middle , and end and how the person feels and perceives the situation is critical as the process continues . transition could result in a feeling of displacement and lack of control over their lives . the situation and time span vary and may consist of short periods or months and years ; an example of transition is hospitalization for an acute injury or illness . the study has been approved by the northern ethical committee in sweden ( d - number 07 - 159 ) . the first author informed and asked the nurses about participation in the study in accordance with verbal and written criteria . they were informed about confidentiality and their rights to withdraw their participation without giving reason . as the aim of the study was to describe and illuminate the transition process between icu and general ward , qualitative content analyses were considered [ 25 , 26 ] . the data were also used in a larger study that aimed to generate theories about main concerns in icu transitional care . data were collected between 2008 and 2010 in two hospitals located in sweden with different sizes . the participants were recruited in three icus and five general wards specializing in surgical , medical , or general fields . one of the hospitals had a step - down unit , and the second had no step - down unit . all interviews were conducted by the first author . there were totally 15 individual interviews : seven icu nurses from three icus in sweden , one anesthesiologist , one en ( enrolled nurse ) from an icu , and six rns working in different surgical or medical wards in two hospitals . the participants had different genders and lengths of experience ( 125 years ) ; 3 were males and 12 were women . their ages varied between 25 and 62 years . the staff in general wards was registered nurses and the nurses who worked in the icu had a one - year specialist certification in intensive care . the interviews were performed in a quiet place at the unit in the hospital and lasted between 30 and 50 minutes . the focus in this study was on how to organize patient transfer to the ward for further care and rehabilitation and not on patients transferred for palliative end - of - life care . could you please tell me about how the transfer process for icu patients is organized at your unit and your feelings about it ? clarifying questions such as what do you mean , when , why , and can you tell me more about that were asked when necessary to encourage interviewees to narrate their experiences . the data analyses were inspired by elo and kyngs ' ( 2008 ) description of content analysis . , the interview text was read several times to get a sense of the whole . the manifest analyses started with sorting the data in content areas : before , during , and after transfer from the icu to a general ward . this was followed by an open coding with handwritten notes and headings in the text , using as many headings as possible , close to the text , in order to describe all aspects of the content . with further abstraction , the codes were divided into subcategories and categories with similar incidents named with content - characteristic words that were all relevant to the aim and the research topic . the results show that the categories secure , encourage , and collaborate are strategies used in the three phases of the icu transitional care process before , during , and after transfer from the icu ( table 1 ) . the main category , a safe , interactive rehabilitation process , illustrates how all strategies were characterized by an intention to create and maintain safety during the process . it also included attempts to help the patient reach the best possible condition in the different phases so that their recovery and the rehabilitation proceeded as planned . one nurse stated : the goal for our care in this process is that the patients are going to be better in all ways , both physical and physiological . the goal for our care in this process is that the patients are going to be better in all ways , both physical and physiological . the main category and the strategies also illustrated a three - way interaction in the process : between staff and patient / families , between team members and involved units , and between patient / family and environment . the first category , secure , included activities that aimed at preserving patient safety during the transfer and preventing adverse events immediately after transfer or later . the second category , encourage , included activities focused on strengthening , inspiring , and giving hope and courage to patients and their families . the third category , collaborate , included activities aimed at intertwining the process : coordination , cooperation , and communication between the icu and the general wards . the first phase in icu transitional care included secure , optimizing vital signs and reducing and adjusting the intensive care , encourage , promoting self - ability and customizing information , and collaborate , communicating and coordinating with the ward and arranging a pretransfer meeting . optimize vital functions . securing the patients ' care and preserving patient safety during icu transitional care one essential issue was to optimize the patient 's vital functions prior to transfer , a key component to minimize risk for adverse and readmissions , which were commonly referred to in the interviews . the icu staff interacted with technology and other senses to assess and perform clinical judgments . the patient 's pain relief and vital signs ( especially respiration and hemodynamic ) were the focus and a point of decision in how and when the patient could be transferred out of intensive care.the most important in the transfer process is that the patients ' vital signs are stable enough ; we observe this extremely carefully . the most important in the transfer process is that the patients ' vital signs are stable enough ; we observe this extremely carefully . the decision for transfer mostly by anesthesiologists in consultation with the responsible physician from the ward . however , the icu nurses felt that they were involved and interacted with the physicians in the decision by either confirming that the patients were stable enough or postponing the transfer if the patient had respiratory issues , fever , or some other problematic clinical symptoms . if the patient was fragile or weak , it was seen as important to have a couple of extra days in the icu or , if possible , in an intermediary unit reassuring that intensive care was not needed anymore . this was expressed not only as one important strategy to prevent readmission but also as something that often could not be done . timing for transfer was crucial , and the interviews revealed that patients should not remain either too long or too short time in the icu . the nurses expressed that both compromised patient safety ; if they were transferred too early , they were not stable enough , and if they were transferred later than needed , it made them more immobilized and susceptible to infections and other symptoms . reduce and adjust . to secure patient care and to prevent adverse events later in the icu transitional care process , it was necessary to reduce technology and adjust how medication was administrated . weaning and timing of extubation was one important part , which was expressed as often time - consuming and difficult for less experienced nurses . the staff stated that patients should have time to recover from the state of full respiratory support , proceed to minor support ( noninvasive respiratory support ) , and finally manage to breathe at least 24 hours without respiratory support . it was also expressed as important to reduce and adjust the care for patient safety , both for the prevention for central line infections and for suiting the upcoming level of care and competence at the ward . not all ward nurses , for example , managed multilumen central lines and the like . in the interview , it was also apparent that technology should be reduced and only used when needed in the icu since the environment in the icu could cause trouble for the individual patient . instead , the use of technology should follow the need for hemodynamic assessment so that the patients and their families became used to fewer observations and monitoring once the patients were transferred . adjustments to care were often done in an interaction between ens , rns , and physicians and needed to be documented in the patient record to maintain consistency . it was also considered essential that administration of medications was altered to prepare the patients for the upcoming level of care , for example , shifting to sleeping pills instead of infusions prior to transfer.i think that patients that we try to wean that have been here a long time also should be used to not have full monitoring often , there are enough with a pulse oximetry part of the day . i think that patients that we try to wean that have been here a long time also should be used to not have full monitoring often , there are enough with a pulse oximetry part of the day . this included supporting the patient 's own initiative and promoting self - ability , since the icu period often had a negative impact on the patient 's muscle mass and initiative ability . it was expressed that the patient 's self - ability in this phase influenced the following phases in the transfer process . the staff meant that the patient 's self - ability should be strengthened prior to transfer as a part of their rehabilitation process and as a step toward managing the altered level of care . promoting the patient 's own ability required a lot of persuasion and sensitive interaction with the patient . the icu staff tried to support the patients to manage small things , for example , to use the bed alarm so they would be familiar with it later on to receive help at the ward . the staff described how they tried on a daily basis to encourage patients to sit up at the bedside and in chairs so that they would not be immobilized in bed at all times . it was also considered essential and helpful if the patients got early and frequent physiotherapy at the icu.we help the patient to drink water which includes helping them grip the glass and set it to the mouth we have to tell them that everything is better than doing nothing ; you can do it in your own way . we help the patient to drink water which includes helping them grip the glass and set it to the mouth we have to tell them that everything is better than doing nothing ; you can do it in your own way . encouragement based on providing repeated and customized information to patients and their families was considered by the staff to be an important tool in organizing icu transitional care . it was important to adjust and customize the information to the specific patient needs and inform the families about time for the upcoming transfer . it was expressed as essential to interact with the families and inform them several times about the progress , care plans , and goals . the icu and general ward staff expressed that anxiety could be minimized if patients and their families were encouraged and informed repeatedly that their condition was stabilized and that a transfer would soon occur . some patients and families needed to hear the information often and asked repeatedly about things related to their medical status and upcoming transfer while others asked nothing . it was expressed as important to give them consistent responses that did not differ , which could include visual information about the environment and routines at the ward preferably in form of a brochure or something similar . the staff experienced that the patients ' and/or families ' experience of encouragement and participation in their care depended on how well and how clearly the information was given.i think we can do even better , especially to work more with individualized information for the families . i think we can do even better , especially to work more with individualized information for the families . the staff described the coordination between the icu and the department to which the patient would be transferred as a foundation for collaboration , especially when the patient had been cared for in the icu for a long time . collaborating involved communicating and coordinating between the care units for transferred patients . the communication involved information about the patient needs and preparing the unit about what they should consider when the patient arrived at the ward . in some cases , it also involved care - planning meetings with families and different physicians . this made a common view for the patients ' planning , and it was expressed as a wish for mutuality between the icu and the ward , allotting responsibility and planning for the patients ' transfer . coordination was described as being better if it was done as early as possible , especially if the patient had a long length - of - stay in the icu . one unit used a liaison nurse a person with a specific responsibility for collaboration which was experienced in facilitating communication and planning for specific needs . the nurses expressed that if the patient had specific needs , they must be planned for and taken care of a strategy to manage care must be prepared . collaboration could also include planning for what room the patient should be placed in at the ward , considering individual needs and , if possible , what nurse should be responsible . one icu nurse said : it is important to communicate early and tell them ( the staff at the ward ) about every need that the patient may have and not cover or exclude anything . especially if the patient has been a long time in icu it is important to communicate early and tell them ( the staff at the ward ) about every need that the patient may have and not cover or exclude anything . especially if the patient has been a long time in icu arrange a pretransfer meeting . collaborating and arranging a pretransfer meeting between ward staff and the patients and their families before transfer was expressed as one appreciated strategy . this was described as extra important when the patient or the family was anxious or if the patient had been cared for in the icu for a long time and had extended needs . the ward nurses meant that this pretransfer meeting was valuable for them and for the patient , since it was an opportunity to create a contact with the patient and family before transfer . the meeting also provided a possibility to ask questions and get a picture about of the patient 's care needs . the meeting was described in two ways : at the icu or in the general ward , depending on the patient 's status . the nurses expressed that when a pretransfer meeting was organized , this helped to intertwine the healthcare chain.right now , for example , we have a gentleman at our ward that was 4 - 5 months in icu ; he has probably a length - of - stay of 8 months in the hospital ! and before he came up , we were down there and introduced us , told him that we would be responsible at the ward ! right now , for example , we have a gentleman at our ward that was 4 - 5 months in icu ; he has probably a length - of - stay of 8 months in the hospital ! and before he came up , we were down there and introduced us , told him that we would be responsible at the ward ! the strategies in this phase included secure , assess and summarize , hand over responsibility , and arrange a safe transfer , encourage , give confidence , and collaborate , negotiate and facilitate . the staff expressed that it was important prior to transfer to take time to assess and summarize the patient 's discharge status and the different nursing phenomena and actions that had been or should be taken for the patient . if the transfer was not planned in advance , this phase also included examining all the equipment that the patient still had and considering removing measurement and technology specific to intensive care and that were not wanted or needed at the general ward.i think it 's easier when we have someone who is responsible and keeps up the documentation and prepares for discharge we really want the patient 's record to be clear and that it is obvious what is planned . i think it 's easier when we have someone who is responsible and keeps up the documentation and prepares for discharge we really want the patient 's record to be clear and that it is obvious what is planned . the report was mentioned by the staff to be an essential tool to maintain continuity of care and was experienced as the way to hand over the patient without any loose ends . the staff from the general ward tried to prepare themselves by taking part of patient documents before they got to the icu , but they did not always have this opportunity.i try to keep up reading about the patient before the transfer so i do n't have to start from zero knowledge . for example , what surgery they 've done , drains , and also quickly check the latest values i try to keep up reading about the patient before the transfer so i do n't have to start from zero knowledge . for example , what surgery they 've done , drains , and also quickly check the latest values the handover often included a nurse - to - nurse report intended to focus briefly on the history and more specifically on the actual status and the planned care . the icu nurses expressed that their handover should be adjusted with adequate information but they wanted to tell as much as possible so that no loose ends would be left . some ward nurses expressed that they wanted less history and more of the actual status with planned x - rays , medications , and iv - fluids as well as more of a description of the patient 's own ability and problems . sometimes , the nurses from the general ward felt insecure but did not dare to ask the icu nurses about the patient , since they did not want to seem incompetent . most icu patients are fragile , and a safe transfer that maintains patient safety was expressed as essential . the patient often received oxygen during the transfer to avoid desaturation , and , sometimes , portable monitors were used to control vital signs . in the interviews , one nurse described an example of adverse events related to a transfer : well , we do n't want something critical to happen during the transfer but i will always remember one time i got to get a patient from icu i directly saw that she was hugely fragile and wondered if we really should move her from icu . but both our physician and icus physician insisted , and we took the patient and went away immediately when we came out from the elevator , the patient 's vital signs were deteriorating . we hurried as soon we could into the ward to get help and had to alarm for assistance . well , we do n't want something critical to happen during the transfer but i will always remember one time i got to get a patient from icu i directly saw that she was hugely fragile and wondered if we really should move her from icu . but both our physician and icus physician insisted , and we took the patient and went away immediately when we came out from the elevator , the patient 's vital signs were deteriorating . we hurried as soon we could into the ward to get help and had to alarm for assistance . the staff experienced that it was essential to give confidence prior to and during transfer and to talk in a positive way with patients and their families . the nurses expressed that some patients were anxious and showed feelings of disconnectedness when leaving the environment where they felt safe and staff they knew . the staff felt that the patients ' families also needed to feel that the transfer was a step forward and that it was a positive sign.when the day of the transfer comes , it may not be too ridden with anxiety for the patient either - but you have to play down the whole and try to make it easier it might be a grief to leave the place where you ( have ) been so long and become better . that you try to focus on that this is an important positive sign that the patient has to be transferred at a ward , emphasize that now is the worst of the crisis over ! when the day of the transfer comes , it may not be too ridden with anxiety for the patient either - but you have to play down the whole and try to make it easier it might be a grief to leave the place where you ( have ) been so long and become better . that you try to focus on that this is an important positive sign that the patient has to be transferred at a ward , emphasize that now is the worst of the crisis over ! collaboration was described by the staff as essential on the day of transfer and included negotiating a suitable time for transfer and facilitating the transfer for all involved staff members . it was important to seek resolutions for problems and to see each other as equal . the staff expressed that barriers could occur for collaboration that instead complicate the transfer , for example , blaming each other and not trying to understand each other 's work situation . hence , interprofessional respect between the staff at the units was essential and facilitated the process . the actual day of the transfer was expressed as more or less planned , depending on the acute situation at the unit . the nurses on general wards said that it was obvious that the icu , with rapid decision - making , led to rapid transfers , sometimes unplanned . they wished to be involved in deciding the time of transfer if possible , a time that suited staff from both the general ward and the icu . the staff at the general ward considered it extremely important to have the opportunity to prepare when they were about to get a patient from the icu , since it was often time - consuming . if they knew in time , they could plan their work , which made the process feel easier and safer . when the staff communicated about a suitable time for transfer , they also were informed about specific equipment needed , such as oxygen.most of the times , we can not influence the time for transfer , but we think that it more often should be possible to communicate about it , make it suit us all . most of the times , we can not influence the time for transfer , but we think that it more often should be possible to communicate about it , make it suit us all . the third phase included the strategies secure , take charge , encourage , create a good encounter and instill hope and courage , and collaborate , prepare discharge and follow up . when the patient arrived at the general ward , the staff stated that it was important to get a grip of the whole situation and to take charge of the patient . it involved an overall view from clinical judgments to take control over the paperwork and plan future care actions that were described as time - consuming . depending on the patient 's status , the ward nurses spent more or less time on continuing care , for example , supplying oxygen ; controlling wounds , drains , and drain holes ; monitoring feeding tubes and intravenous lines ; calculating fluid balance ; and checking vital signs . the nurses expressed that it also was vital that the physician at the ward checked out the ordinations and wrote an updated status in the patient 's record so that they had a tool for managing their care.it's not just ( that ) the patient must be in good condition when he or she is discharged from the icu ; the care must also continue with high quality at the general ward . it 's not just ( that ) the patient must be in good condition when he or she is discharged from the icu ; the care must also continue with high quality at the general ward . sometimes , it was necessary to change equipment and time for medication to suit the care at the ward . some of the ward nurses expressed that they felt that it was more often patients with a shorter length - of - stay at the icu who were hemodynamically unstable and readmitted . according to their experience , patients with longer length - of - stay more often were better optimized in their vital functions . the ward nurses mentioned that their control of vital signs sometimes required them to alarm a specific outreach team . this team included staff from the icu ( nurse / physician ) who could initiate treatment for the patient at the ward or decide to readmit the patient to the icu again . they expressed that the outreach team was a helpful tool and made them feel safer in their care but also meant that it was important to have personal clinical judgment.there are always some warning clocks when you are checking vital signs and something is wrong . sometimes , you can feel that something is breaking out , something will be wrong with the patient i think it depends of a combination of ( your ) own intuition and the patient 's history . there are always some warning clocks when you are checking vital signs and something is wrong . sometimes , you can feel that something is breaking out , something will be wrong with the patient i think it depends of a combination of ( your ) own intuition and the patient 's history . instill hope and courage . creating a good encounter with the patients and their families at the first meeting ( either in the icu or when arriving at the general ward ) a good encounter included a personal meeting , introducing the responsible staff , and interacting and supporting individual needs . the ward nurses wanted to have time to communicate and sense needs at the first encounter so that they could calm the patients and their families . they expressed that families often displayed anxiety and had questions about the differences in the environments . it was also important to inform the patient and other family members that staff members were available at the ward even though they were not physically present at the patient 's room all the time . it took some time to establish hope and courage , and some were more difficult than others.the patients and their families can be in a shock reaction when they arrive , and they still try to process and understand what happened according to my experience , this is more often seen if the intensive period were short . the patients and their families can be in a shock reaction when they arrive , and they still try to process and understand what happened according to my experience , this is more often seen if the intensive period were short . the ward nurses expressed that early mobilization in the icu was vital for how the patient 's rehabilitation process proceeded . the nurses expressed that the patient 's own ability to take initiative needed to be supported . depending on diagnosis , this ability differed , but the ward nurses expressed that the care in the icu often made patients immobilized and used to others taking care of their hygiene . nurses in the ward felt that the patients had become apathetic and then stepped aside , as others still were responsible for their body.some can stand on their legs immediately , but some must first be mobilized in the bed but you can really tell that they 've been working with early mobilization in icu . some can stand on their legs immediately , but some must first be mobilized in the bed but you can really tell that they 've been working with early mobilization in icu . collaboration in this phase included planning for further discharge for the patient , which depended on the individual patient 's status and home situation . sometimes , patients needed extensive planning and interaction with community nurses while other patients could be directly transferred at home without any extended help . the nurses explained that this planning often took time and was an important part of the patient 's healthcare chain . the icu and general ward staff often continued their collaboration around the patient following transfer a strategy that intertwined the icu transitional care process . sometimes , a ward visit was made by the icu staff primarily not only to meet the patient again but also to give the staff an opportunity to ask questions and to give medical advice if needed . in one of the hospitals , patients were further followed up within postintensive care meetings after they had been discharged and left the hospital.we try to go up and visit the patient at the ward a few days after the transfer , if we have time ; it 's appreciated by both the patient and the staff since they may ask if there is something missed . we try to go up and visit the patient at the ward a few days after the transfer , if we have time ; it 's appreciated by both the patient and the staff since they may ask if there is something missed . the main category of this study showed that strategies used by the staff before , during , and after transfer aimed to contribute to a safe , interactive rehabilitation process . the results showed how the strategies secure , encourage , and collaborate could be used to enhance and organize icu transitional care . patient safety and interaction with patient , family , environment , and other team members are essential for icu transitional care . the main category also illustrates that there is a three - way interaction in the process : between staff and patients / families , between different team members and involved units , and between patient / family and environment . the interaction between environment and patients has also been illuminated by theorists such as rogers , king , and nightingale , all of whom claimed that the patient is in constant interaction with his or her environment . the icu staff interacted with technology as a tool to determine if patients were stable enough for weaning and , further on , for transfer . the staff felt that the environmental differences between the icu and general wards are often a cause of concern for patients and their families . according to meleis therefore , we assumed that the care must be individualized even if there are routines and procedures to follow . transition stands for a change in health status , role relationships , expectations , or abilities . the transition event is dependent on , for instance , suddenness , personal meaning , and level of well - being . hence , it can be argued as logical to strive for a strengthening process that considers these needs and involves an active plan for a smoother transition . it is important to clarify and explain to the patient and family that observations and monitoring are reduced and adapted to the patient 's current health status , which may reduce the perceived stress during the transitional phase . overall , the results indicate that the transition experience is dependent on preparation of both patient and families for the first contact with the team at the ward and the time of transfer ; therefore , the planning must be taken seriously . timing seemed to be a critical point , and this is confirmed in a study by garland and connors ( 2013 ) . their study indicated that 30-day mortality increased not only if the patients had to leave earlier than planned from the icu but also if the transfer was delayed so they had to leave later than was optimal . this is in accordance with other researches and confirms that weaning is vital for patient safety . ( 2003 ) showed that readmission was more often seen when the time between extubation and discharge from the icu was short . a recent study including patients with traumatic brain injuries showed that reintubations within 48 hours ( i.e. , extubation failure ) were significantly associated with lengthened hospital stays , increased frequency of tracheotomy and of pulmonary complications , worsened functional outcomes , and increased mortality . intermediary units can be used as a way to reduce care and to prepare patients for the altered level [ 32 , 33 ] , but not all hospitals have this kind of unit . however , icus can identify beds at the unit aimed at intermediary care where patient care can proceed but with less technology and staff . the necessity of avoiding miscommunication during clinical handovers is also described in several other studies . miscommunication can lead to risk of prolonged stay , lack of continuity of care , suboptimal patient flow , readmissions , and patient dissatisfaction reports [ 34 , 35 ] . in a systematic review of foster and manser ( 2012 ) , handovers and transfer of patients in acute care were studied . the results showed that the handover process is multifacetted and can therefore be difficult to compare and evaluate , but they also show that standard report pages are one way to facilitate reporting . boutilier ( 2007 ) concluded that the most important thing in the icu transitional care process was to systematically communicate the necessary information to the receiving device so that patient safety was not threatened and to ensure the necessary continuity of care . the second strategy found in our study , encourage , is often mentioned in nursing . to encourage meant to instill hope , identity , and confidence for the patients and their families . the results of this study illustrate the importance of pep talk and supportive strategies to encourage the patient to manage the transition and recover from critical illness . according to the nurses , patients need to feel safe , a result also described by hupcey ( 2000 ) as a psychosocial need for icu patients . one characteristic of transition is disconnectedness associated with disruption of the links on which the person 's feelings of security depend . the nurses in this study wanted to be able to offer customized information prior to transfer since they thought that anxiety could be reduced with information . this can be compared with the result from strahan and brown ( 2004 ) , who found that patients often are anxious over how the transfer process will be done and wish to receive detailed information on what will happen . the nurses expressed that the patients and their families should be a part of the transfer . presence from relatives can affect the patient 's sense of participation and contribute to feelings of being cared for and of safety . it is important to minimize experiences of a gap between the icu and general wards . health and illness transitions include the sudden role change that can result from moving from a well state to an acute illness . the results of this study indicate that the patient 's role changes when arriving at the general ward from being more or less passive to being seen as active . furthermore , patients have a desire for normality and independence and wish to be able to have personal contacts also seen in a study by mckinney and deeny ( 2002 ) . the results also illustrate the importance of the team at the receiving ward having the opportunity to devote time to the patient and family when they first arrive . the ward nurses in our study expressed that patients showed mixed feelings about the transfer to the general ward and that they needed to instill hope and courage . our study indicated that both an immediate and later follow - up after leaving the icu could be useful , helping patients identify problems and find routes for rehabilitation and support . a study by schandl ( 2011 ) actually showed that multidisciplinary icu follow - up and the first six months after discharge are most important for follow - up . the results in this study showed that icu transitional care is a complex , multidisciplinary process that involves collaboration both within the icu and within other units involved . the findings of our study show that collaboration includes respectful , functional communication between units and different actions aimed to intertwine the healthcare chain . effective teamwork and coordination among staff can improve the icu patient discharge process and also reduce the gap separating icu and the general ward [ 14 , 21 ] . our study indicates that team members in their own unit and in other units need to treat each other with respect so everyone ask questions without fear or ridicule contempt . the results of their study showed , as did this study , that respect and equality are important . previous studies have focused on how a specific liaison nurse can be used to facilitate collaboration . a liaison nurse coordinates the transfer and is helpful for the patients and their families [ 43 , 44].the results of this study indicate that there are benefits to a function like that . many of the strategies during the process focus not only on identifying and minimizing risks for complications , such as pneumonia and central line infections , but also on strengthening mind and body . this process is described as an active , unique , nonlinear process with stages and phases . the results of this study follow a model called chime : connectedness , hope and optimism , identity , meaning in life , and empowerment . one important part of icu transitional care is to promote patients ' self - care capability and encourage patients ' autonomy . this is confirmed by chick and meleis ( 1986 ) , who also claim that transitions are linked to shifts in self - care capability . orem ( 1980 ) writes that independence was recognized by early nursing theorists as important for patients well - being and closely connected to their ability to perform daily activities and meet their own care needs . however , the nature of intensive care and its environment makes independence and autonomy difficult . hughes ( 2004 ) emphasized that the degree of autonomy is connected not only to the ability to actually be independent but also to the healthcare staff 's perception and understanding of the need for this assessment . the nurses in this study expressed that early mobilization was felt important not least for the patients ' ability to do things for themselves and manage the upcoming care . this can be compared with the results of an intervention study by korupolu et al . they saw that early mobilization in the icu and a strategy for whole - body rehabilitation in the early stage of critical illness showed better functional outcomes at hospital discharge , a shorter duration of delirium , and more ventilator - free days compared with standard care . mcfetridge ( 2011 ) discussed the importance of a structured , patient - centered rehabilitation program that patients can follow on their journey from critical care to ward and , finally , through discharge from hospital . mcfetridge also claims that interaction in care is essential so as to prevent actions taken from becoming fragmented . hence , the process should be seamless and transparent for all persons that are involved in the patient 's care journey and include a multidiscipline approach and a family perspective . this study illustrates how the strategies secure , encourage , and collaborate summarize specific actions that can be used as a basis for a patient - centered guideline for icu transitional care . however , the fact that guidelines on their own are not a solution to minimize the gap between icu and general wards must be discussed . a cultural gap has been identified between icu and general wards , and a study by van sluisveld et al . ( 2013 ) implies that there are social and cultural barriers to the implementation of guidelines and effective icu discharge . to summarize , no guidelines in clinical practice are useful if there is low adherence . also important are resources ( time , staffing ) and knowledge essential components in order to manage the organization of a safe transfer process from the icu to a general ward . nurses ' intention to support patients in icu transitional care often is balanced against the organization 's demands , work stress , and time restraint . a recent study showed that nursing care hours per patient in the icu and skill that mix significantly contribute to prevention of bloodstream infections and a shorter length - of - stay in the icu . ( 2011 ) found that caring for an increasing number of patients with complex problems caused stress for nursing staff already facing work overload in wards . qualitative content analysis was used in this study and was well suited since the aim was to describe experiences of intensive care and general ward staff . the results were derived from data from three icus and six general wards and , therefore , other hospitals may have other strategies for icu transitional care that have not been described in this study . the first and second author have their own experiences as rns from both icu and general wards , and their preconceptions have been bridled by thorough data analysis and discussion , by being open as possible to new perspective . this study involved staff with different professions from different hospitals , in order to enhance the variety of experiences . both authors were familiar with chosen method . during the analysis they were both part of the process and discussed how to label the codes , subcategories , and categories until agreement . by presenting the process of the analysis and presenting the result with quotes from the text , so it is possible for the readers to create their own value , important for the study 's credibility . the results of this study conclude that secure , encourage , and collaborate , called the sec - model , illustrates essential strategies suggested to use when organizing the care before , during , and after transfer from the icu to a general ward . the result is in concordance with other researches on meeting the needs of patients and families . a recent review described patients ' and families ' experiences about transfers from icu ; the themes were physical responses , psychological responses , information and communication , safety and security , and the needs of relatives all of which have been addressed in the sec - model developed through this study . the significance of this study is also strengthened by a study by lin et al . ( 2009 ) , who claim that clarification of guidelines and standardization of discharge decision - making and handover are needed . research also indicates that the care of acute ill ward patients is suboptimal which implies that this crucial link needs to be safer . according to massey et al . ( 2008 ) , suboptimal care at the wards depends on failure to seek advice , failure to appreciate clinical urgency , lack of knowledge , failure of the organization , and lack of supervision . to ensure safe and effective care transitions , strategies are needed to improve shared situational awareness , teamwork , patient flow , and resource efficiency in icu transitional care [ 59 , 60 ] . the result indicates that a successful icu transitional process aims to create an interactive , safe , forward - thinking process influenced by actions that preserve patient safety and promote individualized care . to sum it up , a safe transition involves coordination , optimal timing , early mobilization , participation , and a multidiscipline approach . also relatives perceive safety and a continuing care as very important in the icu transitional care process and want to participate . a recommendation for future studies is to explore safe transitions out of the family perspective in a systemic way .

background . organizing and performing patient transfers in the continuum of care is part of the work of nurses and other staff of a multiprofessional healthcare team . an understanding of discharge practices is needed in order to ultimate patients ' transfers from high technological intensive care units ( icu ) to general wards . aim . to describe , as experienced by intensive care and general ward staff , what strategies could be used when organizing patient 's care before , during , and after transfer from intensive care . method . interviews of 15 participants were conducted , audio - taped , transcribed verbatim , and analyzed using qualitative content analysis . results . the results showed that the categories secure , encourage , and collaborate are strategies used in the three phases of the icu transitional care process . the main category ; a safe , interactive rehabilitation process , illustrated how all strategies were characterized by an intention to create and maintain safety during the process . a three - way interaction was described : between staff and patient / families , between team members and involved units , and between patient / family and environment . discussion / conclusions . the findings highlight that icu transitional care implies critical care rehabilitation . discharge procedures need to be safe and structured and involve collaboration , encouraging support , optimal timing , early mobilization , and a multidiscipline approach .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusions

microglia are macrophage - like resident immune cells in the central nervous system ( cns ) and possess both neurotoxic and neuroprotective function . microglia accumulate in the lesions of a variety of neurodegenerative disorders , such as alzheimer 's disease ( ad ) , parkinson 's disease , and multiple sclerosis , and are thought to play both toxic and protective functions for neuronal survival . microglia are considered to be a first line defense and respond quickly to various stimuli . when activated , microglia undergo morphological changes to ameboid , proliferate , migrate toward injured areas , and release many soluble factors and phagocytosis of foreign substances or unwanted self - debris . appropriate migration of microglia to damaged area is controlled by chemokines and nucleotide atp [ 2 , 3 ] . phagocytosis seems to be important to prevent the senile plaque expansion in ad by removing amyloid ( a ) deposit . microglia not only engulf the a protein but also phagocytose apoptotic cells and degenerated neuronal debris . phagocytosis of apoptotic or degenerated neuronal debris is crucial to reduce inflammation and maintain healthy neuronal networks . another type of phagocytosis , phagocytosis with inflammation , occurs in chronic inflammatory - related neurodegenerative disorders including alzheimer disease [ 57 ] . degenerated neurons releases several signaling molecules , including nucleotides , cytokines , and chemokines , to recruit microglia and enhance their activities [ 8 , 9 ] . the phenomenon are now termed as find - me , eat - me , and help - me signals . in this paper , we focused on find - me , and eat - me signals from degenerated neurons to microglia . most distinguished and examined eat - me signal is phosphatidylserine ( ps ) , which is a component of cellular membrane and is everted on apoptotic cell membrane [ 10 , 11 ] . nucleotides are also considered as the eat - me signal lately ; microglia expresse various p2x and p2y receptors , nucleotide receptors , which regulate not only chemotaxis but also phagocytosis [ 8 , 12 ] . microglia express many other surface receptors , which have direct interaction with the target to initiate phagocytosis , including ps receptor , lipopolysaccharide ( lps ) receptor cd14 , the scavenger receptor cd36 , the purine receptor p2y6 , and the toll - like receptors ( tlrs ) ( table 1 , figure 1 ) . another surface receptor , the cx3c chemokine fractalkine receptor cx3cr1 , is almost exclusively expressed in microglia throughout the cns , which is involved in progression of neurodegenerative disease by altering microglial activities [ 16 , 17 ] ( figure 2 ) . deletion of cx3cr1 expression in microglia results in progressive neuronal cell death in an animal model of neurodegenerative disease , by inducing microglial dysfunction . it has been identified recently that neurons themselves produce cytokine and chemokine , such as fractalkine . as shown in figure 2 , we previously reported that interleukin-34 ( il-34 ) , a newly discovered cytokine , is produced by neurons , and that its receptor , colony - stimulating factor 1 receptor , is primarily expressed on microglia . fractalkine and il-34 might be important mediator between neurons and microglia , and it is important to clarify this cellular crosstalk signaling pathways for seeking future therapeutic target of neurodegenerative diseases including ad . in the following sections , we will discuss about recent advances of microglial chemotaxis and phagocytosis and their implications for ad therapy . the expression levels were increased under pathological conditions , which seem to facilitate the recruitment and trafficking of glial cells to the damaged area . microglia constitutively express several chemokine receptors ( table 1 ) , which are implicated in the recruitment and accumulation microglia in ad lesions . when exposed to a , microglia are induced to produce several chemokines , such as ccl2 , ccl4 , and cxcl12 . ccr2 , the receptor of ccl2 , deficiency resulted in reduction of microglial accumulation and higher brain a levels in mouse model of ad , which might be mediated via suppression of anti - inflammatory molecule , transforming growth factor ( tgf- ) . however , there is a conflicting report showing increased tgf- signaling in microglia surrounding a plaques in ccr2 knockout in ad model mouse ( appswe / ps1/ccr2/ ) . this ad model has been shown to have accumulation of oligomeric a and memory impairment . ccl2 expression level is also related to another neurodegenerative disorder , multiple sclerosis ( ms ) ; ccl2 level is downregulated in cerebrospinal fluid from ms patients . ccl21 triggers chemotaxis of microglia through cxcr3 , but not ccr7 which implicated in peripheral lymphoid organs . neuronal ccl21 upregulates p2x4 receptor , the nucleotide receptor , expressed in microglia . this cascade is implicated in pathophysiology of tactile allodynia to cause chronic neuropathic pain . a-induced microglial activation is mediated through p2x7 receptor that is reported as proinflammatory response conductive receptor . atp predominantly induced microglial migration among nucleotides through p2y receptors , especially p2y12 [ 2 , 37 ] . following cns injury , expression of p2y12 in microglia drastically reduced after microglial activation , suggesting that p2y12 is a primary and temporary receptor to induce microglial chemotaxis at early stages of the local cns injury . the other nucleotide udp increases mainly microglial phagocytosis ( uptake of microspheres ) via p2y6 receptor . in the condition brain damage by kainic acid administration , the cx3c chemokine cx3cl1 ( fractalkine , also called as neurotactin ) , which has been identified as two forms , soluble or membrane - anchored forms , plays a pivotal role in signaling between degenerating neurons and microglia . cx3cl1 and its receptor cx3cr1 are highly expressed in brain tissue , particularly in neurons and microglia [ 4042 ] . cx3cl1 directly induces various microglial functions including migration , proliferation , inhibition of fas - ligand - induced cell death and glutamate - induced neurotoxicity [ 45 , 46 ] , and inhibition of proinflammatory cytokine production [ 42 , 47 ] . recently , we have shown that soluble form of cx3cl1 also directly enhances microglial clearance of degenerated neuronal debris , which is mediated through phosphatidylserine ( ps ) receptor and production of milk fat globule - egf factor 8 protein ( mfg - e8 ) ( figure 2 ) . the membrane - anchored cx3cl1 is cleaved by several proteases including a disintegrin and metalloprotease ( adam ) family , adam-10 and adam-17 [ 4850 ] , and cathepsin s . when neurons are injured or exposed to glutamate , shedding of cx3cl1 occurs immediately [ 41 , 52 ] . however , little is known about direct connection with a-induced neuronal toxicity and the cx3cl1-shedding . another important function of adam family enzyme , except for cx3cl1 shedding , is an -secretase . it cleaves app in the centre of the a domain , and generated -app is considered to have neurotrophic function [ 5356 ] . cathepsin s is expressed predominantly in microglia and implicated in microglial activation of neuropathic pain . therefore , these cx3cl1-shedding proteases may regulate the microglial phagocytosis directly or indirectly through cx3cl1 expression , and may also play a role on pathogenesis of ad . there are some reports showing that ccl2 activates cx3cr1 expression , and the induction of cx3cr1 expression by ccl2-ccr2 axis is mediated through p38 mapk activation . cx3cr1 deficiency increases susceptibility to neurotoxicity in mouse models of parkinson 's disease , amyotrophic lateral sclerosis , and systemic lps administration . cx3cl1-induced neuroprotection in a rat model of parkinson 's disease has also been reported recently . in addition , there are some reports showing that pathologic features of ad mouse model are worsened by knockout of cx3cr1 [ 17 , 61 ] . the receptor leading to inflammatory status includes cd14 , cd36 , the receptor for advanced glycation end products ( rages ) , and toll - like receptor ( tlr ) 1 , tlr2 , tlr4 , and tlr6 . the receptor leading to anti - inflammatory status includes triggering receptor expressed on myeloid cells 2 ( trem2 ) and ps receptor ( psr ) , mfg - e8 . microglia also express many other phagocytosis - related receptors which are not yet unclear to the correlation of inflammatory status . cr3microglia express classical phagocytosis - related receptor , the m 2 integrin complement - receptor-3 ( cr3 ; mac-1 ; cd11b / cd18 ) , and scavenger - receptor ( sr)-ai / ii ( sr - ai / ii ; cd204 ) . cr3 synergistically activated sr - ai / ii - mediated myelin phagocytosis by microglia [ 63 , 64 ] . cr3 is implicated in clearance of bacteria through induction of major histocompatibility complex class ii ( mhc ii ) antigens . cr3 is involved in endocytosis in normal conditions and mhc ii antigens in an inflammatory state . microglia express sr - ai / ii and sr - bi during neonatal period , while in adult they lack the expression . microglia express classical phagocytosis - related receptor , the m 2 integrin complement - receptor-3 ( cr3 ; mac-1 ; cd11b / cd18 ) , and scavenger - receptor ( sr)-ai / ii ( sr - ai / ii ; cd204 ) . cr3 synergistically activated sr - ai / ii - mediated myelin phagocytosis by microglia [ 63 , 64 ] . cr3 is implicated in clearance of bacteria through induction of major histocompatibility complex class ii ( mhc ii ) antigens . cr3 is involved in endocytosis in normal conditions and mhc ii antigens in an inflammatory state . microglia express sr - ai / ii and sr - bi during neonatal period , while in adult they lack the expression . cd14cd14 is the lps receptor which is also considered as classical phagocytosis - related receptor in macrophage and microglia . cd14-mediated phagocytosis does not require expression of ps receptor and possibly induces inflammatory conditions through activation of cd14 signaling . the authors suggested that it might be due to reduction of insoluble , but not soluble , a . cd14 is the lps receptor which is also considered as classical phagocytosis - related receptor in macrophage and microglia . cd14-mediated phagocytosis does not require expression of ps receptor and possibly induces inflammatory conditions through activation of cd14 signaling . the authors suggested that it might be due to reduction of insoluble , but not soluble , a . fcreceptorexposure of fibrillar a to microglia induces phagocytosis through the receptors distinct from those used by the classical phagocytosis : immunoglobulin receptors ( fcri and fcriii ) or complement receptors . exposure of fibrillar a to microglia induces phagocytosis through the receptors distinct from those used by the classical phagocytosis : immunoglobulin receptors ( fcri and fcriii ) or complement receptors . cd36 and cd47a directly interacted with microglial cell surface receptor complex comprising the class b scavenger receptor cd36 , 6 1 integrin , and integrin - associated protein cd47 , all of them involved in migration and phagocytosis of microglia [ 7072 ] . cd36 is required for fibrillar a-induced chemotaxis and proinflammatory molecules including reactive oxygen species ( ros ) , tnf , il-1 , and several chemokines in microglia . a activates microglial recruitment to amyloid deposition site through cd36-dependent signaling cascade involving the src kinase family members , lyn and fyn , and the erk1/2 . cd47 is a membrane glycoprotein , broadly expressed in the various cell types in the cns , including neurons and microglia . signal regulatory protein- ( sirp ; cd172a ) is a receptor binding to cd47 , which is also expressed on neurons and myeloid cells . sirp is an inhibitory molecule of cd47 that downregulates mapk phosphorylation which is a downstream pathway of cd47 . sirp interacts with the protein tyrosine phosphatases shp-1 and shp-2 , which are predominantly expressed in neurons , dendritic cells , and macrophages . therefore , cd47 functions normally as a marker of self to protect intact body component [ 76 , 77 ] . a directly interacted with microglial cell surface receptor complex comprising the class b scavenger receptor cd36 , 6 1 integrin , and integrin - associated protein cd47 , all of them involved in migration and phagocytosis of microglia [ 7072 ] . cd36 is required for fibrillar a-induced chemotaxis and proinflammatory molecules including reactive oxygen species ( ros ) , tnf , il-1 , and several chemokines in microglia . a activates microglial recruitment to amyloid deposition site through cd36-dependent signaling cascade involving the src kinase family members , lyn and fyn , and the erk1/2 . cd47 is a membrane glycoprotein , broadly expressed in the various cell types in the cns , including neurons and microglia . signal regulatory protein- ( sirp ; cd172a ) is a receptor binding to cd47 , which is also expressed on neurons and myeloid cells . sirp is an inhibitory molecule of cd47 that downregulates mapk phosphorylation which is a downstream pathway of cd47 . sirp interacts with the protein tyrosine phosphatases shp-1 and shp-2 , which are predominantly expressed in neurons , dendritic cells , and macrophages . therefore , cd47 functions normally as a marker of self to protect intact body component [ 76 , 77 ] . ragein has been reported that the direct interaction of a peptide with the receptor for rage is important in ad pathophysiology . in ad brains , rage expression is increased , and rage directly induces neurotoxicity by production of oxidative stressors and indirectly by activating microglia . rage increases macrophage - colony stimulating factor ( m - csf ) from neurons via nuclear - factor-b- ( nf-b- ) dependent pathway and released m - csf induced interaction of cognate receptor c - fms on microglia which prolongs survival of microglia . rage recognizes multiple ligands other than a peptide , such as advanced glycation end products ( ages ) , ps , and high - mobility group box 1 protein ( hmgb1 ) [ 80 , 81 ] . these molecules act as an agonist of rage on microglia , by inducing proinflammatory molecules , such as no , tnf- , il-1 , and il-6 , via map - kinase - kinase ( mek ) and phosphatidylinositol 3-kinase ( pi3k ) pathways . activation of rage leaded to nf-b and mapk - mediated signaling to propagate and perpetuate inflammation status . rage also mediates the transport of peripheral a into the brain across the blood - brain barrier ( bbb ) . in has been reported that the direct interaction of a peptide with the receptor for rage is important in ad pathophysiology . in ad brains , rage expression is increased , and rage directly induces neurotoxicity by production of oxidative stressors and indirectly by activating microglia . rage increases macrophage - colony stimulating factor ( m - csf ) from neurons via nuclear - factor-b- ( nf-b- ) dependent pathway and released m - csf induced interaction of cognate receptor c - fms on microglia which prolongs survival of microglia . rage recognizes multiple ligands other than a peptide , such as advanced glycation end products ( ages ) , ps , and high - mobility group box 1 protein ( hmgb1 ) [ 80 , 81 ] . these molecules act as an agonist of rage on microglia , by inducing proinflammatory molecules , such as no , tnf- , il-1 , and il-6 , via map - kinase - kinase ( mek ) and phosphatidylinositol 3-kinase ( pi3k ) pathways . activation of rage leaded to nf-b and mapk - mediated signaling to propagate and perpetuate inflammation status . rage also mediates the transport of peripheral a into the brain across the blood - brain barrier ( bbb ) . cd200rcd200 is a transmembrane glycoprotein and is expressed on many different cell types including neurons , endothelial cells , lymphocytes , and dendritic cells . the receptor of cd200 , cd200r expression , is predominant in myeloid cells , macrophages , and microglia [ 84 , 85 ] . as in the case of sirp-cd47 , cd200 exerts inhibitory effect on cd200r , so that cd200-cd200r interaction can downregulate activity of microglia . in retina , it has been shown that activation of cd200r in microglia does not show direct effect on migration , but cd200-cd200r signaling restores lps / ifn-induced loss of migration . cd200 knockout leads to an expansion of the myeloid population in several tissues and increased expression of the activation markers in microglia , including the signaling adaptor protein dnax - activating protein of 12 kda ( dap12 ) , cd11b , cd45 , cd68 , and inducible no synthase ( inos ) . these observations suggest that cd200-cd200r signaling leads to anti - inflammatory state and protection against neurotoxic stimuli . cd200 and cd200r expression levels ( neurons and microglia , resp . ) are decreased in ad hippocampus and inferior temporal gyrus , indicating that inhibition of cd200-cd200r axis contributes to ad pathology . cd200 is a transmembrane glycoprotein and is expressed on many different cell types including neurons , endothelial cells , lymphocytes , and dendritic cells . the receptor of cd200 , cd200r expression , is predominant in myeloid cells , macrophages , and microglia [ 84 , 85 ] . as in the case of sirp-cd47 , cd200 exerts inhibitory effect on cd200r , so that cd200-cd200r interaction can downregulate activity of microglia . in retina , it has been shown that activation of cd200r in microglia does not show direct effect on migration , but cd200-cd200r signaling restores lps / ifn-induced loss of migration . cd200 knockout leads to an expansion of the myeloid population in several tissues and increased expression of the activation markers in microglia , including the signaling adaptor protein dnax - activating protein of 12 kda ( dap12 ) , cd11b , cd45 , cd68 , and inducible no synthase ( inos ) . these observations suggest that cd200-cd200r signaling leads to anti - inflammatory state and protection against neurotoxic stimuli . cd200 and cd200r expression levels ( neurons and microglia , resp . ) are decreased in ad hippocampus and inferior temporal gyrus , indicating that inhibition of cd200-cd200r axis contributes to ad pathology . trem2trem2 , a recently identified innate immune receptor , and its adaptor protein dap12 are expressed on microglia and cortical neurons , but not on hippocampal neurons , astrocytes , and oligodendrocytes . their expression correlates with clearance of apoptotic neurons by microglia without inflammation [ 9092 ] . however , endogenous ligand or specific agonist of trem2 had not been identified until recently . heat shock protein 60 ( hsp60 ) is a mitochondrial chaperone , which interacts with trem2 directly . hsp60-induced phagocytosis is only found in microglia which have robust expression of trem2 . in ad model mouse , trem2 expression was highest in the outer zone in a plaques , and the expression level correlated with the size of a plaque . forced expression of trem2 positively regulated microglial phagocytosis , the ability of microglia to stimulate cd4 t - cell proliferation , tnf- and ccl2 production , but not ifn production . trem2 , a recently identified innate immune receptor , and its adaptor protein dap12 are expressed on microglia and cortical neurons , but not on hippocampal neurons , astrocytes , and oligodendrocytes . their expression correlates with clearance of apoptotic neurons by microglia without inflammation [ 9092 ] . however , endogenous ligand or specific agonist of trem2 had not been identified until recently . heat shock protein 60 ( hsp60 ) is a mitochondrial chaperone , which interacts with trem2 directly . hsp60-induced phagocytosis is only found in microglia which have robust expression of trem2 . in ad model mouse , trem2 expression was highest in the outer zone in a plaques , and the expression level correlated with the size of a plaque . forced expression of trem2 positively regulated microglial phagocytosis , the ability of microglia to stimulate cd4 t - cell proliferation , tnf- and ccl2 production , but not ifn production . tlrs are class of pattern - recognition receptors in the innate immune system to induce inflammatory responses . 13 tlrs have been identified in human and mouse to date , except for tlr10 which is expressed only in human [ 95 , 96 ] . cd11b , a marker of macrophages and microglia , has been shown to interact with tlr signaling . cd11b knockout mouse exhibited enhanced tlr - mediated responses and subsequent more susceptibility to endotoxin shock . among the tlrs , lps receptor tlr4 potently activates microglia in various aspects , such as a phagocytosis and proinflammatory molecules production [ 98 , 99 ] . activation of tlr1 , tlr2 , tlr3 , and tlr9 by each selective agonist also increased phagocytosis and several cytokines and chemokine production [ 98 , 100 ] . the response of microglia to fibrillar a is mediated via cd14 , which act together with tlr4 and tlr2 to bind fibrillar a and induced microglial activation through p38 mapk . deficiency of cd200 induces the expression of tlr2 and tlr4 in glial cells and proliferation of cd11b / mhcii / cd40 activated microglia . these mice show memory impairment , suggesting that dual activation of tlr2 and tlr4 may induce an inflammatory phenotype of microglia which negatively regulate synaptic plasticity in the ad model . a triggers the inflammatory status in microglia via heterodimer of tlr4 and tlr6 , which is regulated by cd36 . therefore , cd14-tlr2-tlr4 and cd36-tlr4-tlr6 signaling are crucial to a-induced inflammatory response , and also in microglial phagocytosis . bacterial dna containing motifs of unmethylated cpg dinucleotides ( cpg - dna ) is a ligand of tlr9 , which is initially identified to activate microglia and strongly induces tnf- and il-12 production . however , we have shown that cpg activated microglia to produce neuroprotective molecule , such as hemeoxygenase-1 ( ho-1 ) and matrix metalloproteinase 9 ( mmp-9 ) without producing neurotoxic molecules , such as tnf- , glutamate and nitric oxide ( no ) , and enhanced a clearance to protect memory disturbance in vivo . there are the discrepancies about cpg effect between aforementioned two reports despite using the same origin cells ( mouse primary microglia ) . it may be due to concentrations of this tlr9 ligand used : higher concentration ( 10 m ) in former report , whereas lower concentrations ( 1 to 100 nm ) in latter report . moreover , the latter report revealed the difference on neuroprotective effect of cpg among synthetic oligodeoxynucleotides ( odns ) classes ( a to c ) . class a cpg did not activate microglia , but classes b and c cpgs increased microglial neuroprotective effect through induction of clearance of a and production of neuroprotectant . cpg increased chemokine ccl9 and its receptor ccr1 expression in macrophages and microglia via tlr9/myd88 signaling involving erk , p38 mapk , and pi3k pathways . phagocytotic cells recognize apoptotic cells by several mechanisms , including recognition of ps expressed on the cells . the receptors of ps ( psrs ) had not been clarified for a long time but have uncovered in recent years . these include mfg - e8 ( the lactadherin homolog in humans ) and t - cell immunoglobulin mucin domain 4 ( tim4 ) [ 107 , 108 ] . these act as a bridge between ps - expressing apoptotic cells and psr expressing phagocytes and trigger engulfment of cellular debris . mfg - e8 is expressed on various macrophage subsets in the periphery and on microglia in the cns . recently , we have shown that cx3cl1 induces mfg - e8 expression in primary mouse microglia to lead to the microglial clearance of degenerated neuronal debris . others also reported the induction of mfg - e8 by cx3cl1 in macrophages and rat microglia [ 109 , 110 ] . mfg - e8 bridges ps and integrins v 3 or v 5 on the surface of phagocytes [ 107 , 111 ] . high - mobility group box 1 protein ( hmgb1 ) is an intracellular protein that activates transcriptional factors , including p53 and nf-b . hmgb1 reportedly suppresses the interaction between mfg - e8 and v 3 integrin in macrophage and inhibits the phagocytosis of apoptotic cells through erk - mediated signaling pathway . mfg - e8 may also be involved in a phagocytosis , since its expression is reduced in ad . therefore , mfg - e8 may possibly lead to targeted clearance of unwanted molecules , such as a , without inflammation . the other well - studied psr , tim4 , is expressed in mac-1 cells in various mouse tissues , including spleen , lymph nodes , and fetal liver . among the other tim family members , tim1 , but neither tim2 nor tim3 , also specifically binds to ps . it has been shown recently that rage also recognizes ps and induces apoptotic cell clearance . however as mentioned previously , rage - guided intracellular signaling pathway induces prolonged inflammatory status . -secretase is a protein complex of four essential membrane proteins : aph-1 , pen-2 , nicastrin , and presenilin . a recent study suggests that presenilin increases microglial phagocytosis of a , and this -secretase has dual role for ad pathogenesis : one is cleavage of amyloid precursor protein ( app ) to produce pathologic a , and the other is reduction of microglial phagocytosis of a by -secretase inhibitor . vertebrates have two presenilin genes , psen1 ( located on chromosome 14 in humans ; encoded presenilin 1 ) and psen2 ( located on chromosome 1 ; encoded presenilin 2 ) . there is a report that presenilin 2 is identified as the predominant -secretase in mouse microglia ( but not presenilin 1 ) , which repressed microglial activation via its function as a -secretase , and its expression is increased by inflammatory stimuli ( ifn- ) . in order to explore the detailed mechanism how -secretase regulates microglial activity , further studies are needed since -secretase is a therapeutic target for ad . in traumatic injury of the brain , presenilin and nicastrin expressions -secretase mainly cleaves app to lead to accumulation of a 1 - 42 , which then results in aggregation of a protein to worsen ad pathology . microglia express a wide array of receptors characteristic to immune cell , such as cd molecules , integrins , chemokine receptors , and psr ( figure 1 ) . chemokine receptors not only induce migration of microglia but also contribute directly to ad pathogenesis through regulation of phagocytosis and neuroprotective activity . ps acts as eat - me signal , and psr - mediated phagocytosis so far is regarded as inducing anti - inflammatory responses . however , according to some recent reports , rage interacts with psr and facilitates phagocytosis with robust inflammation status [ 80 , 120 ] . therefore , if the other phagocytosis - related receptors including tlrs interact with psr , microglia would be activated to outbreak excessive phagocytosis with robust inflammation . microglia from old app / ps1 mouse , but not from younger ones , show the reduction of sra , cd36 , rage , and the a-degrading enzymes including insulysin , neprilysin , and mmp9 . thus , it is important to consider the microglial status depending on the disease stage , to treat ad effectively . as shown in figure 2 , fkn and il-34 may be an intrinsic neuroprotectant for damaged but still surviving neurons through activation of microglia .

microglia are multifunctional immune cells in the central nervous system ( cns ) . in the neurodegenerative diseases such as alzheimer 's disease ( ad ) , accumulation of glial cells , gliosis , occurs in the lesions . the role of accumulated microglia in the pathophysiology of ad is still controversial . when neuronal damage occurs , microglia exert diversified functions , including migration , phagocytosis , and production of various cytokines and chemokines . among these , microglial phagocytosis of unwanted neuronal debris is critical to maintain the healthy neuronal networks . microglia express many surface receptors implicated in phagocytosis . it has been suggested that the lack of microglial phagocytosis worsens pathology of ad and induces memory impairment . the present paper summarizes recent evidences on implication of microglial chemotaxis and phagocytosis in ad pathology and discusses the mechanisms related to chemotaxis toward injured neurons and phagocytosis of unnecessary debris .

1. Introduction 2. Microglial Chemotaxis in CNS Injury 3. CX3CL1-CX3CR1 4. Microglial Receptors Involving Phagocytosis with or without Inflammation: Possible Implication to AD Pathology 5. TLRs 6. PSR 7. 8. Concluding Remarks

we conducted a qualitative study during september and november 2006 , in a peri - urban community on the outskirts of cape town , south africa . this township emerged during the last two decades because of rapid in - migration and is today a permanent fixture of formal and informal dwellings , where many of the younger generation are born in the township , but still maintain strong bonds to the rural areas of their family origin . generally , most people are living under very poor conditions with high unemployment rates , as well as high levels of crime , alcohol and drug use ( 23 , 24 ) . this qualitative study was undertaken in conjunction with a larger quantitative survey among men at high risk of hiv . the quantitative survey employed respondent - driven sampling ( rds ) to recruit participants ( 25 ) . rds is a chain - referral method that requires a pre - determined number of initial contacts and subsequent recruits to enlist a maximum of three new participants from their social network . the inclusion criteria for the quantitative survey were men older than 18 years , who had had more than one female sexual partner in the past 3 months , where at least one of these sexual partners was younger than 24 years , or 3 or more years younger than the participant . close to 56% had some high school education and 17.2% were unemployed . the majority ( 94.7% ) purposive sampling to identify individuals who were willing to participate in in - depth individual interviews was used among the participants in the quantitative study . twenty participants were selected and asked to participate in the qualitative component while they were waiting to complete the quantitative survey . interviews were conducted during weekends , as it was difficult to attract men to participate during the week . however , screening of the participants before the interviews was necessary to ensure that they were not under the influence of narcotics or alcohol , which is a common problem during weekends in the study setting . each interview took approximately one and a half hours and was conducted by the first author ( ar ) . a trained local interpreter was present during the interviews and translated the conversations into english and/or isixhosa when necessary . all interviews were audiotaped , transcribed verbatim and cross - checked with the initial recordings to ensure the quality of the transcription . participants were given a cellular telephone voucher worth 30 rand ( approximately us$4 ) as a token of appreciation . a thematic question guide ( tqg ) with open - ended questions was used during the interviews . the tqg provided a structure for the interviews while simultaneously allowing the interviewer to freely explore , probe and ask questions that would expand on or clarify particular topics . the tqg themes included sexual behaviours , social and sexual networks , masculinity and risk reduction strategies . the transcripts were analysed according to a latent content analysis suggested by graneheim and lundman ( 26 ) . descriptive , explicit areas of content with little attempt at interpretation were extracted first . these were examined for underlying meaning and situated in sub - themes that cut across categories . sub - themes were then grouped into overarching themes that expressed the latent content of the transcripts . data was discussed in detail by the research team to identify different themes for further analysis and to facilitate the uncovering of aspects of the underlying meaning , which contributed to the increased validity of the analysis . ethical clearance was obtained from the health sciences faculty research ethics committee at the university of cape town . this research was funded by the swedish research council , the swedish international development agency ( sida)/sarec and the national research foundation ( nrf ) , south africa . self - perceived social identities among hard - to - reach men in the study 's informal urban context suggested dominant masculine ideals and ways to legitimise specific behaviours that created a high - risk environment for sexually transmitted hiv . one common category among the study participants described reasons for having multiple , female sexual partners . themes that emerged and that promoted power imbalances in sexual relationships were lack of trust in women , disempowerment and biological determinism . the predominant ideal male identity was conceptualised in one word , the player , characterised by two symbols of status : wealth and women . thus , the player incorporated symbols believed to be important in the make - up of a hegemonic masculinity and clearly helped these men to position themselves in the community . money and material goods that could be visualised was crucial and seemed to override other status symbols such as education or societal position.it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king.you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king . you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . symbols such as cellular telephones , sunglasses and trendy clothes were important in overtly portraying economic status , and were used to position these men in relation to other men in the study context . in this manner , new and fashionable items became important in communication with other men and were key to forming hierarchical systems that incorporated specific forms of ideal masculinities . these symbols of material wealth also played an important role in men 's strategies to access sexual networks and sexual partners . besides displays of overt economic status , a player would be expected to show he could afford and handle several women at the same time . having multiple , often young female sexual partners further enhanced men 's social position and women served as a marker of both sexual and financial power.i can make an example about me because i have about eight girlfriends . it is my style and what i wear , my clothes and my money , because i have money.yes i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . it is my style and what i wear , my clothes and my money , because i have money . i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . seemingly , women responded to these strategies and aided in the socialisation of men 's sexual identities , with new gender power dynamics contrary to many of the previous norms in traditional initiation schools . furthermore , strong peer pressure to have many concurrent , young sexual partners played an important part in the creation of the masculine ideal within male social groups , manifesting in large sexual networks.i will take a lot of pressure from the boys . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all.other people will think that you do not have a game ( if not having multiple girlfriends ) . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all . other people will think that you do not have a game ( if not having multiple girlfriends ) . the pressure to live up to set norms further reinforced the meaning and status of the player . if a man adopted an alternative form of masculine ideal , he would risk being emasculated and thought not to have what it takes to be a real man according to prevalent norms in this specific context and group of men . it would seem that the masculinity norms that have evolved in the study context are making use of new symbols to express power that facilitate the redefinition of men in relation to other men thereby reinforcing risky sexual behaviours . the attributes suggested for a successful man also indicate that the representations of a man , like the player , exist and are integrated into a new urban form of masculinity . to explore forms of identities , underlying and associated reasons on different levels are needed to further explain why certain types of masculinity evolve . two themes closely associated with the player were biological determinism and frustration over a self - perceived disempowerment . the construction of the player not only involved the manifestation of material wealth and multiple girlfriends , but also offered a set of underlying reasons for gender - related power structures . irrespective of whether women had long - term concurrent or temporary sexual encounters , strong and negative views about them were revealed . a woman who crossed the strict boundaries that existed for gender relations was often labelled a bitch ; a term also sometimes used to describe women in general.they get a negative label , they are called bitches . they lose their respect and dignity.they look at them in a funny way . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . they get a negative label , they are called bitches . they lose their respect and dignity . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . by assessing women 's behaviour and what was perceived as unacceptable , men automatically felt that they had certain rights , which further legitimised a negative power play between men and women . applying restrictive social norms to certain female behaviours was not the only way to define men 's masculinity and to promote power imbalances . women were also considered physically vulnerable when engaging in sexual intercourse , especially with several sexual partners . biological determinism legitimised sexual risk taking among men , such as men having a greater need for several sexual partners , and being built for sexual encounters.the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished.it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it.because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished . it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it . because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . these biological explanations further reinforced strong , negative perceptions of women and female sexuality , which helped polarise men 's interpretation of gender constructions . the fact that women often challenged predominant gender stereotypes , by engaging in what was perceived to be normative male sexual behaviour , was used as an excuse for degrading attitudes and behaviours towards women . this could explain the rubbish can metaphor used for the vagina , and men 's association of women as being destroyed or thus , behaviours and the biology of women were themes that helped men to structure relations with women , which also promoted high - risk sexual behaviours in the form of multiple and concurrent sexual relations . another theme that occurred was that of an underlying frustration among these men that also reflected their position in society at large . bitches or women as passive victims , but rather as active agents who strategically played their cards . thus , a certain ambivalence was revealed in men 's views of women and the opposition between sexes.they are players like us because we think we play them and they are playing also their cards , they are playing us also.they ( women ) got so much power now . we got less power . [ ] because the women rights are too much . they are players like us because we think we play them and they are playing also their cards , they are playing us also . we got less power . [ ] because the women rights are too much . this underlying distress and insecurity among these men can be seen as a sign of a situation where the traditional hegemonic masculinity is contested , allowing new forms to evolve to maintain a certain power ( im)balance . the urban context characterised by lack of money and men 's self - perceived disempowerment in relation to women ( and society in general ) , created a situation where manhood was constantly questioned . many of the men believed that women actively engaged in concurrent transactional relationships for economic benefits . this interpretation of women as active agents in relationships created a deeply rooted insecurity among men that alienated them from women . the commonly expressed negative perceptions of women were thus multilayered and reflected an intricate power play that included lack of control and distrust towards women.according to me they can not keep one partner anymore because they do n't trust , that is why they are going around.they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . if i do not give you money they will go to the next person . according to me they can not keep one partner anymore because they do n't trust , that is why they are going around . they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . men 's distrust and a perceived disempowerment in relation to women supported the formation of large sexual networks characterised by unequal power dynamics . sexual relationships within these networks were often based on direct economic reciprocity , which is common in urban and peri - urban townships where people struggle to meet basic needs . the predominant ideal male identity was conceptualised in one word , the player , characterised by two symbols of status : wealth and women . thus , the player incorporated symbols believed to be important in the make - up of a hegemonic masculinity and clearly helped these men to position themselves in the community . money and material goods that could be visualised was crucial and seemed to override other status symbols such as education or societal position.it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king.you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . it is because these guys have a lot of money and they can afford things yes , and if you have money , you are the king . you must be well dressed and you must look brand new everyday even if your clothes are not new , and you must always have money and when she asks for something sometime , you must be able to take care of that . symbols such as cellular telephones , sunglasses and trendy clothes were important in overtly portraying economic status , and were used to position these men in relation to other men in the study context . in this manner , new and fashionable items became important in communication with other men and were key to forming hierarchical systems that incorporated specific forms of ideal masculinities . these symbols of material wealth also played an important role in men 's strategies to access sexual networks and sexual partners . besides displays of overt economic status , a player would be expected to show he could afford and handle several women at the same time . having multiple , often young female sexual partners further enhanced men 's social position and women served as a marker of both sexual and financial power.i can make an example about me because i have about eight girlfriends . it is my style and what i wear , my clothes and my money , because i have money.yes i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . it is my style and what i wear , my clothes and my money , because i have money . i can say that these guys are looked upon , even when ladies see that you like girls and have a lot of girls and you have friends who are girls . they will bring you more girls even if they know that you have many girls already . seemingly , women responded to these strategies and aided in the socialisation of men 's sexual identities , with new gender power dynamics contrary to many of the previous norms in traditional initiation schools . furthermore , strong peer pressure to have many concurrent , young sexual partners played an important part in the creation of the masculine ideal within male social groups , manifesting in large sexual networks.i will take a lot of pressure from the boys . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all.other people will think that you do not have a game ( if not having multiple girlfriends ) . they will tease and make funny jokes and tell me that having one girlfriend is the same as having no one at all . other people will think that you do not have a game ( if not having multiple girlfriends ) . the pressure to live up to set norms further reinforced the meaning and status of the player . if a man adopted an alternative form of masculine ideal , he would risk being emasculated and thought not to have what it takes to be a real man according to prevalent norms in this specific context and group of men . it would seem that the masculinity norms that have evolved in the study context are making use of new symbols to express power that facilitate the redefinition of men in relation to other men thereby reinforcing risky sexual behaviours . the attributes suggested for a successful man also indicate that the representations of a man , like the player , exist and are integrated into a new urban form of masculinity . to explore forms of identities , underlying and associated reasons on different levels are needed to further explain why certain types of masculinity evolve . two themes closely associated with the player were biological determinism and frustration over a self - perceived disempowerment . the construction of the player not only involved the manifestation of material wealth and multiple girlfriends , but also offered a set of underlying reasons for gender - related power structures . irrespective of whether women had long - term concurrent or temporary sexual encounters , strong and negative views about them were revealed . a woman who crossed the strict boundaries that existed for gender relations was often labelled a bitch ; a term also sometimes used to describe women in general.they get a negative label , they are called bitches . they lose their respect and dignity.they look at them in a funny way . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . they get a negative label , they are called bitches . they lose their respect and dignity . they call them bitches , it is a girl who is always sleeping with different partners , this is the girl i always have sex with , a girl that i use . by assessing women 's behaviour and what was perceived as unacceptable , men automatically felt that they had certain rights , which further legitimised a negative power play between men and women . applying restrictive social norms to certain female behaviours was not the only way to define men 's masculinity and to promote power imbalances . women were also considered physically vulnerable when engaging in sexual intercourse , especially with several sexual partners . biological determinism legitimised sexual risk taking among men , such as men having a greater need for several sexual partners , and being built for sexual encounters.the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished.it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it.because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . the thing is that you have lots of girls , you finish the girls , but the girl who has many boys is stupid because she is the one who is being finished . it is because of our different sexual orientation where guys deposit and ladies receive . because this , ( the vagina ) , looks like a rubbish can where we throw everything in it . because the girl 's body is destroyed pretty easy when she has a lot of men , and the guy 's body does not deteriorate that easy with many girlfriends . these biological explanations further reinforced strong , negative perceptions of women and female sexuality , which helped polarise men 's interpretation of gender constructions . the fact that women often challenged predominant gender stereotypes , by engaging in what was perceived to be normative male sexual behaviour , was used as an excuse for degrading attitudes and behaviours towards women . this could explain the rubbish can metaphor used for the vagina , and men 's association of women as being destroyed or thus , behaviours and the biology of women were themes that helped men to structure relations with women , which also promoted high - risk sexual behaviours in the form of multiple and concurrent sexual relations . another theme that occurred was that of an underlying frustration among these men that also reflected their position in society at large . the informants did not view bitches or women as passive victims , but rather as active agents who strategically played their cards . thus , a certain ambivalence was revealed in men 's views of women and the opposition between sexes.they are players like us because we think we play them and they are playing also their cards , they are playing us also.they ( women ) got so much power now . we got less power . [ ] because the women rights are too much . they are players like us because we think we play them and they are playing also their cards , they are playing us also . we got less power . [ ] because the women rights are too much . this underlying distress and insecurity among these men can be seen as a sign of a situation where the traditional hegemonic masculinity is contested , allowing new forms to evolve to maintain a certain power ( im)balance . the urban context characterised by lack of money and men 's self - perceived disempowerment in relation to women ( and society in general ) , created a situation where manhood was constantly questioned . many of the men believed that women actively engaged in concurrent transactional relationships for economic benefits . this interpretation of women as active agents in relationships created a deeply rooted insecurity among men that alienated them from women . the commonly expressed negative perceptions of women were thus multilayered and reflected an intricate power play that included lack of control and distrust towards women.according to me they can not keep one partner anymore because they do n't trust , that is why they are going around.they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . if i do not give you money they will go to the next person . according to me they can not keep one partner anymore because they do n't trust , that is why they are going around . they are also players , because the thing is unemployment rate , if that can be organized to get jobs , maybe they can start their lives themselves . if i do not give you money they will go to the next person . men 's distrust and a perceived disempowerment in relation to women supported the formation of large sexual networks characterised by unequal power dynamics . sexual relationships within these networks were often based on direct economic reciprocity , which is common in urban and peri - urban townships where people struggle to meet basic needs . we found challenges to hiv prevention among men living in an urban south african township that urgently need to be addressed . the dominant masculine ideal , the player , thrived on money , multiple concurrent sexual relations and casual sex . strong social pressure within male core groups to pursue and maintain these concurrent sexual relationships and temporary sexual encounters existed , and helped legitimise specific behaviours that the player represented . the common use of derogatory words attributed to women or their genitals , such as bitch and rubbish can , dehumanised women and restricted female sexuality in order to retain , and in some instances , reclaim male superiority . women were perceived as too empowered and could not be trusted , making men feel alienated and lacking control in ( sexual ) relationships . the lack of trust in women 's fidelity was stated as an important reason for engaging in concurrent sexual relationships as well as casual sexual encounters , which is known to be a key driver of hiv transmission ( 10 ) . our findings thus support previous research showing that dominant masculinities can be characterised by large sexual networks as a means to express manhood and as a response to societal changes , unemployment and poverty , low self - esteem and perceived disempowerment ( 27 ) . the representation of a man and his associated attributes has evolved and been re - shaped into new sets of meanings , where traditional social expectations of conservative , restrained sexuality have largely changed . in this particular context , the ways in which manhood is defined has clearly put men and women at increased risk for sexually transmitted hiv as these gender structures profoundly influence men 's sexual identity , and how sexuality is used to manifest power , not least between men themselves . an urban and modern way of sexual socialisation that incorporates an ideal masculinity as the player , poses a clear risk that needs to be addressed . one potential way in which this risk could be addressed is during traditional initiation rituals that are key to the sexual socialisation of boys into men . the practice continues to be an important part of many young men 's transition to adulthood and should thus be revisited for its potential in integrating gender - related hiv prevention . previous research and designed interventions show weak support for the scaling up of traditional male circumcision as a biomedical intervention ( 28 ) , but that medical circumcision together with traditional initiation could be promising ( 29 ) . in the global debate , the potential benefits of circumcision in relation to hiv infection has largely had a biomedical focus , thereby ignoring the very important core of traditions and the context in which it is stipulated . by taking into account the traditional importance of rites of passage , the power of a successful intervention might not be solely in the removal of the foreskin , but rather in the development of structures in which boys can be sexually and gender socialised into responsible men . the potential in bridging traditional systems with medical interventions has shown promising results and is currently recommended ( 30 ) . however , further research is required to examine the effectiveness of bridging medical and traditional interventions as well as to assess potential harm reduction associated with , for example , circumcision ( 28 ) . traditional structures could be one such entry point as they are important and give meaning to people . this research was based on a selected group of men at high risk of sexually transmitted hiv in one specific urban environment in south africa . self - reported data does not fully explore underlying structures of social norms . however , these in - depth interviews gave the interviewees an opportunity to describe the quality of their social and sexual relationships and shed light on the normative systems that legitimise their behaviours in this peri - urban settlement . these norms represent a masculine ideal supported by males and accepted in society at large . this was a unique study in terms of the high - risk context in which it was conducted , and we believe we managed to reach males who are normally difficult to research , but whose behaviours are key to explaining the extremely high hiv prevalence in south african townships . although findings might only be representative of this group of men , we believe that this unique opportunity to reach men in this harsh urban setting provides important new knowledge on how contemporary masculine ideals affect gender dynamics that need to be addressed in hiv prevention . our results highlight the need to more firmly address sexuality and gender dynamics among men in the growing informal urban areas with strikingly high hiv prevalence in south africa . an understanding of a dominant urban masculinity and how key characteristics of that masculinity affect hiv transmission , as well as more innovative interventions that can help endorse alternative norms and behaviours is urgently needed . traditional and new structures might potentially serve as focal entry points for future preventive actions , where prevention efforts should focus on changing underlying masculine ideals and gender relations that promote and maintain concurrent and temporary sexual relationships as well as other high - risk behaviours . future research should try to explore the potential for using traditional structures in intervention strategies by testing innovative intervention models . the authors have not received any funding or benefits from industry to conduct this study .

backgroundthe perspectives of heterosexual males who have large sexual networks comprising concurrent sexual partners and who engage in high - risk sexual behaviours are scarcely documented . yet these perspectives are crucial to understanding the high hiv prevalence in south africa where domestic violence , sexual assault and rape are alarmingly high , suggesting problematic gender dynamics.objectiveto explore the construction of masculinities and men 's perceptions of women and their sexual relationships , among men with large sexual networks and concurrent partners.designthis qualitative study was conducted in conjunction with a larger quantitative survey among men at high risk of hiv , using respondent - driven sampling to recruit participants , where long referral chains allowed us to reach far into social networks . twenty in - depth , open - ended interviews with south african men who had multiple and concurrent sexual partners were conducted . a latent content analysis was used to explore the characteristics and dynamics of social and sexual relationships.resultswe found dominant masculine ideals characterised by overt economic power and multiple sexual partners . reasons for large concurrent sexual networks were the perception that women were too empowered , could not be trusted , and lack of control over women . existing masculine norms encourage concurrent sexual networks , ignoring the high risk of hiv transmission . biological explanations and determinism further reinforced strong and negative perceptions of women and female sexuality , which helped polarise men 's interpretation of gender constructions.conclusionsour results highlight the need to address sexuality and gender dynamics among men in growing , informal urban areas where hiv prevalence is strikingly high . traditional structures that could work as focal entry points should be explored for effective hiv prevention aimed at normative change among hard - to - reach men in high - risk urban and largely informal contexts .

Methods Results The player rules a masculine ideal promoting high-risk behaviours The rubbish can the influence of biological determinism Player and bitches disempowerment, frustration and lack of trust Discussion Conclusion Conflict of interest and funding

in the previous issue of critical care , chase and coworkers reported on their implementation into clinical practice and evaluation of the specialized relative insulin nutrition table ( sprint ) . this is an improved protocol in the form of a wheel - based system to control blood glucose levels and nutritional intakes in intensive care patients , which was developed a few years ago . blood glucose has become a key biological parameter in critical care since publication of the study conducted by van den berghe and colleagues , who demonstrated decreased mortality in surgical intensive care patients in association with tight glycaemic control ( tgc ) , based on intensive insulin therapy . however , two negative studies were recently reported , which were interrupted early because of high rates of severe hypoglycaemia , namely the visep study and the as yet unpublished glucontrol trial . hence , there is currently much debate regarding the actual benefits of such a strategy in intensive care patients in terms of outcomes . it is also uncertain whether the results of the ongoing multicentre , open label , randomized controlled trial nice sugar of the effects of blood glucose management on 90-day all - cause mortality in a heterogeneous population of intensive care unit ( icu ) patients will resolve remaining concerns about tgc in the icu . included among these concerns is the key issue of what is the most appropriate algorithm to achieve the desired blood glucose range . the major focus of the study conducted by chase and colleagues was on the method to achieve a predetermined blood glucose range by modulating both insulin infusion rate and nutritional inputs . as with the other reported studies comparing protocols , efficacy was evaluated by comparison with historical control patients . however , although the study reported by chase and coworkers was conducted with great care and rigour , it is but another case - control retrospective comparative study . nevertheless , there is a clear need to introduce efficient tools that will help clinicians and nursing staff to control blood glucose levels in icu patients , because hyperglycaemia superior to 10 mmol studies are required to provide clinicians with recommendations on the evaluation and comparison of the various protocols currently in use or that are soon to become available . benchmarking of tgc protocols must take in account all the dimensions of efficiency : performance , risk for severe hypoglycaemia , practical aspects ( ease of use , training time and required materials prior to implementation , error rate ) , integrated continuous monitoring , nursing workload ( evaluated on the mean time between controls ) . furthermore the best way to compare performance is controversial : is it the time with glucose within a common target range , the hyperglycaemia index , the recently described glycaemic penalty index , or the variability that would be associated with outcome ? this raises the question of whether the efficacy results from instructions regarding nutritional intake , allowing insulin infusion rates to be limited to a level lower than usual , or from the intrinsic quality of the algorithm used , which is based on the glucose - insulin regulatory system model ( capturing insulin utilization rate , insulin losses and saturation dynamics ) . also , sprint is apparently associated with few severe hypoglycemia events , which contrasts with the high rate of severe hypoglycaemic episodes reported in the second leuven study . finally , sprint should be relatively simple to implement in numerous icus as a paper - based protocol , presented in an original form using a wheel , without need for computational resources . weaknesses of sprint rest in its inability to monitor parameters related to the quality of glucose control as sprint is a paper - based protocol . most importantly , despite the favourable subjective opinions of care givers , sprint may not reduce workload because it requires measurements every hour or 2 hours . ultimately , evaluation of any tgc protocol must also include an assessment of its ability to be implemented easily and safely in another icu that did not participate in its development . the monocentric study of chase and coworkers may not ensure the ' exportability ' of their tgc protocol . the debate continues about the real benefits of tgc , with numerous questions being asked . what is the optimal target range ? which patients will benefit the most ? when during the icu stay should tgc be applied and to derive which benefits ? which is the best method to control glucose level intensive insulin therapy , and/or limitation of nutritional intakes during acute phase , and/or antidiabetic drugs ? however , the competition to develop the ideal tool with which to control blood glucose levels in the icu and perhaps throughout the hospital stay has begun , involving multidisciplinary teams of physicians and engineers who have specialized in control systems ( feedback control or model predictive control ) . icu = intensive care unit ; sprint = specialized relative insulin nutrition table ; tgc = tight glycaemic control . pk declares that he holds shares of lk2 ( saint - avertin , france ) .

the report by chase and coworkers in the previous issue of critical care describes the implementation into clinical practice of the specialized relative insulin nutrition table ( sprint ) for tight glycaemic control in critically ill patients . sprint is a simple , wheel - based system that modulates both insulin rate and nutritional inputs . it achieved a better glycaemic control in a severely ill critical cohort than their previous method for glycaemic control in a matched historical cohort . reductions in mortality were also observed .

None Abbreviations Competing interests

foot and mouth disease ( fmd ) is one of the highly contagious diseases of domestic animals , caused by foot - and - mouth disease virus ( fmdv ) , a member of the family picornaviridae , which has a colossal global impact , due to the huge number of animals affected . the fmd endemic countries collectively contain three - quarters of the world 's population ( robinson et al . , 2011 ) , indicating the global economic significance of the disease . at the national level in india , annual total economic loss due to fmd ranges from rs 12,000 crore to rs 14,000 crore ( singh et al . , 2013 ) . in spite of all the control measures taken , fmd continues to be an economically important disease in india due to poor surveillance and inadequate control programs . understanding of the mechanism of foot - and - mouth disease virus ( fmdv ) infection and replication of this virus is important to the control of this worldwide menace ( alexandersen et al . , 2003 , longjam et al . , 2011 ) . a vital question that has yet to be addressed concerns the role of viral receptors in the pathogenesis of fmd . integrins are the biologically important set of proteins used by the cells to bind and respond to the extracellular matrix which belong to a large family of integral membrane receptors that is required for cell adhesion . functionally active integrins consist of two noncovalently bound transmembrane glycoprotein subunits viz , alpha ( ) and beta ( ) ( springer , 2002 ) . evidence suggested that virus binding and infection of the integrin 6 ( itgb6 ) transfected cells are mediated through an rgd - dependent interaction ( baxt and becker , 1990 , jackson et al . , 2000 , mason et al . , 1994 ) . considering the role of host itgb6 receptor gene , the aim of the present study was to screen genetic variation within a snp ( rs136500299 ) at exon 14 region among different indigenous cattle . the polymorphism t / c is located at position 2145 of the reference itgb6 mrna and produces a missense change phe / ser in the position 667 of the polypeptide . previously , this polymorphism has been reported at least ten times : ss250661448 , ss263658018 , ss415630763 , ss420604742 , ss422472086 , ss679762826 , ss752601716 , ss828200981 , ss907724252 , and ss942564461 . in this study , we describe an accurate method on the basis of competitive polymerase chain reaction ( pcr ) so called tetraplex arms pcr , for identification of the snp among different animals . for analyzing the scenario of targeted snp among different zebu cattle , a total of 148 animals of different indigenous breeds of cattle { sahiwal ( 51 ) , kankrej ( 48 ) , ongole ( 38 ) and gir ( 11 ) } from different agricultural zones were used in the study . blood samples were collected from all the animals by jugular vein puncture using sodium heparin ( 10 iu / ml ) as an anticoagulant . immediately after collection , blood samples were stored in a portable refrigerator at 4 c , transported to the laboratory , and stored at 80 c until dna extraction . chloroform extraction method ( sambrook and russell , 2001 ) and the purity of genomic dna was assessed spectrophotometrically . the tetra - primer pcr procedure ( ye et al . , 2001 ) was used for genotyping the snp ( rs136500299 ) at exon 14 region of itgb6 receptor gene . the method employs four primers to amplify a fragment from dna containing the snp and amplicons representing each of the two allelic forms . primers can be designed to amplify fragments of differing sizes for each allele band in order for them to be easily resolved using agarose gel electrophoresis . details of the primer used for the present study and the amplicon size for different genotypes were shown in table 1 . pcr was performed in a total volume of 25 l containing approximately 50 ng dna , 2.5 l of 10x buffer , 2.0 mm mgcl2 , 0.2 mm dntps , 5 pmol of each outer primers , 10 pmol of each inner primers and 1 u of taq polymerase ( sigma aldrich , usa ) . the polymerase chain reaction ( pcr ) protocol was 94 c for 5 min , followed by 35 cycles of 94 c for 30 s , annealing at 55 c for 30 s and 72 c for 30 s , and a final extension at 72 c for 10 min . the pcr products were separated on 1.0% agarose gel ( sigma aldrich , usa ) including 0.5 g / ml of ethidium bromide and photographed under gel documentation system ( alpha imager ep ) . validation of the three different genotypes was analyzed by cloning and sequencing the 433 bp pcr products for each genotype . gene ( allele ) and genotype frequencies of itgb6 receptor gene were calculated by direct counting method . the four populations were tested for hardy weinberg equilibrium using proc allele ( sas inst . cary , nc ) . the chi - square ( ) test was used to find the difference in genotype frequencies in different breeds . the present study was aimed to develop a single tube tetraplex pcr based genotyping assay for snp ( rs13650029 ) at exonic region of bovine itgb6 receptor gene . snp genotyping techniques depend on amplification of the target dna using pcr technique , but differ in the means of discerning between the different alleles , which involves significant post - amplification manipulations . for instance , the restriction fragment length polymorphism typing method based on digestion of amplified pcr products with suitable restriction endonuclease . another extensively used snp typing technique allele specific oligonucleotide ( aso ) melting involves lengthy blotting and hybridization techniques . tetra - primer arms pcr method described in the present context circumvents the limitations of the earlier mentioned techniques . the technique used in the present study involves a single step pcr protocol with two sets of primers to detect the different banding pattern , without the downstream processing like re digestion or hybridization . the primers were designed in such a way to amplify fragments of differing sizes for each allele , in order to resolve differentially in agarose gel electrophoresis . the method described in the present study is a simple , swift and cost - effective method for snp genotyping in large number of individuals ( ye et al . , 2001 ) . correspondence between arms pcr and sequencing confirmed three genotype pattern of the targeted snp at exon 14 region of bovine itgb6 gene among different zebu cattle breeds ( fig . 1 ) . genotyping revealed that , the genotype tt is widely distributed among the targeted zebu cattle breeds . the study revealed that the frequency of t allele was higher in indigenous populations compared to the c allele . this trend was mostly seen in all the breeds except sahiwal where the heterozygotic frequency was comparatively higher , even though the difference is not statistically significant ( table 2 ) . the test for hw equilibrium among different populations showed that all the breeds were under equilibrium with respect to the itgb6 receptor gene indicating the absence of aggressive selection i.e. selection primarily based on this gene ( p > 0.05 ) . the frequency of animals having cc genotype was lowest in all the four breeds . in order to study the variation in the genotype frequencies among different indigenous breeds of cattle , the result showed in table 3 indicates that the genotype frequencies were not statistically different ( = 7.900 , p = 0.2455 ) among the breeds indicating that all the populations are having similar genetic constitution with regard to the itgb6 receptor gene . the snp ( rs136500299 ) t / c located at position 2145 of the reference itgb6 mrna produces a missense change phe / ser . serine is a polar amino acid with its smaller size , whereas phenylalanine is an aromatic amino acid with complex structure . thus it may be presumed that changes of amino acid may alter the conformational changes of the itgb6 receptor coding polypeptide , which however , needs to be confirmed through further studies . the results of the present study suggest that t allele is widely distributed among the indigenous breeds of cattle which can be associated with the resistance to fmd virus , as the susceptibility to fmd virus is lower than the tauras breeds . however , studies with larger sample sets and wide range of cattle breeds are still needed to confirm the exact genetic distribution pattern of the snp .

the present study was aimed to screen genetic variation within a snp ( rs136500299 ) located at exon 14 region of bovine itgb6 gene among different zebu cattle breeds . the genotyping method describe in the present study is a tetraplex arms pcr , which offers extremely fast , economical , and simple detection tool . the distribution of the itgb6 genotypes among the different breeds studied suggested that the populations were under hardy weinberg equilibrium . our findings revealed that tt genotypes are widely distributed among different zebu cattle breeds , which can be associated with the resistance to fmd virus , as the bos indicus are more resistant to fmd virus in comparison to bos taurus .

Introduction Materials and methodology Results and discussion

huntington 's disease ( hd ) is a progressive , autosomal dominantly inherited neurodegenerative disorder , characterized by impaired motor control , cognitive decline , and occasional psychiatric illness . hd results from the expansion of a cag repeat in exon 1 of the huntingtin ( htt ) gene , yielding a protein with a polyglutamine ( polyq ) expansion tract near its amino terminus . this polyq - expanded huntingtin protein has a propensity to misfold , making it resistant to proteasomal and autophagy - mediated degradation . earlier age of disease onset and more rapid disease progression correlate with increasing cag repeat length , with hd manifesting in patients carrying 40 or more cag repeats . hd is the most common polyq repeat disease , with a prevalence of at least 1 in 10,000 in the usa and europe . in hd , polyq - expanded huntingtin protein is expressed throughout the brain , with the most severe degeneration occurring in striatal medium spiny neurons and cortical projection neurons that extend to the striatum . currently , hd research is conducted in rodent models , rodent primary neurons , and non - neural human cell lines , but there are questions as to whether such systems yield findings that are truly relevant to the mechanistic basis of human hd pathogenesis . about six years ago , takahashi and yamanaka developed a method for reprogramming human somatic cells so that they entered a pluripotent state , allowing for further differentiation into other cell types , including neurons . this induced pluripotent stem cell ( ipsc ) approach has ushered in a new era of human neurodegenerative disease modeling . two recent publications , one from the hd ipsc consortium and another by an et al . , respectively , reported the generation and characterization of ipsc - derived models for hd and the genetic correction of a disease - causing cag repeat expansion mutation in ipscs from individuals with hd . together , these two studies provide important insights into the utility and limitations of ipsc modeling of neurodegenerative disease . one of the greatest challenges facing the ipsc modeling field is the enormous variability of different ipsc lines and the neurons derived from them . as generation and characterization of ipsc lines can be laborious and costly , one obvious solution to assure the validity of ipsc disease models is to create research teams that work together on a particular disorder , sharing ipsc lines and derived neurons . this very strategy was applied in the hd ipsc consortium study , in which eight different groups produced ipscs from three individuals with hd and three control individuals and used them to assay a wide range of phenotypes . these included gene expression , cell adhesion , bioenergetics , glutamate toxicity , cell death , calcium flux , and trophic factor withdrawal . these phenotypes were assessed in neural stem cells ( nscs ) grown in spherical aggregates under defined growth factor conditions , as well as in differentiated striatal - like neurons obtained using both long and short differentiation protocols , which differed in the growth factors that were added . this comprehensive inventory of cellular and molecular assays revealed that certain phenotypes correlate with hd disease cag repeat length , whereas other phenotypes do not . the common results that were recapitulated across the various laboratories highlight the integrity of the developed ipsc lines , making the hd ipsc consortium study an impressive blueprint that is worthy of emulation . the study by an et al . had quite a different goal : to perform genetic correction in an hd - ipsc line , thereby creating an isogenic revertant ipsc line that would carry two normal - length htt alleles . these ' corrected ' controls had a genetic background that was identical to that of the ' uncorrected ' ipsc line , so any phenotypic or expression differences between the two could be solely attributed to the cag expansion . the ' corrected ' line was created successfully using a standard recombination approach , allowing the comparison of a number of cellular and molecular characteristics between the mutant progenitor line and the normalized derivative line . this study also demonstrated that the genetically corrected hd - ipsc line could be used to produce nscs using a protocol described previously by this group . furthermore , these nscs could be successfully transplanted into the striatum of hd mice , were able to populate this crucial brain region , and then underwent proper differentiation into neurons and glia . thus , the stage is set for the potential future application of this strategy as a therapeutic intervention in hd . in tandem , these papers catalogue a variety of phenotypes that were observed in hd - ipsc - derived neural progenitors and in medium spiny neurons . both groups performed gene expression analysis to compare hd - ipscs and their neuronal derivatives . the hd ipsc consortium findings implicated genes that are involved in cell signaling , cell cycling , axon guidance and neuronal development as dysregulated in nscs ( figure 1 ) . importantly , medium cag repeat expansions and longer cag repeat expansions expressed most of these genes differently . an and co - workers performed transcript expression analysis , although they did this in ipscs rather than in ipsc - derived nscs . they observed that , in comparison with corrected controls , hd - ipscs showed increased expression of genes that are involved in tgf- signaling and decreased expression of genes involved in cadherin signaling ( figure 1 ) . notably , the expression differences between the hd - ipscs and corrected hd - ipscs were of an order of magnitude less than those between non - related control ipscs and hd - ipscs . hd - ipscs , corrected ipscs , and ipsc - derived neurons reveal key disease - associated phenotypes . an et al . also generated corrected hd - ipscs by homologous recombination and showed that , when compared to corrected hd - ipscs , hd - ipscs have increased expression of genes that are induced by the tgf- signaling pathway and decreased expression of cadherin pathway genes . hd - ipsc lines developed by the hd ipsc consortium were further differentiated to form npcs and darpp-32-positive striatal - like neurons . these npcs yielded evidence for the altered expression in hd of genes that are involved in cell signaling , cell cycling , axon guidance , and neurodevelopmental pathways . as delineated here , future studies might compare these two datasets and thus could evaluate the expression of the target genes of those transcription factors known to interact with huntingtin , examine mitochondrial dysfunction in hd and investigate the exact identity of the dying cells in hd . npc , neural progenitor cell ; ros , reactive oxygen species ; tf , transcription factor . comparison of the two studies also highlights the shared relevance of certain cellular and molecular phenotypes , such as cell death and mitochondrial dysfunction ( figure 1 ) . initial studies had previously indicated that hd - ipsc - derived nscs display increased caspase activity , and that both hd - ipscs and derived neurons exhibit increased lysosomal activity . an important finding that is shared by the two groups relates to defects in energy metabolism . mitochondrial dysfunction , resulting in impaired cellular bioenergetics , is an emerging hallmark of hd . the hd ipsc consortium reported decreased intracellular atp levels in hd neural progenitor cells ( npcs ) , and an et al . both of these phenotypes revealed defects in energy metabolism in the hd models , but the importance of mitochondrial dysfunction requires further investigation . another hd - associated phenotype that was observed by both groups is increased cell death . the hd ipsc consortium used a variety of assays , including longitudinal survival tracking , nuclear condensation assays , and caspase 3/7 activation , to detect increased cell death in hd - nscs and striatal - like derivatives . an and colleagues also observed cag repeat - length - dependent cell death in similar assays . both groups demonstrated a more severe cell death phenotype upon withdrawal of brain - derived neurotrophic factor ( bdnf ) , adding to the mounting evidence to support a role for bdnf - associated toxicity in hd . bdnf is a secreted neurotrophin , which undergoes transcriptional repression in certain hd models , and there are indications that over - expression of bdnf can ameliorate the hd phenotype . cell death in the striatum and cortex are hallmarks of hd ; hence , this shared observation is significant , as it recapitulates in vitro what is seen in vivo . an et al . have taken an initial step toward the application of corrected hd - ipsc - derived neurons in cell replacement therapy . they showed that differentiated striatal - like neurons can survive transplantation into mouse brain , but they did not report whether transplanted r6/2 mice exhibited any recovery in phenotype . despite this uncertainty , it is quite clear that the newly derived hd - ipsc models hold great potential for use in drug screening . as illustrated in figure 1 , future studies should examine effects on htt interactor target genes , and compare the results so obtained with the data sets from these two studies . the observations from both studies regarding gene expression and mitochondrial dysfunction support findings generated in other models . the huntingtin protein is known to interact with a number of transcription factors and co - activators , including creb - binding protein ( cbp ) , sp1 , tafii130 , and ppar co - activator 1 ( pgc-1 ) . follow - up analysis of the expression of the target genes that encode these already - implicated transcription factors might be worthwhile ( figure 1 ) . diminished pgc-1 function appears to be a major contributor to hd pathogenesis , so the hd - ipsc model could be used to evaluate the mitochondrial phenotypes seen in hd mice , including decreased mitochondrial complex activities , decreased mitochondrial number , and increased oxidative stress . pgc-1 overexpression can rescue hd - associated phenotypes in mice by inducing transcription factor eb ( tfeb ) , a master regulator of autophagy . validation of the role of pgc-1 and tfeb dysfunction in hd - ipsc models would be worthwhile and could facilitate the development of new therapies . finally , a missing piece of the puzzle is the exact identity of the dying cells in hd . the hd ipsc consortium used a method for tracking live cells through longitudinal studies , which allowed them to identify the dying cells as being morphologically similar to neurons , although the specific neural type was unspecified . as hd patients with longer cag repeats suffer more widespread neuron cell death , it will be important to determine if the vulnerability of ipsc - derived neurons differs between neural cell types ( figure 1 ) . amelioration of cell death in a relevant ipsc - derived neural lineage could be a useful indicator of potential drug efficacy . once the hd field homes in on viable lead compounds , hd - ipsc models could serve as invaluable tools for validation and should accelerate therapeutic development . bdnf : brain - derived neurotrophic factor ; hd : huntington 's disease ; htt : huntingtin gene ; ipsc : induced pluripotent stem cell ; nsc : neural stem cell . the authors acknowledge funding support from the nih ( r01 ns065874 ) for their huntington 's disease research .

deconstructing the mechanistic basis of neurodegenerative disorders , such as huntington 's disease ( hd ) , has been a particularly challenging undertaking , relying mostly on post - mortem tissue samples , non - neural cell lines from affected individuals , and model organisms . two articles recently published in cell stem cell report first the generation and characterization of induced pluripotent stem cell ( ipsc)-derived models for hd , and second , the genetic correction of a disease - causing cag expansion mutation in ipscs from individuals with hd . taken together , these two studies provide a framework for the production and validation of ipsc materials for human neurodegenerative disease research and yield crucial tools for investigating future therapies .

Application of stem cell modeling to Huntington's disease: the time is now A plethora of high-quality and high-quantity HD-iPSC models unveiled Candidate pathways revealed by gene expression profiling and by cellular and molecular assays HD-iPSC models offer opportunities for finding new therapies Abbreviations Competing interests Acknowledgements

in early december 2013 , during the zikv outbreak , a 44-year - old man in tahiti had symptoms of zikv infection : asthenia , low grade fever ( temperature from 37.5c to 38c ) and arthralgia . eight weeks later , he described a second episode of symptoms compatible with zikv infection : temperature from 37.5c to 38c , headache on days 13 , and wrist arthralgia on days 57 . the patient did not seek treatment , thus biological samples were not collected during the first 2 periods of illness . the patient fully recovered from the second episode , but 2 weeks later he noted signs of hematospermia and sought treatment . because the patient had experienced symptoms of zikv infection some weeks before , he was referred to our laboratory in the institut louis malard , papeete , tahiti for zikv infection diagnostic testing . the medical questionnaire revealed no signs of urinary tract infection , prostatitis , urethritis , or cystitis , and the patient stated that he did not had any recent physical contact with persons who had acute zikv infection . we extracted rna using the nuclisens easymag system ( biomrieux , marcy letoile , france ) from 200 l of blood and from 500 l of semen and urine ; both were eluted by 50 l of elution buffer . we tested blood and semen rna extracts using real - time reverse transcription pcr ( rrt - pcr ) as described using 2 primers / probe amplification sets specific for zikv ( 3 ) . the rrt - pcr results were positive for zikv in semen and negative in blood , and confirmed by sequencing of the genomic position 8581138 encompassing the prm / e protein coding regions of zikv . the generated sequence ( genbank accession no . km014700 ) was identical to those previously reported at the beginning of the zikv outbreak ( 3 ) . three days later , we collected a urine sample , then a second set of blood and semen samples . semen and urine from this second collection were not found to contain traces of blood by both direct and macroscopic examinations . rrt - pcr detected zikv rna in the semen and urine , but not in the blood sample . we quantified zikv rna loads using an rna synthetic transcript standard that covers the region targeted by the 2 primers / probe sets . rna loads were : 2.9 10 copies / ml and 1.1 10 copies / ml in the first and second semen samples , respectively , and 3.8 10 copies / ml in the urine sample . we cultured semen and urine as described for dengue virus cultured from urine ( 6 ) . briefly , 200 l of each sample diluted in 200 l of 1% fetal calf serum ( fcs ) minimum essential medium ( mem ) were inoculated onto vero cells and incubated for 1 h at 37c ; inoculum was then removed and replaced by 1 ml of culture medium . we also inoculated a negative control ( 200 l of 1% fcs - mem ) and a positive control ( 5 l of a zikv - positive serum diluted in 200 l of 1% fcs - mem ) . the presence of zikv in the culture fluids was detected by rrt - pcr as described . replicative zikv particles were found in the 2 semen samples but none were detected in the urine sample . ( 7 ) , but perinatal transmission ( 8) and potential risk for transfusion - transmitted zikv infections has also been demonstrated ( 9 ) . ( 10 ) , who described a patient who was infected with zikv in southeastern senegal in 2008 . after returning to his home in colorado , united states , he experienced common symptoms of zikv infection and symptoms of prostatitis . four days later , he observed signs of hematospermia , and on the same day , his wife had symptoms of zikv infection . because the wife of the patient had not traveled out of the united states during the previous year and had sexual intercourse with him 1 day after he returned home , transmission by semen was suggested . zikv infection of the patient and his wife was confirmed by serologic testing , but the presence of zikv in the semen of the patient was not investigated . infectious organisms , especially sexually transmitted microorganisms including viruses ( human papillomavirus or herpes simplex virus ) , are known to be etiologic agents of hematospermia ( 11 ) . to our knowledge , before the report of foy et al . ( 10 ) and this study , arbovirus infections in humans had not been reported to be associated with hematospermia , and no arboviruses had been isolated from human semen . we detected a high zikv rna load and replicative zikv in semen samples , but zikv remained undetectable by rrt - pcr in the blood sample collected at the same time . these results suggest that viral replication may have occurred in the genital tract , but we do not know when this replication started and how long it lasted . the fact that the patient had no common symptoms of zikv acute infection concomitantly to hematospermia suggests that the viremic phase occurred upstream , probably during the first or second episode of mild fever , headache , and arthralgia . the detection of zikv in both urine and semen is consistent with the results obtained in a study of effects of japanese encephalitis virus , another flavivirus , on boars . the virus was isolated from urine and semen of experimentally infected animals , and viremia developed in female boars that artificially inseminated with the infectious semen ( 12 ) . flaviviruses have also been detected in urine of persons infected with west nile virus ( wnv ) . wnv rna was detected in urine for a longer time and with higher rna load than in plasma ( 13 ) . wnv antigens were detected in renal tubular epithelial cells , vascular endothelial cells , and macrophages of kidneys from infected hamsters ( 14 ) , suggesting that persistent shedding of wnv in urine was caused by viral replication in renal tissue . dengue virus ( denv ) rna and denv nonstructural protein 1 antigen were also detected in urine samples for a longer time than in blood , but infectious denv has not been isolated in culture . concluded that the detection of denv by rrt - pcr was useful to confirm denv infections after the viremic phase ( 6 ) . also , yellow fever virus rna was isolated from the urine of vaccinated persons ( 15 ) , and saint louis encephalitis viral antigens , but not infective virus , have been detected in urine samples from infected patients ( 10 ) . furthermore , the observation that zikv rna was detectable in urine after viremia clearance in blood suggests that , as found for denv and wnv infections , urine samples can yield evidence ofzikv for late diagnosis , but more investigation is needed .

in december 2013 , during a zika virus ( zikv ) outbreak in french polynesia , a patient in tahiti sought treatment for hematospermia , and zikv was isolated from his semen . zikv transmission by sexual intercourse has been previously suspected . this observation supports the possibility that zikv could be transmitted sexually .

The Study Conclusions

irritable bowel syndrome ( ibs ) is a common gastrointestinal disorder affecting approximately 1015% of the people in western world . the patient group is very heterogeneous and characterized by abdominal pain , discomfort and abnormal bowel habits . the pathophysiology of ibs is unknown , it would appear that the due to the heterogeneous patient material there are multiple explanations for the symptom development . currently , patients are subdivided according to their main symptom that have been proposed in the so called rome criteria ; ibs with diarrhea ( ibs - d ) , ibs with constipation ( ibs - c ) or ibs with mixed bowel habits ( ibs - m ) . no single unifying cause for ibs has been reported but several factors have been associated with the disease etiology , including genetic predisposition , anxiety and depression as well as female gender . moreover , there is strong evidence indicating that changes in the intestinal microbiota and their function could be one of the contributing factors in the development of ibs . several studies have reported differences in the intestinal microbiota composition of ibs patients and healthy controls , however the results show little overlap from study to study . this is likely caused by different methodological approaches in the analysis steps as well as the heterogeneity of the ibs patients . regardless of the lack of coherent changes in the microbial composition , there is clear evidence for the role of the microbiota in the pathogenesis of ibs . for one , the patients report improved symptoms after consuming agents targeted to modify the intestinal microbiota , including probiotics , antibiotics or changing diet , such as avoidance of fodmap carbohydrates . moreover , the strongest predictor for ibs is a prior gastroenteritic episode , which will increase the risk of developing ibs by 7-fold . it has been estimated that about 10% of the ibs cases begin after an episode of gastroenteritis , causing post - infectious ( pi ) ibs , indicating a cause and effect relationship . this is in line with a previously proposed model in which an event , such as an infection , affects the intestinal microbiota and generates an alternative stable state . recently , a number of bistable microbial groups , i.e. microbes with two stable homeostatic states , have been identified that could act as tipping points in this well known ecological process that is associated with resilience . in our recent study by jalanka and colleagues the differences in the microbial composition of pi - ibs patients were addressed by analyzing the faecal microbiota of the study subjects with a previously validated and benchmarked phylogenetic microarray , detecting the abundance of over 1000 bacterial phylotypes found in the human intestine . the studied cohort consisted of 57 british subjects divided into 5 study groups ; patients with a pi - ibs diagnosis ( group 1 , rome ii ) , patients whom 6 months after the gastroenteritic episode had a persisting bowel dysfunction , which however did not qualify for ibs ( pi - bd ) ( group 2 ) or did not experience any bowel dysfunction anymore ( pi - nonbd ) ( group 3 ) . the results were benchmarked against ibs - d patients ( group 4 , rome ii ) and healthy controls ( group 5 ) . our aim was to determine the microbial compositional differences and to address associations between the faecal microbiota and the clinical features of ibs from patients with a varying degree of symptoms . as the development of ibs is multifactorial , we hypothesized that the observed symptoms and development of pi - ibs may arise from the interplay between the faecal microbiota , the host immune response , and psychological factors . therefore , we analyzed in parallel the detailed records of the participants ' psychological well - being , intestinal symptoms , systemic immunological markers as well as the levels of host gene expression on rectal mucosa . this integrated approach , addressing the associations between clinical phenotype and the faecal microbiota allowed us to address the complex relationships between the host and microbiota in the pathogenesis of ibs and pi - ibs . since the intestinal microbiota is very heterogeneous and subject - specific , we hypothesized that instead of limiting the comparative microbiota analysis to individual taxa , it would be more relevant to search for a microbial signature that potentially differs between the patients and healthy controls . by using a novel bioinformatic approach ( bootstrap aggregated rda method ; baggedrda ) we identified a combination of 27 genus - like bacterial groups that significantly separated healthy controls and patients with ibs symptoms . moreover , we found that the microbial profile of the pi - ibs and pi - bd groups resembled those suffering from ibs - d . the major difference in the patients ' microbiota was the 12-fold increase of bacteroidetes phylum and the 35-fold decrease of uncultured clostridiales belonging to the firmicutes phylum . furthermore , when all participants were ranked according to the abundance of the discriminating bacterial taxa , a specific ordering was obtained , the so - called index of microbial dysbiosis ( imd ) . this rank ordering reflected the patient 's health status as it correlated with the increase of several immunological markers and intestinal complaints , where as no such associations were observed when subdividing the patients according to their clinical status . interestingly , there were no significant correlations between the imd and any of the psychological symptoms often associated with ibs . our results corroborate the previous reports that the microbial composition could be used to further stratify ibs patients and thus complement the clinical subgroupings . in a recent study it was estimated that circa 33% of the ibs patients exhibit anxiety and 13% suffer from depression . in addition , it was found that somatisation was significantly increased in the ibs patients compared to controls with gastrointestinal symptoms but without ibs . although the intestinal microbiota of these patients was not studied , in the light of the recent evidence it could be hypothesized that the etiology of the disease in the patients with psychological symptoms differs from those with microbial aberrations . taken together , these results suggest that the discriminant microbial profile could be used as an objective measure of disturbed bowel functions and therefore a potential novel way of stratifying this heterogeneous patient material in future studies and in clinical practise . to verify our findings in a larger patient material we combined the microbiota data form the british subjects with that of a swedish pi - ibs cohort reported recently by sundin and colleagues . both studies were conducted with an identical analysis pipeline , including the well established mechanical lysis for extraction of faecal dna , the hitchip phylogenetic microarray platform , and the normalization and analysis software suite . the combined data aimed to determine whether the pi - ibs patients from 2 countries share similarities in their microbiota compared to healthy controls . the swedish cohort introduced new subjects to both healthy controls ( n=16 ) and pi - ibs patients ( n=13 , rome iii ) therefore doubling the sample size to a total of 51 subjects . remarkably , the significant difference in the microbiota profiles between the healthy and pi - ibs patients was replicated ( p < 0.05 , fig . 1 ) . the strongest difference was observed with the significant increase in the bacteroides ssp . and decrease of members of the uncultured clostridiales in the pi - ibs patients . interestingly , both bacteroides fragilis and uncultured clostridiales have been identified as bistable microbial groups and predicted to act as tipping points of the gut ecosystem in opposite ways . however , not all of the imd taxa reported in the original study appeared as significantly different between the patients and controls , and there were new taxa that were significantly increased in the combined pi - ibs patient group . one of them was dialister , which was recently identified as bistable i.e. , being either abundant or nearly absent in contrast to most other bacteria that show gradual abundance distribution . this highlights the need of conducting the future research in large cohorts in order to verify the separating species between pi - ibs and healthy controls . figure 1.intestinal microbiota of pi - ibs patients is significantly different form that of healthy controls ( hc ) . the difference was measured with baggedrda that was used to identify a microbial signature that separates hc from pi - ibs patients from the two study cohorts . abundance of the discriminating taxa creates the so - called index of microbial dysbiosis ( imd ) . intestinal microbiota of pi - ibs patients is significantly different form that of healthy controls ( hc ) . the difference was measured with baggedrda that was used to identify a microbial signature that separates hc from pi - ibs patients from the two study cohorts . abundance of the discriminating taxa creates the so - called index of microbial dysbiosis ( imd ) . the role of bacteroides spp . in the pathophysiology of ibs is controversial , probably reflecting the heterogeneity of the patients as well as the genus bacteroides . previous studies have reported opposing results regarding the relevance of bacteroides genera in ibs , as there is evidence for both increased as well as decreased abundance in the patients . interestingly , elevated levels of bacteroides spp were among the microbiota changes that have been associated with susceptibility to enteric pathogens in mice . it has been suggested that susceptibility to infections could increase due to a imbalanced microbial composition , since complex interactions between the microbiota and host immune system might reduce the resistance to episodes of gastroenteritis . recently , similar observations were made in humans by dicksved and colleagues who followed abattoir workers who have a high risk of contracting c. jejuni infection . the participants donated faecal samples at the start of employment and were then regularly followed . the subjects who became c. jejuni infected had increased levels of bacteroides species prior the infection , as opposed to those individuals who did not contract the disease during the follow - up period . similar to these findings , it was recently shown that the subjects contracting a gastroenteritis had increased levels of bacteroides spp . and decreased levels of firmicutes immediately after the infection . although the infectious agents in this study were diverse , the differences in the microbiota profile were similar to what we found . altogether , these data from several studies allow to hypothesize that the subjects with a microbial profile resembling the imd could be more susceptible for contracting a gastrointestinal infection and later developing pi - ibs . more studies on patients before and after gastroenteritis are required to verify this hypothesis . moreover recent experiments in mice have indicated that innate and mucosal immune system play a deterministic role in shaping the gut microbiota . hence , the observed imd may be a reflection of the activity of the host 's immune system . while this alternative explanation may offer new insight in the etiology of ibs , it confirms the significance and application potential of imd in stratifying ibs subjects . to further analyze the crosstalk between the host and the microbiota , we concentrated on the 27 bacterial taxa of imd and separately studied the associations between bacterial abundance and the host gene expression in mucosal surface of rectum , the last part of large intestine . we mapped the microarray - derived gene expression data from rectal biopsies to known biological functions using gene set analysis ( gsa ) . there were numerous statistically significant associations between the abundance of the ibs - type microbiota and the level of expressed host genes that suggested an association between the discriminant microbiota and the physical barrier integrity of the host . the most prominent negative correlation was observed between 7 bacteroides ssp and the expression of glycine , serine and threonine metabolism pathway . in general , glycine , serine and threonine are known to be important in the maintenance of gut integrity and the structure of the mucin layer . for example , the majority of the dietary threonine is utilized for synthesising the secretory mucin and its dietary restriction results into impaired gut barrier . moreover , the mucus structure and the extent of its glycosylation shape the microbiota composition and similar compositional changes have also been previously associated with barrier function abnormalities . fucosyltransferase-2 is involved in the formation of the abo - blood groups and its activity also creates adhesion receptor for several microbes . therefore , it is interesting that fut2 deficient mice ( fut2 ) have been shown to display similar differences in their microbiota composition to what was found in our study , i.e. increased amounts of several bacteroides species and decreased levels of clostridiales . in addition to alterations in the mucosal barrier genes , we identified several other pathways , including those that regulate cell junctions and inflammatory responses , to be associated with the imd bacterial taxa . the results led us to hypothesize that both the physical and immunological gut barrier of subjects with an ibs - type microbiota has been compromised resulting in a low - grade inflammation , a feature of some ibs patients . changes in the inflammatory response , barrier function and microbiota composition could therefore lead into changes in the other host parameters such as gut - brain axis receiving signals from these systems . moreover , we observed that the imd correlated with the ibs symptoms and clinical features whereas the scores of anxiety and depression did not correlate with the imd , indicating that patients with a more psychological basis for the disease have a rather undisturbed microbiota . this is in line with previous findings and of great significance as it allows proposing a model in which some subjects with a relatively healthy microbiota report ibs symptoms while others with a microbiota resembling that of the ibs patients have no complaints as they have compensating factors that overcome the microbial component and hence are scored healthy ( fig . 2 ) . the pi - ibs patients can be stratified according to the imd ( or more generally the microbial composition ) to those with ibs - like microbiota ( red ) and those with a composition resembling healthy individuals ( turquoise ) . objective measurements such as low - grade inflammation and altered gene expression correlated with the imd . psychological aspects , such as depression or anxiety do not correlate with the imd and these patients , though diagnosed with ibs , show a healthy - like microbiota profile . the correlations between host response and microbiota aberration identified in our study are indicated in bold . proposed model on how microbial aberrations could explain the disease etiology in ibs . the pi - ibs patients can be stratified according to the imd ( or more generally the microbial composition ) to those with ibs - like microbiota ( red ) and those with a composition resembling healthy individuals ( turquoise ) . objective measurements such as low - grade inflammation and altered gene expression correlated with the imd . psychological aspects , such as depression or anxiety do not correlate with the imd and these patients , though diagnosed with ibs , show a healthy - like microbiota profile . the correlations between host response and microbiota aberration identified in our study are indicated in bold . ibs has a heavy impact on the health care system and currently the patient diagnosis is based on subjective symptoms and exclusion of other gastrointestinal diseases . results from our study as well as from others suggest that diagnosis based on symptoms alone is not optimal and should be accompanied with objective markers , such as the microbiota composition . we introduced an index of microbial dysbiosis ( imd ) that can potentially be used to objectively stratify patients into smaller groups where the patients with psychological basis of the syndrome could be separated from those with a peripheral gut abnormality . by using a relatively small but carefully phenotyped cohort we identified several associations between the imd taxa , hosts ' gene expression and clinical markers suggesting that impairment of intestinal barrier may underlie both immunological and microbiological deviations often associated with pi - ibs . here we show that the microbial signatures that were observed in our british pi - ibs cohort were reproduced in an independent swedish pi - ibs cohort ( fig . 1 ) . this testifies for the robustness of these signatures in different cohorts and countries as well of the used microbial analysis pipeline . the main observed microbial aberration included an increased level of bacteroides spp and decreased level of uncultured clostridiales , both recently identified as contrasting tipping point taxa that reflect alternative stable states of the gut ecosystem . recently , similar bacterial signatures have been also detected in subjects prior to gastroenteritis , suggesting that perhaps the microbial composition already at the time of the initial infection predisposes certain subjects for pi - ibs . this is of significance as it allows not only to diagnose these subjects but also may provide basis for treatment . dietary corrections represent the major approach in managing the ibs symptoms and it is expected that their efficacy depends on the individual 's gut microbiota as microbial activity on food metabolism directly or indirectly contributes to many ibs symptoms . this hypothesis is supported by a pilot study among pediatric ibs patients in us . of note , those children whose symptoms decreased during the test diet had lower abundance of bacteroides and dialister spp . that we found to typify the pi - ibs microbiota , further stressing the relevance of these organisms in ibs . we believe that the proposed model ( fig . 2 ) built on microbiota - based patient stratification will not only give insight into the observed microbiota heterogeneity among ibs patients as well as its partial overlap with healthy controls , but also represents a concrete step toward improved diagnostics and novel treatment options with improved efficacy . we are grateful to our collaborators , notably prof robin spiller md and prof robert jan brummer md , for helpful discussions . this work was partly supported by the finland academy of sciences ( grants 137389 , 141140 and 1272870 ) and the unrestricted spinoza of the netherlands organization for scientific research .

irritable bowel syndrome ( ibs ) is a multifactorial and heterogeneous disorder estimated to affect over 10% of the western population . a subset of the patients reports the start of the disease after an episode of gastroenteritis . the alterations in the intestinal microbiota of the post - infectious ibs ( pi - ibs ) patients were recently investigated in a british cohort and shown to differentiate from the healthy controls and resemble that of diarrhea - predominant ibs ( ibs - d ) patients . the altered 27 genus - like groups created a microbial signature , which could be used to objectively stratify patients and healthy controls . in this addendum , we combine the microbiota data derived from the british cohort with that of a recently reported swedish pi - ibs cohort . remarkably , robust and reproducible microbiota signatures were observed in these pi - ibs patients . we discuss these results with attention on the emerging role of microbiota in the classification , development and treatment of pi - ibs .

Introduction Microbial Signatures and IMD Associations Between Microbiota and Host Gene Expression Conclusions Disclosure of Potential Conflicts of Interest Acknowledgments Funding

intracranial dural arteriovenous fistula ( davf ) is abnormal arteriovenous shunts that occur within the leaflets of the dura mater and comprised 10 - 15% of intracranial arteriovenous malformation8 ) . in a meta - analysis of 377 patients with davf reported before 1989 , tentorial davf was only 8% ( 32/ 377 ) of all intracranial davf , but tentorial davf had the most aggressive neurological behavior , with 97% ( 31/32 ) causing hemorrhage or progressive focal neurological deficits110 ) . unlike the less aggressive davfs of the lateral and cavernous sinuses that drain into venous sinuses , tentorial davf drains into pial veins11 ) . ct and mr imaging revealed an acute hemorrhage at left tectum of mid brain and fourth ventricle ( fig . cerebral angiography showed a davf around torcular fed by meningeal branch of ascending pharyngeal artery , transosseus branch of the right occipital artery and drained into cortical vein of left occiput and basal vein of rosenthal ( fig . an intravenous bolus of 3000 iu of heparin was administered after placement of a 6-fr guiding sheath in the right femoral artery . a 6-fr guiding catheter ( envoy , cordis endovascular , miami lakes , fl , usa ) was placed at external carotid artery . we planned to superselective catheterization of meningeal branch of ascending pharyngeal artery but failed due to tortous curve at cranial base . we could access to fistula point through transosseus branch of occipital artery using microcatheter ( apollo , ev3 inc . , the volume of 0.7 ml onyx-18 ( ethyl vinyl alcohol , ev3 inc . , irvine , post - embolization angiogram demonstrated onyx occupying the retrograde drainage of cortical vein and obliteration of davf ( fig . the patient was discharged 7 days later and his postoperative course was uneventful . a 74-year - old man presented with progressive headache and dementia symptom for several months . seven years ago , he had spontaneous hemorrhage in 4th ventricular and been treated for davf by transarterial embolization using n - butyl cyanoacrylate ( nbca ) . both middle meningeal arteries ( using 33 and 50% glue ) and transosseus branch of left occipital artery ( using 20% glue ) were selected for embolization . although arteriovenous shunting flow was not completely abolished , retrograde cortical flow was remarkably decreased . he refused further treatment and did not seek hospital since then . by this time of visit , he has been well . ct and mr image showed a prominent , tortous vascular structure around medial tentorium and multiple fine vessels engorgements at both thalamic and putamen areas ( fig . angiogram demonstrated recanalization and recruitment from both middle meningeal arteries and posterior choroidal arterial supply from vertebral artery . the retrograde leptomeningeal drain of infratentorial vermian vein showed dilated and saccular change ( fig . we embolized davf only through the right middle meningeal artery and inject onyx-18 1.0 mg as described above . korea mini - mental state examination ( k - mmse ) score was improved from 14 to 28 . ct and mr imaging revealed an acute hemorrhage at left tectum of mid brain and fourth ventricle ( fig . cerebral angiography showed a davf around torcular fed by meningeal branch of ascending pharyngeal artery , transosseus branch of the right occipital artery and drained into cortical vein of left occiput and basal vein of rosenthal ( fig . an intravenous bolus of 3000 iu of heparin was administered after placement of a 6-fr guiding sheath in the right femoral artery . a 6-fr guiding catheter ( envoy , cordis endovascular , miami lakes , fl , usa ) was placed at external carotid artery . we planned to superselective catheterization of meningeal branch of ascending pharyngeal artery but failed due to tortous curve at cranial base . we could access to fistula point through transosseus branch of occipital artery using microcatheter ( apollo , ev3 inc . , the volume of 0.7 ml onyx-18 ( ethyl vinyl alcohol , ev3 inc . , irvine , post - embolization angiogram demonstrated onyx occupying the retrograde drainage of cortical vein and obliteration of davf ( fig . a 74-year - old man presented with progressive headache and dementia symptom for several months . seven years ago , he had spontaneous hemorrhage in 4th ventricular and been treated for davf by transarterial embolization using n - butyl cyanoacrylate ( nbca ) . both middle meningeal arteries ( using 33 and 50% glue ) and transosseus branch of left occipital artery ( using 20% glue ) were selected for embolization . although arteriovenous shunting flow was not completely abolished , retrograde cortical flow was remarkably decreased . he refused further treatment and did not seek hospital since then . by this time of visit , he has been well . ct and mr image showed a prominent , tortous vascular structure around medial tentorium and multiple fine vessels engorgements at both thalamic and putamen areas ( fig . angiogram demonstrated recanalization and recruitment from both middle meningeal arteries and posterior choroidal arterial supply from vertebral artery . the retrograde leptomeningeal drain of infratentorial vermian vein showed dilated and saccular change ( fig . we embolized davf only through the right middle meningeal artery and inject onyx-18 1.0 mg as described above . korea mini - mental state examination ( k - mmse ) score was improved from 14 to 28 . davf associated with retrograde leptomeningeal venous drainage exhibit a high incidence of aggressive behavior3912 ) and those with only leptomeningeal venous drainage was classified type iii by borden et al.2 ) . in tentorial davf patients , 22/22 cases ( 100%)14 ) , 26/31 cases ( 84%)10 ) and 13/14 cases ( 93%)6 ) were classified borden type iii and needed prompt treatment . the tentorial davf is a rare type of dvaf and should be treated even though without remarkable clinical symptoms because natural history is very disappointing . to our knowledge , tentorial davf is located in the tentorial dura mater and fed primarily from branches of the meningohypophyseal trunk , middle meningeal arteries , and occipital artery . the multitude of small dural arteries , a variety of venous pathways , and high flow may prevent spontaneous closure11 ) . davf usually has multiple fistula points by multiple feeding arteries but drains into single vein . the aim of the davf treatment with retrograde leptomeningeal venous drainage is complete and permanent obliteration of the all arterial supply or occlusion of proximal draining vein . therapeutic options for treating davf include transvenous and/or transarterial embolization and surgical excision of the davf nidus , ligation of the draining vein . usually the transvenous embolization was preferred when the venous anatomic features were open and not too tortuous for catheterization of the draining vein and fistula13 ) . these combinations of transvenous and transarterial embolization results in high obliteration rates for most davf , but tentorial davf are somewhat different . more importantly , tentorial davf often drain exclusively to subarachnoid veins rather than to their associated sinus ( borden type iii ) , which prevents transvenous access261011 ) . huang et al.6 ) , reported complete cure of tentorial davf in 78.6% ( 11/14 ) by transarterial embolization using onyx . in case of partial obliteration of the arterial supply , patients with borden type ii or iii lesions should considered surgery because they may experience temporary palliation of their symptoms but can not provide protection from the risk of hemorrhage or progression of the effects of venous hypertension414 ) . hwang et al.7 ) and lawton et al.10 ) recommended surgical option for incomplete embolization of tentorial davf by simple interruption of draining vein . surgical interruption of simple venous drainage is analogous to transarterial and/or tranvenous embolization of outlet from the nidus without effect of normal venous drainage . the surgical interruption of proximal venous drain could be considered according to types of tentorial davf by location and drain vein . the tentorial sinuses drain the supra- and infratentorial veins as well as the deep veins of the brain stem . lasjaunias et al.9 ) , divided tentorial davf into torcular , basal tentorium and marginal tentorium by their location and draining veins . lesions in marginal tentorium drain into tentorial vein , vein of rosenthal , and mesencephalic veins . picard et al.12 ) , divided tentorial davf to 3 groups by draining veins as medial , lateral and marginal tentorial sinus group . lesions in the medial tentorial sinus group are situated adjacent to the torcular and drain into it or the lateral or straight sinus . they primarily receive venous drainage from the cerebellar hemispheres and vermis ( infratentorial drainage ) . lesions in the lateral tentorial sinus group lie adjacent to the lateral sinus and receive supratentorial drainage from the lateral and inferior surfaces of the temporal and occipital lobes . lesions in the marginal tentorial sinus group lie along the free edge of the tentorium and receive venous drainage from the basilar and lateral mesencephalic veins . fistulae at this location may have infra- or supratentorial drainage or drain into spinal veins . case 1 was torcular type by lasjaunias et al.9 ) and lawton et al.10 ) , and then marginal tentorial sinus group by picard et al.12 ) which was located around tocular area and drained to supratentorial cotical vein into basal vein of rosenthal and causes brain stem hemorrhage . case 2 was medial tentorial sinus group by picard et al.12 ) and straight sinus type by lawton et al.10 ) which was located around midline torcular and drained to infratentorial vermian vein and causes venous hypertension . various embolic agents have been used including particles , platinum coils , nbca and onyx for arterial approach . in case of nbca , solidification time of the material should be matched to the local flow velocity and to the angioarchitectural features of the lesion . although nbca is usually considered as permanent embolic agent but may result in recanalization as our case in delayed fashion5 ) . furthemore posterior choroidal artery could not be selected for embolization of complete angiographic cure by nbca . recently due to the superior ability of penetration and controllability , onyx is used as the treatment of choice for transarterial embolic agent of davf and very effective for obliteration of multiple fistula point and proximal draining vein by single transarterial access . contrast to nbca , in case 2 , onyx could penetrate deeper venous sac and reach posterior choroidal arterial supply from middle meningeal artery and results in complete angiographic cure . tentorial davf should be treated aggressively and transarterial embolization with onyx can be prior option when transvenous access is difficult . transarterial approach using onyx also can be applied for delayed recanalization after embolization with nbca . if partially treated tentorial davf by embolization procedure , simple surgical interruption of proximal draining vein should be considered according to the location and drain veins .

tentorial dural arteriovenous fistula ( davf ) is a rare vascular disease , which has high risk of intracranial hemorrhage . we present two cases of tentorial davf which were successfully treated with single trial of transarterial embolization using onyx . we briefly reviewed the types of the tentorial davf and strategies of treatment .

INTRODUCTION CASE REPORT Case 1 Case 2 DISCUSSION CONCLUSION

over the last 3 decades , population - based studies aiming to determine the true burden of neurological disorders have increasingly been acknowledged . nevertheless , in developing countries , the limited access of neurologists to rural areas and the logistic and economic limitations to perform neuroepidemiological studies have significantly complicated their implementation.1 however , a series of epidemiological studies was conducted in upper egypt in the last 2 decades . epidemiologic research has progressed in egypt , particularly over the last 6 years , through two major projects to evaluate the incidence and prevalence of different neurological disorders in al kharga district , new valley and al quseir city , red sea governorate , upper egypt . this project aimed to evaluate the incidence and prevalence of epilepsy and stroke ; the incidence of bell s palsy ; and the prevalence of dementia , chorea , athetosis , dystonia , parkinson s disease , cerebellar ataxia , multiple sclerosis , migraine , trigeminal neuralgia , cerebral palsy , and spinal cord disorders . the project was designed to assess major neurological disorders in al quseir city as a representative coastal city lying on the red sea . al quseir city has a long history , dating from the pharaonic era when it was named thagho . at the time of the batlamic and romanic eras it was named leukos limen and poptus albus , respectively . its population is representative of native inhabitants and immigrants from different governorates who reside there to participate in tourist activities , fishing , and mining.2 all eligible inhabitants who had been living in al quseir city for at least 6 months at the time of the interview were included in the survey . the study duration extended from july 1 , 2009 to june 31 , 2012 through different stages of data collection , a preparatory stage , screening , data entry , statistical manipulation , and tabulation . all households ( n = 33,285 ; 7497 families ) and both sexes , with 49.4% males ( n = 16,428 ) and 50.6% females ( n = 16,857 ) , were screened by three neurologists , in addition to 15 female social workers ( for sociodemographic data collection ) via a door - to - door survey . a standardized questionnaire , the sensitivity and specificity of which are 96% and 93.2% , respectively,3 was applied by three neurologists to every member of each family ( children and elderly were questioned through their caregivers ) . a total population of 31,754 ( 95.4% ) were free from any neurological disorder , versus 1531 ( 4.6% ) of both sexes ( 3.9% [ n = 647 ] males and 5.2% [ n = 884 ] females ) had different neurological disorders . full history data and examinations ( general , systemic , and neurological ) were carried out by the three neurologists in collaboration with nine other senior staff members from neurology departments in assiut , al - azhar , sohag , and qena universities . all neurological disorders were finally diagnosed after evaluation by the three neurologists , each separately , as well as specific investigatory tools . diagnoses of different neurological disorders were based on the accepted definition and diagnostic criteria given by the world health organization.4,5 the definition of incidence rate , prevalence , and lifetime prevalence in this study are according to those provided by abramson.6 ethical approval for the study was obtained from the research ethics committee of assiut university and from the ministry of health to carry out this project in al quseir city , red sea governorate . each participant provided written informed consent ( children , illiterate , and disabled individuals consented through the responsible person in the family or their caregivers ) . data management was carried out by two specialists in data entry and three medical statisticians using spss software ( v 16 ; ibm corporation , armonk , ny , usa ) , excel ( microsoft corporation , redmond , wa , usa ) , and epicalc 2000 ( microsoft corporation ) . the project was designed to assess major neurological disorders in al quseir city as a representative coastal city lying on the red sea . al quseir city has a long history , dating from the pharaonic era when it was named thagho . at the time of the batlamic and romanic eras it was named leukos limen and poptus albus , respectively . its population is representative of native inhabitants and immigrants from different governorates who reside there to participate in tourist activities , fishing , and mining.2 all eligible inhabitants who had been living in al quseir city for at least 6 months at the time of the interview were included in the survey . the study duration extended from july 1 , 2009 to june 31 , 2012 through different stages of data collection , a preparatory stage , screening , data entry , statistical manipulation , and tabulation . all households ( n = 33,285 ; 7497 families ) and both sexes , with 49.4% males ( n = 16,428 ) and 50.6% females ( n = 16,857 ) , were screened by three neurologists , in addition to 15 female social workers ( for sociodemographic data collection ) via a door - to - door survey . a standardized questionnaire , the sensitivity and specificity of which are 96% and 93.2% , respectively,3 was applied by three neurologists to every member of each family ( children and elderly were questioned through their caregivers ) . a total population of 31,754 ( 95.4% ) were free from any neurological disorder , versus 1531 ( 4.6% ) of both sexes ( 3.9% [ n = 647 ] males and 5.2% [ n = 884 ] females ) had different neurological disorders . full history data and examinations ( general , systemic , and neurological ) were carried out by the three neurologists in collaboration with nine other senior staff members from neurology departments in assiut , al - azhar , sohag , and qena universities . all neurological disorders were finally diagnosed after evaluation by the three neurologists , each separately , as well as specific investigatory tools . diagnoses of different neurological disorders were based on the accepted definition and diagnostic criteria given by the world health organization.4,5 the definition of incidence rate , prevalence , and lifetime prevalence in this study are according to those provided by abramson.6 ethical approval for the study was obtained from the research ethics committee of assiut university and from the ministry of health to carry out this project in al quseir city , red sea governorate . each participant provided written informed consent ( children , illiterate , and disabled individuals consented through the responsible person in the family or their caregivers ) . data management was carried out by two specialists in data entry and three medical statisticians using spss software ( v 16 ; ibm corporation , armonk , ny , usa ) , excel ( microsoft corporation , redmond , wa , usa ) , and epicalc 2000 ( microsoft corporation ) . the incidence rates of epilepsy , stroke , and bell s palsy compared to worldwide rates are illustrated in table 1 . the prevalence of the neurological disorders studied in al quseir city is illustrated in tables 24 . table 5 shows a record of the prevalence of different neurological disorders studied in al quseir city and worldwide . the increase in the number of different major projects to study neurological disorders worldwide during the last 3 decades will help all of us to construct a map of neurological disorders throughout the world and clarify the burden of neurological disorder among different countries . this study is a new one in the series of large epidemiological studies carried out in our country to assess the burden of neurological disorders at different localities in egypt . screening was carried out through a door - to - door survey , including every household , by using a standardized questionnaire,7 with subsequent clinical evaluation of all suspected cases by neurologists ( three specialists as well as nine senior staff members ) in al quseir general hospital and assiut university hospital . the limitations of this study include : the stigma of some neurological disorders ( eg , the view of epilepsy as an evil attack and dementia as a normal aging process ) necessitates extraneous effort from the project team to clarify the nature of these disorders and their suitability to treat . noncompliance by the patients with the prescribed treatments due to scarce financial resources and lack of health care systems suited for long term needs of chronic neurological disorders . these limitations required an extra effort on the project team to encourage participation of most inhabitants through : detailed explanations of the project goals by the members of the project team and responsible authorities in the defined area of the study . recruitment of well - known key persons , as well as female social workers , among native inhabitants , who accompanied the screening team to facilitate family interviews and encourage participation . a capture - recapture method for those who were not present at their homes during the first visit , the incidence rate of epilepsy ( 48/100,000 per year ) in al quseir city is within the international rate and lower than that recorded in sub - saharan africa10 and northern tanzania.11 meanwhile , the incidence rate of epilepsy ( 48/100,000 ) in this study was higher than that recorded in new york , ny , usa ( 16/100,000)12 and in the netherlands ( 29/100,000).13 although the incidence rate of stroke in al quseir city was lower than that recorded in france14 and other areas of egypt ( al kharga district , new valley),7 it is similar to that recorded in sohag , upper egypt.15 the incidence rate of bell s palsy in al quseir city was higher than that recorded worldwide.7,1618 the great variation in the incidence rate of epilepsy , stroke , and bell s palsy in al quseir city in comparison to incidence rates worldwide could be attributed to the different genetic characteristics of the population in al quseir city , where the population contains a mixture of native inhabitants and immigrants from different adjacent governorates , and thus a variable ( not distinct ) genetic predisposition for various neurological disorders . study of age - specific prevalence of some neurological disorders in al quseir city revealed that dementia ( 3.83% of those aged 60 years ) was the most frequent , followed by migraine ( 2.8% ) , stroke ( 6.2/1000 ) , epilepsy ( 5.5/1000 ) , parkinson s disease ( 452.1/100,000 ) , and cerebral palsy ( 3.6/1000 ) . however , less frequent neurological disorders were spinal cord injury ( 63.0/100,000 ) , dystonia ( 39.1/100,000 ) , cerebellar ataxia ( 30.0/100,000 ) , trigeminal neuralgia ( 28/100,000 ) , chorea ( 21.0/100,000 ) , athetosis ( 15.0/100,000 ) , and multiple sclerosis ( 13.7/100,000 ) . macdonald et al s uk study found that stroke was the most frequent neurological disorder , followed by epilepsy , then parkinson s disease , and , finally , multiple sclerosis.9 on the other hand , in a mexican rural community in 2011 , quet et al found that headache was the most frequent neurological disorder , followed by epilepsy , stroke , parkinson s disease , and cerebellar ataxia.19 in the al kharga district , the order of frequency of prevalence of the neurological disorders studied was as follows : dementia , epilepsy , stroke , parkinson s disease , cerebral palsy , cerebellar ataxia , chorea , dystonia , and athetosis.7 these variable results of different epidemiological studies might be attributed to differences in the methodology and types of neurological disorders studied ; the impact of different environmental factors in diverse geographic areas ; and different genetic compositions of the studied populations . the prevalence of multiple sclerosis ( 13.7/100,000 ) and spinal cord disorders ( 63/100,000 ) ( 18/100,000 traumatic and 45/100,000 nontraumatic causes ) in al quseir city are the first results of a community - based epidemiological study in egypt . while radhakrishnan et al recorded a lower prevalence of multiple sclerosis in libya ( 5.9/100,000)20 than that recorded in our study , macdonald et al recorded a much higher prevalence ( 200/100,000 ) in the uk.9 on the other hand , an intermediate figure ( 33.9/100,000 ) for the prevalence of multiple sclerosis was recorded by elik et al in the metropolitan area of edrine city , turkey.21 this wide variation in the prevalence of multiple sclerosis worldwide could be attributed to differences in climate , race , immigration , and emigration , as well as varied genetic predispositions of the studied sample frame . this study clarified important issues regarding the epidemiology of the studied neurological disorders , which can be summarized as follows : the high prevalence of parkinson s disease ( 452.1/100,000 ) in comparison with worldwide studies and those in other localities in egypt , which could be attributed to environmental pollution by heavy metals ( manganese , lead , and phosphorus ) in this area . the higher prevalence of cerebral palsy ( 3.6/1000 ) than in worldwide records and previously recorded data in al kharga district , egypt could be explained by a lack of antenatal , perinatal , and postnatal care in al - quseir city . the lower prevalence of chorea in al - quseir city ( 21.03/100,000 ) in comparison to the other two studies conducted in our country ( al kharga district : 31.96/100,000;7 assiut : 62.00/100,00022 ) for rheumatic chorea could be attributed to governmental efforts for the eradication of rheumatic activities in the last few years . door - to - door surveys , with direct personal interviews with both health - seeking and non - health - seeking individuals , gives a better chance for diagnosing rare neurological disorders . it remains the gold standard method of epidemiological studies , especially in the absence of existing coded data due to a lack of medical registry systems and health insurance coverage . this study had the aim of evaluating the prevalence of trigeminal neuralgia , multiple sclerosis , and spinal cord disorders for the first time in egypt . the study shows a high prevalence of parkinson s disease , cerebral palsy , and stroke in al quseir city compared with other cities worldwide . the study shows a marked decline in the prevalence of chorea in egypt , compared to a previous study.22

a door - to - door survey , including every household , was conducted for all inhabitants of al quseir city ( 33,283 ) , red sea governorate , egypt by three specialists of neurology as well as nine senior staff members of neurology and 15 female social workers to assess the epidemiology of major neurological disorders . over six phases , from july 1 , 2009 to january 31 , 2012 , screening of all eligible people in the population was carried out , by which case ascertainment of all major neurological disorders included in the study was done according to the accepted definitions and diagnostic criteria of the world health organization . the order of frequency of prevalence of the studied neurological disorders was dementia ( 3.83% for those aged > 60 years ) , migraine ( 2.8% for those aged > 8 years ) , stroke ( 6.2/1000 for those aged > 20 years ) , epilepsy ( 5.5/1000 ) , parkinson s disease ( 452.1/100,000 for those aged > 40 years ) , cerebral palsy ( 3.6/1000 among children < 18 years ) , spinal cord disorders ( 63/100,000 ) dystonia ( 39.11/100,000 ) , cerebellar ataxia ( 30.01/100,000 ) , trigeminal neuralgia ( 28/100,000 for those aged > 37 years ) , chorea ( 21.03/100,000 ) , athetosis ( 15/100,000 ) , and multiple sclerosis ( 13.74/100,000 ) . the incidence rates of stroke , epilepsy , and bell s palsy were 181/100,000 , 48/100,000 , and 98.9/100,000 per year , respectively .

Introduction Methods Study area Ethics Statistical analysis Results Discussion Conclusion

group a included patients of arm with ru fistula who had undergone posterior sagittal anorectoplasty ( psarp ) without closure of ru fistula , from february 2006 to january 2010 . the rest of the psarp procedure was the same as conventionally done , the only difference being that we did not close the ru fistula after separating it from the rectum . we just separated the rectum from the urethra and left the urethral fistula as it is without closing it . group b included 34 previous successive patients who had undergone psarp before january 2006 in whom the ru fistula was closed using interrupted sutures . all the patients in both the groups had undergone staged repair of arm and not primary psarp . micturating cystourethrogram ( mcu ) and distal colostogram was done in all these patients prior to psarp . all the patients were evaluated during follow - up both clinically and with investigations like mcu and cystoscopy . the patients were studied for parameters like urinary stream , urinary dribbling , urinary tract infections , and recurrent ru fistula . moreover , all patients had undergone urethrocystoscopy three months after psarp to check for the status of the urethra and bladder . patients who had sacral agenesis were excluded from the study group because such congenital sacral defects can lead to a neurogenic bladder . the following were the observations in group a ( a ) in the immediate postoperative period , there was no urinary leakage , urinary retention , or any other complication ; thus all these patients had an uneventful recovery . ( b ) urinary stream was normal ; there was no evidence of urinary dribbling or retention , urinary tract infection , and recurrent rectourinary fistula during follow - up . mcu showed normal urethra , without any evidence of stenosis or stricture , urethro - ejaculatory duct / vasal reflux , or diverticulum in any of the cases . however , in group b , complications like urethral stenosis , urethral diverticulum , and neurogenic dysfunction were seen . a comparative analysis of the two groups was done and overall complications were listed [ table 1 ] . during psarp , urological injuries in male patients are known complications.[13 ] excessive traction on the urethra during dissection leads to transection or injury to the urethra . it is extremely important for the surgeon to bear in mind that in arm with ru fistula , the rectum is intimately attached to the urethra and that meticulous dissection and separation are necessary . urethral stenosis can occur due to traction on the ru fistula , that is , indirect traction on the urethra during separation and closure of fistula . urethral stenosis can be avoided by applying less traction on the fistula during separation and avoiding the closure of fistula ; we have seen in our series that urethral stenosis was not seen in any of the cases of group a , where the ru fistula was not closed . closing another point to note is that if we separate the rectum from the urethra very near the urethral wall and use interrupted sutures for its closure , it also increases the chances for urethral stenosis . urethral stenosis due to ligation or closure of the ru fistula may result in recurrent epididymo - orchitis . urethral diverticulum is the result of a segment of the rectum left attached to the urethra and the separated end closed . such patients usually present with recurrent urinary tract infections , stone formations in diverticulum again leading to dysuria , urinary tract infections , and so on . this complication can be avoided by separating the rectum away from the urethra without leaving any segment of the rectum attached and leaving the fistula as it is without closing it , so that nothing like a pouch / diverticulum is formed . neurogenic dysfunction after psarp has been reported in the form of neurogenic bladder , impotence , or loss of ejaculation . postoperatively , a neurogenic bladder may reflect a poor surgical technique with denervation of bladder and bladder neck during repair . by avoiding the closure of the ru fistula , we can avoid excessive traction on the fistula and hence on the urethra , and also prevent the excessive dissection during fistula closure and minimize the chance of neurogenic dysfunction . damage to the external vesical sphincter has also been reported during ligation or closure of the fistula . thus by avoiding closure of the fistula , we avoid this complication also and , hence , neurovesical dysfunction . thus we have seen that by not doing something , that is , by not closing the ru fistula during psarp , we can avoid many complications ; so , not doing something is preferable here .

aim : to study the effect of nonclosure of rectourethral ( ru ) fistula and to do a comparative analysis of the complications with and without nonclosure of ru fistula during posterior sagittal anorectoplasty ( psarp ) in anorectal malformation cases ( arm).materials and methods : a total of 68 cases of arm were included in the study group , of which 34 cases were those in whom ru fistula was not closed ( group a ) during psarp . another 34 successive cases were included in study group b in whom the ru fistula was closed as is conventionally done by using interrupted sutures.results:comparatively , group a had none or minimum urological complications as compared to group b.conclusion:ru fistula closure is not mandatory during psarp and nonclosure avoids urological complications . it especially avoids urethral complications , which are 100% preventable .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

the pathogenesis of middle ear cholesteatoma has been debated for a century , but there are four basic theories : invagination , basal cell hyperplasia , epithelial ingrowth and squamous metaplasia . during last century , etiopathogenicity of middle ear cholesteatoma raised many controversies and still a unanimous concept does n't exist . from a histological point of view this pathological tissue the interaction between cellular components is essential for the major histopathological features of cholesteatoma - hyperproliferation and bone destruction . in 1964 , the american anatomist gray referred to middle ear cholesteatoma as " skin in the wrong place " ( 1 ) . this simplified misdefinition stuck into literature and has been cited profusely in many scientific papers . the metaplasia theory ( 2 ) and existence of so - called " congenital " cholesteatoma put into question the " skin origin " of middle ear cholesteatoma and gave etiopathogenetical evidences against the simplified definition " skin in the wrong place " . the list of histological differences includes the absence of cutaneous adnexa and specialized dermal structures . neither hypodermal structures , nor blood vessels , or nerve endings have been ever described in cholesteatoma . the only similarity that is left between them is the presence of multistratified squamous epithelium , but in cholesteatoma this epithelium is with hyperproliferative properties . experimental models are critical for studying pathogenesis of middle ear cholesteatoma . in vivo models were tried to establish by chemical injection into the middle ear ( 3 ) , ligation of external auditory canal ( 4 ) and transplantation of skin ( 5 ) . keratinocytes of middle ear cholesteatoma were cultured by tissue culture ( 6 ) , passaged subculture ( 7 ) and air - liquid interface system ( 8) . however , stable experimental models of middle ear cholesteatoma are not established until now . our study has aimed to create an " adequate " in vitro model , based on the cell - to - cell interaction between keratinocytes and fibroblasts and to assess the grade of homogeneity between cholesteatoma and skin keratinocytes by focusing on the cytokeratin profile . enzymatic disaggregation protocol for simultaneous isolation of primary keratinocytes and fibroblasts from cholesteatoma tissue has been developed . the biopsy samples were harvested from patients undergoing surgical treatment in the department of otolaryngology , ajou university hospital , korea . an informed consent form approved by the institutional review board of ajou university hospital cholesteatoma keratinocytes were cultured in defined keratinocytes serum free medium ( gibco , brl , gaithersburg , md , usa ) supplied with growth supplement and 5 mg / ml gentamicin ( gibco , brl ) according to the manufacture instructions . cholesteatoma primary fibroblasts were successfully cultured in dulbeco modified eagle medium ( gibco , brl ) supplemented with 10% fetal bovine serum ( fbs , clonetics , walkersville , md , usa ) and 1% penicillin - streptomycin mixture ( gibco , brl ) . primary keratinocytes ( passage 1 ) and fibroblasts ( between passages 1 - 5 ) were used for establishment of the co - cultured system . hacat cell line was kindly provided from the department of dermatology in ajou university hospital . feeder layer from cholesteatoma fibroblasts louis , mo , usa ) in concentration 0.25 g / ml . fibroblasts were incubated overnight at 37 , 5% co2 in humidified atmosphere . on the next day the feeder layer was subsequently trypsinized from the substrate and transferred on polyester membrane in transwell ( costar , cambridge , ma , usa ) system to develop a subconfluent feeder layer . cholesteatoma keratinocytes and hacat cells were seeded at density 210 cells / ml over the feeder layer , co - cultured for 3 - 5 days and air - exposed for a period of 14 days . transwell membranes were fixed in 4% formaldehyde overnight at 4 , dehydrated in graded alcohols , cleared in histoclear ( national diagnostics , atlanta , georgia ) and embedded in paraffin . six m - thick sections were prepared and subjected to routine hematoxylin - eosin staining . polyacrylamide gel electrophoresis has been used for protein separation and staining with coomassie blue was performed to compare the protein profile between cholesteatoma keratinocytes and hacat cells . blocking of nonspecific staining was accomplished with membrane incubation with 5% non - fat skim milk for 1 hr at room temperature ( rt ) . membranes were then incubated with primary mouse monoclonal anti - cytokeratin antibody clone k 8.12 ( sigma ) which is immunospecific for cytokeratin 13 ( ck 13 ) and cytokeratin 16 ( ck 16 ) , with dilution factor 1:100 , for 1 hr at rt . after washing membranes were incubated with goat anti - mouse horseradish peroxidase conjugate ( zymed , san francisco , ca , usa ) . the positive reaction was visualized with ecl western blotting detection system ( amersham , bucks , uk ) . the localization of cytokeratin 13 and 16 in middle ear cholesteatoma tissue was accomplished by ihc study as described before ( 9 ) . five middle ear cholesteatoma samples were compared with the corresponding retroauricular skin samples obtained during surgery . six m thick formalin - fixed paraffin - embedded tissue sections were processed with abc - method . primary mouse monoclonal anticytokeratin antibody ( sigma ) at concentration 1:20 was incubated overnight at rt . vectorlab elite abc kit ( vector laboratories , burlingame , ca , usa ) was used for detection of positive reaction using diaminobenzidine substrate . enzymatic disaggregation protocol for simultaneous isolation of primary keratinocytes and fibroblasts from cholesteatoma tissue has been developed . the biopsy samples were harvested from patients undergoing surgical treatment in the department of otolaryngology , ajou university hospital , korea . an informed consent form approved by the institutional review board of ajou university hospital cholesteatoma keratinocytes were cultured in defined keratinocytes serum free medium ( gibco , brl , gaithersburg , md , usa ) supplied with growth supplement and 5 mg / ml gentamicin ( gibco , brl ) according to the manufacture instructions . cholesteatoma primary fibroblasts were successfully cultured in dulbeco modified eagle medium ( gibco , brl ) supplemented with 10% fetal bovine serum ( fbs , clonetics , walkersville , md , usa ) and 1% penicillin - streptomycin mixture ( gibco , brl ) . primary keratinocytes ( passage 1 ) and fibroblasts ( between passages 1 - 5 ) were used for establishment of the co - cultured system . hacat cell line was kindly provided from the department of dermatology in ajou university hospital . feeder layer from cholesteatoma fibroblasts louis , mo , usa ) in concentration 0.25 g / ml . fibroblasts were incubated overnight at 37 , 5% co2 in humidified atmosphere . on the next day the feeder layer was subsequently trypsinized from the substrate and transferred on polyester membrane in transwell ( costar , cambridge , ma , usa ) system to develop a subconfluent feeder layer . cholesteatoma keratinocytes and hacat cells were seeded at density 210 cells / ml over the feeder layer , co - cultured for 3 - 5 days and air - exposed for a period of 14 days . transwell membranes were fixed in 4% formaldehyde overnight at 4 , dehydrated in graded alcohols , cleared in histoclear ( national diagnostics , atlanta , georgia ) and embedded in paraffin . six m - thick sections were prepared and subjected to routine hematoxylin - eosin staining . polyacrylamide gel electrophoresis has been used for protein separation and staining with coomassie blue was performed to compare the protein profile between cholesteatoma keratinocytes and hacat cells . blocking of nonspecific staining was accomplished with membrane incubation with 5% non - fat skim milk for 1 hr at room temperature ( rt ) . membranes were then incubated with primary mouse monoclonal anti - cytokeratin antibody clone k 8.12 ( sigma ) which is immunospecific for cytokeratin 13 ( ck 13 ) and cytokeratin 16 ( ck 16 ) , with dilution factor 1:100 , for 1 hr at rt . after washing membranes were incubated with goat anti - mouse horseradish peroxidase conjugate ( zymed , san francisco , ca , usa ) . the positive reaction was visualized with ecl western blotting detection system ( amersham , bucks , uk ) . the localization of cytokeratin 13 and 16 in middle ear cholesteatoma tissue was accomplished by ihc study as described before ( 9 ) . five middle ear cholesteatoma samples were compared with the corresponding retroauricular skin samples obtained during surgery . six m thick formalin - fixed paraffin - embedded tissue sections were processed with abc - method . primary mouse monoclonal anticytokeratin antibody ( sigma ) at concentration 1:20 was incubated overnight at rt . vectorlab elite abc kit ( vector laboratories , burlingame , ca , usa ) was used for detection of positive reaction using diaminobenzidine substrate . the low selectivity and potential damages of cells in case of prolonged incubation were major disadvantages of the procedure . as an inflammatory tissue , cholesteatoma posed some difficulties in isolation of primary keratinocytes , because of the attendant inflammation and possible contamination , which requires an adequate decontamination . the first primary keratinocytes appeared usually 7 - 10 days after trypsinization and generally within the next 10 days keratinocytes reached a confluence . the individual variations in proliferative activity of cholesteatoma keratinocytes were related to cholesteatoma localization and sex of the patient . for instance , attic cholesteatoma frequently resulted in isolation of primary keratinocytes with bigger proliferative ability . another interesting correlation was the fact that cholesteatoma samples obtained from young female patients were characterized by higher proliferative activity in vitro . primary cholesteatoma keratinocytes were uniform in shape and arranged in a typical monolayer pattern ( fig . in passage 2 an abrupt deceleration in proliferation was established , accompanied by morphological alterations such as enlargement and flattening of the cells , as well as vacuolization of the cytoplasm . in passage 3 primary cholesteatoma fibroblasts were steadily maintained without significant changes of doubling time until passage 10 . bigger stratification and proliferation have been observed in the proposed 3d in vitro system when primary cholesteatoma keratinocytes were co - cultured in the presence of fibroblasts ( fig . 3b ) in comparison with the control group without fibroblasts ( fig . 3a ) . 4a ) . disrupted differentiation in the suprabasal layers was revealed when cholesteatoma keratinocytes were air - exposed in the presence of fibroblasts mimicking the parakeratosis state , which is typical for this pathological tissue in vivo ( fig . coomassie blue staining of the protein extracts from primary cholesteatoma keratinocytes and hacat cells revealed significantly different protein profiles ( fig . stronger expression of ck 16 ( 48 kda ) and ck 13 ( 51 kda ) was established in cholesteatoma keratinocytes in comparison with hacat cells ( fig . 5b ) . using mouse monoclonal antibody clone k 8.12 , tissue localization of ck 13 and ck 16 the intensity of the ck 13 , 16-positive staining varied in different areas of the same cholesteatoma sample . the positive control revealed specific expression of ck 13 and 16 confined to the islands of neoplastic squamous cells in laryngeal carcinoma specimens . the low selectivity and potential damages of cells in case of prolonged incubation were major disadvantages of the procedure . as an inflammatory tissue , cholesteatoma posed some difficulties in isolation of primary keratinocytes , because of the attendant inflammation and possible contamination , which requires an adequate decontamination . the first primary keratinocytes appeared usually 7 - 10 days after trypsinization and generally within the next 10 days keratinocytes reached a confluence . the individual variations in proliferative activity of cholesteatoma keratinocytes were related to cholesteatoma localization and sex of the patient . for instance , attic cholesteatoma frequently resulted in isolation of primary keratinocytes with bigger proliferative ability . another interesting correlation was the fact that cholesteatoma samples obtained from young female patients were characterized by higher proliferative activity in vitro . primary cholesteatoma keratinocytes were uniform in shape and arranged in a typical monolayer pattern ( fig . in passage 2 an abrupt deceleration in proliferation was established , accompanied by morphological alterations such as enlargement and flattening of the cells , as well as vacuolization of the cytoplasm . in passage 3 primary cholesteatoma fibroblasts were steadily maintained without significant changes of doubling time until passage 10 . bigger stratification and proliferation have been observed in the proposed 3d in vitro system when primary cholesteatoma keratinocytes were co - cultured in the presence of fibroblasts ( fig . 3b ) in comparison with the control group without fibroblasts ( fig . 3a ) . 4a ) . disrupted differentiation in the suprabasal layers was revealed when cholesteatoma keratinocytes were air - exposed in the presence of fibroblasts mimicking the parakeratosis state , which is typical for this pathological tissue in vivo ( fig . coomassie blue staining of the protein extracts from primary cholesteatoma keratinocytes and hacat cells revealed significantly different protein profiles ( fig . ck 13 ( 51 kda ) was established in cholesteatoma keratinocytes in comparison with hacat cells ( fig . using mouse monoclonal antibody clone k 8.12 , tissue localization of ck 13 and ck 16 was verified both in the basal and suprabasal layers of cholesteatoma ( fig . the intensity of the ck 13 , 16-positive staining varied in different areas of the same cholesteatoma sample . the positive control revealed specific expression of ck 13 and 16 confined to the islands of neoplastic squamous cells in laryngeal carcinoma specimens . the putative interactions between major cellular components of cholesteatoma tissue have been extensively studied in the literatures . we demonstrate a summarized schematic model of possible intercellular " cross - talking " between epithelial - mesenchymal components of cholesteatoma in fig . pro - inflammatory cytokines such as interleukin-1 and interleukin-1 , ( il-1 , il-1 ) released from matrix keratinocytes stimulate keratinocytes growth factor ( kgf ) expression , which in turn induced proliferation of the cholesteatoma keratinocytes ( 10 ) . granulocyte - macrophage colony stimulating factor ( gm - csf ) is another fibroblasts - derived growth factor induced by il-1 , with acknowledged stimulating effects both on proliferation and terminal differentiation of keratinocytes ( 11 ) . keratinocytes - derived parathyroid hormone - related protein ( pthrp ) induces expression of kgf . kgf is then released by perimatrix fibroblasts and accelerates the proliferative response in matrix keratinocytes . a possible connection between pthrp and bone destructive lesions have also been reported ( 12 ) . transforming growth factor alpha ( tgf- ) is constitutively expressed in keratinocytes and regulates keratinocyte proliferation and differentiation in an autocrine manner ( 13 ) . on the other hand , a possible synergism between tgf- and epidermal growth factor ( egf ) was proposed with possible effect on keratinocytes proliferation through epidermal growth factor receptor ( egf - r ) , indicating existence of an alternative paracrine regulatory mechanism ( 14 ) . transforming growth factor - beta ( tgf- ) is expressed in hyperproliferative epithelium and granulation tissue ( 15 ) and influences the epithelial morphogenesis both in an autocrine and paracrine fashion . the above statements about cellular interactions were taken into consideration for creating an adequate in vitro middle ear cholesteatoma model . as it was mentioned before , the proliferative capacity of primary cholesteatoma keratinocytes in vitro is limited and expires within 2 passages . to improve their plating efficiency and survival rate at low density 3t3-mouse embryo fibroblasts that used routinely in cell culture practice display different morphological patterns ( cobble - stone morphology ) , which differs from in vivo state and complicates result interpretation . feeder layer secretes nutrients , growth factors and matrix constituents . as a positive control in our study we choose hacat cell line in order to avoid donor - to - donor and site - to - site variations in proliferative capacity of normal skin keratinocytes . hacat cells are spontaneously immortalized human adult skin keratinocytes raised in reduced calcium level and elevated temperature conditions ( 16 ) . hacat found an extensive application in tissue engineering for development of artificial skin equivalents ( 17 ) . the proposed organotypic in vitro model revealed stronger proliferation and bigger stratification , when hacat cells were co - cultured with fibroblasts , in comparison with the control group without fibroblasts . histopathological diagnosis of cholesteatoma includes : unrestrained proliferation , uncoordinated differentiation , migration , and invasion ( 18 ) . consistent with the above - mentioned observation , the proposed 3d - model disclosed disrupted differentiation and presence of nucleated cells in the upper layers of the cholesteatoma ali with fibroblasts . squamous metaplasia of middle ear epithelium is considered to be an adaptive tissue response towards persistent pathological stimuli such as chronic inflammation and possible etiopathogenetical mechanism for middle ear cholesteatoma . an experiment has been reported on the confirmation of a possible transformation of differentiated middle ear epithelium towards functionally inferior multilayered squamous epithelium ( 19 ) . epithelial cells are characterized by the presence of a specific set of intermediate protein filaments named cytokeratins , specific for different epithelial types . one of our primary objectives was to assess the grade of homogeneity between hacat cells and cholesteatoma keratinocyte . ck 16 was suggested in the literature as a proliferation marker , while ck 13 is considered as a marker of differentiation ( 20 ) . immunohistochemical investigation in our study disclosed non - uniform patterns of expression of ck 13 and ck 16 in middle ear cholesteatoma tissue . variation in intensity of the positively stained areas between different cholesteatoma samples as well as between different areas in the same sample was probably related to the different proliferative capacity of cholesteatoma . the expression of nonepidermal cytokeratins in middle ear cholesteatoma tissue gives arguments against the probable skin origin of mech keratinocytes . protein separation and coomassie blue staining revealed significantly different protein profile between cholesteatoma and hacat cells . precised western - blotting analysis demonstrated comparatively stronger expression of ck 13 and 16 in cholesteatoma keratinocytes with regard to hacat cell line . the different protein and cytokeratin profiles established between cholesteatoma keratinocytes and hacat cells favors metaplasia as a possible cause for development of mech . the proposed in vitro model underlines the importance of fibroblasts as a major component of 3d - in vitro system . our results emphasize the integrity of cholesteatoma as a separated pathological tissue , composed of two closely related functional and structural parts : the matrix and the perimatrix .

objectivesexperimental models are of importance to study the pathogenesis of middle ear cholesteatoma , however , they were not established until now . we aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue . hacat cell line was used as a " skin equivalent " and to compare the grade of homogeneity between cholesteatoma keratinocytes and hacat cells.methodsprimary keratinocytes were isolated from cholesteatoma tissue , co - cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air - exposed . the protein profile of cholesteatoma keratinocytes and hacat cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin ( ck ) 13 and 16 . tissue localization of ck 13 and 16 was accomplished with immunohistochemistry.resultsdifferent protein profile and stronger expression of ck 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with hacat cells . bigger stratification was observed in the 3d - in vitro systems when both cholesteatoma keratinocytes and hacat cells were respectively co - cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts.conclusion3d-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue .

INTRODUCTION MATERIALS AND METHODS Cell culture Morphological study Protein profile Cytokeratin profile Immunohistochemistry (IHC) RESULTS Cell culture Air-liquid interface (ALI) Protein and cytokeratin profile Immunohistochemistry DISCUSSION CONCLUSIONS

dynamic biomolecules often undergo large - scale structural changes and visit distinct conformational states during their biological function . it is of great biological and pharmaceutical interest to characterize their structures and conformational transition pathways . ideally , detailed free energy landscapes are sought to understand the functional mechanisms of biomolecules in a quantitative manner . however , due to large energy barriers , conformational transitions of biomolecules usually take place on time scales of milliseconds or even longer . this has presented a grand challenge for computational molecular dynamics ( md ) simulations that are limited to typically hundreds - of - nanoseconds to tens - of - microseconds . to address this challenge , biasing simulation methods have been found useful in enhanced sampling and free energy calculation of the biomolecules . these methods include umbrella sampling , conformational flooding , metadynamics , adaptive biasing force ( abf ) calculations , and orthogonal space sampling , etc . during the simulations , a potential or force bias is applied along certain reaction coordinates ( or collective variables ) to facilitate the biomolecular conformational transitions across high - energy barriers . typical reaction coordinates include atom distances , torsional dihedrals , root - mean - square deviation ( rmsd ) relative to a reference configuration , eigenvectors generated from the principal component analysis , and so on . the definition of the reaction coordinates , however , often requires expert knowledge of the studied systems . furthermore , the predefined reaction coordinates largely place constraints on the pathway and conformational space to be sampled during the biasing simulations . it often leads to slow convergence of the simulations when important reaction coordinates are missed during the simulation setup . accelerated molecular dynamics ( amd ) is an enhanced sampling technique that works often by adding a non - negative boost potential to smoothen the biomolecular potential energy surface . the boost potential , v , decreases the energy barriers and thus accelerates transitions between the different low - energy states . with this , amd is able to sample distinct biomolecular conformations and rare barrier - crossing events that are not accessible to conventional md ( cmd ) simulations . unlike the previously mentioned biasing simulation methods , amd does not require predefined reaction coordinate(s ) , which can be advantageous for exploring the biomolecular conformational space without a priori knowledge or restraints . amd has been successfully applied to a number of biological systems , ( 10 ) and hundreds - of - nanosecond amd simulations have been shown to capture millisecond - time - scale events in both globular and membrane proteins . while amd has been demonstrated to greatly enhance conformational sampling of biomolecules , it suffers from large energetic noise during reweighting . the boost potential applied in amd simulations is typically on the order of tens - to - hundreds of kilocalories per mole , which is much greater in magnitude and wider in distribution than that of other biasing simulation methods that make use of predefined reaction coordinates ( e.g. , several kilocalories per mole ) . it has been a long - standing problem to accurately reweight amd simulations and recover the original free energy landscapes , especially for large proteins . our recent studies showed that when the boost potential follows near - gaussian distribution , cumulant expansion to the second order provides improved reweighting of amd simulations compared with the previously used exponential average and maclaurin series expansion reweighting methods . the reweighted free energy profiles are in good agreement with the long - time - scale cmd simulations as demonstrated on alanine dipeptide and fast - folding proteins . however , such improvement is limited to rather small systems ( e.g. , proteins with less than 35 amino acid residues ) . in simulations of larger systems , the boost potential exhibits significantly wider distribution and , a gaussian accelerated molecular dynamics ( gamd ) approach is presented to reduce the energetic noise for simultaneous unstrained enhanced sampling and free energy calculation of biomolecules , even for large proteins . gamd makes use of harmonic functions to construct the boost potential that is adaptively added to the biomolecular potential energy surface . a minimal set of simulation parameters is dynamically adjusted to control the magnitude and distribution width of the boost potential . as such , the resulting boost potential follows gaussian distribution and allows for accurate reweighting of the simulations using cumulant expansion to the second order . in this study , gamd is demonstrated on unconstrained simulations of the alanine dipeptide , chignolin folding , and ligand binding to the t4-lysozyme . gaussian accelerated molecular dynamics enhances the conformational sampling of biomolecules by adding a harmonic boost potential to smoothen the system potential energy surface ( figure 1 ) . consider a system with n atoms at positions = { 1, ... ,n } . when the system potential v ( ) is lower than a threshold energy , e , a boost potential is added as1where k is the harmonic force constant . the modified system potential , v * ( ) = v ( ) + v ( ) , is given by2otherwise , when the system potential is above the threshold energy , i.e. , v ( ) e , the boost potential is set to zero and v * ( ) = v ( ) . when the threshold energy is set to the maximum potential ( e = vmax ) , the system potential energy surface is smoothened by adding a harmonic boost potential that follows gaussian distribution . the coefficient k0 in the range of 01 determines the magnitude of the applied boost potential . with greater k0 , higher boost potential is added to the original energy surface in conventional molecular dynamics ( cmd ) , which provides enhanced sampling of biomolecules across decreased energy barriers . in order to smoothen the potential energy surface for enhanced sampling , first , for any two arbitrary potential values v1 ( ) and v2 ( ) found on the original energy surface , if v1 ( ) < v2 ( ) , v should be a monotonic function that does not change the relative order of the biased potential values ; i.e. , v1 * ( ) < v2 * ( ) . by replacing v * ( ) with eq 2 and isolating e , we then obtain3second , if v1 ( ) < v2 ( ) , the potential difference observed on the smoothened energy surface should be smaller than that of the original ; i.e. , v2 * ( ) v1 * ( ) < v2 ( ) v1 ( ) . similarly , by replacing v * ( ) with eq 2 , we can derive4with vmin v1 ( ) < v2 ( ) vmax , we need to set the threshold energy e in the following range by combining eqs 3 and 4:5where vmin and vmax are the system minimum and maximum potential energies . to ensure that eq 5 is valid , vmax vmin + ( 1/k ) and k has to satisfy the following:6we define k k0(1/(vmax vmin ) ) ; then 0 < k0 1 . as illustrated in figure 1 , k0 determines the magnitude of the applied boost potential . with greater k0 , higher boost potential is added to the potential energy surface , which provides enhanced sampling of biomolecules across decreased energy barriers . third , the standard deviation of v needs to be small enough ( i.e. , narrow distribution ) to ensure accurate reweighting using cumulant expansion to the second order:7where vav and v are the average and standard deviation of the system potential energies and v is the standard deviation of v with 0 as a user - specified upper limit ( e.g. , 10kbt ) for accurate reweighting . provided eq 5 gives the range of threshold energy e , when e is set to the lower bound e = vmax , we plug in e and k and obtain8we define the right - hand side in eq 8 as k0 = ( 0/v)((vmax vmin)/(vmax vav ) ) . for efficient enhanced sampling with the highest possible acceleration , k0 can then be set to its upper bound as9the greater v that is obtained from the original potential energy surface ( particularly for large biomolecules ) , the smaller k0 may be applicable to allow for accurate reweighting . alternatively , when the threshold energy e is set to its upper bound e = vmin + ( 1/k ) according to eq 5 , we plug in e and k in eq 7 and obtain10we define the right - hand side in eq 10 as k0 ( 1 ( 0/v ) ) ( ( vmax vmin)/(vav vmin ) ) . when k0 0 with v 0 , k0 can be theoretically set to any value between zero and 1 , although k0 = 1 is applied in this case for the current implementation of gamd ( see appendix a ) . note that a smaller k0 will give higher threshold energy e , but smaller force constant k. when 0 < k0 1 , k0 can be set to either k0 for the highest threshold energy e or its upper bound 1.0 for the greatest force constant k. in this case k0 = k0 is applied in the current gamd implementation . when k0 > 1 , we have to lower the threshold energy e to ensure that 0 < k0 1 ; e.g. , e = vmax for default k0 = 1 . given e and k0 , we can calculate the boost potential as11similar to amd , gamd provides options to add only the total potential boost vp , only dihedral potential boost vd , or the dual potential boost ( both vp and vd ) . the dual - boost simulation generally provides higher acceleration than the other two types of simulations for enhanced sampling . the simulation parameters comprise of the threshold energy values and the effective harmonic force constants , k0p and k0d for the total and dihedral potential boost , respectively . to characterize the extent to which v follows gaussian distribution , its distribution anharmonicity , , is calculated as was done previously:12where v is dimensionless as divided by kbt with kb and t being the boltzmann constant and system temperature , respectively , and smax = ( 1/2 ) ln(2ev2 ) is the maximum entropy of v . reweighting by approximating the exponential average term with cumulant expansion to the second order is able to accurately recover the original free energy landscape ( appendix b ) . as increases , the v distribution becomes less harmonic and the reweighted free energy profile obtained from cumulant expansion to the second order deviates from the original . as demonstrated on the alanine dipeptide , chignolin , and t4-lysozyme in this study , a gaussian distribution of the boost potential is normally achieved in the gamd simulations . the anharmonicity of v distribution serves as an indicator of the enhanced sampling convergence and accuracy of the reweighted free energy . gamd is currently implemented in the gpu version of amber 12 ( see appendix a for implementation details ) . simulations of the alanine dipeptide , chignolin , and t4-lysozyme were performed using the amber ff99sb force field on gpus . , the ligand benzene was removed from the x - ray crystal structure of the leu99ala mutant ( protein data bank ( pdb ) , 181l ) . another four benzene molecules were placed in the bulk solvent at least 40 away from the ligand - binding site in the starting configuration . by solvating the structures in a tip3p water box that extends 810 from the solute surface , the alanine dipeptide system contained 630 water molecules , 2,211 waters for chignolin , and 9,011 waters for t4-lysozyme . the total number of atoms in the three systems are 1,912 ; 6,773 ; and 29,692 for alanine dipeptide , chignolin , and t4-lysozyme , respectively ( table 1 ) . bonds containing hydrogen atoms were restrained with the shake algorithm , and a 2 fs time step was used . weak coupling to an external temperature and pressure bath was used to control both temperature and pressure . the electrostatic interactions were calculated using the pme ( particle mesh ewald summation ) with a cutoff of 8.0 for long - range interactions . the three systems were initially minimized for 2,000 steps using the conjugate gradient minimization algorithm , and then the solvent was equilibrated for 50 ps in an isothermal isobaric ( npt ) ensemble with the solute atoms fixed . another minimization was performed with all atoms free , and the systems were slowly heated to 300 k over 500 ps . final system equilibration was achieved by a 200 ps isothermal isovolumetric ( nvt ) and 400 ps npt run to ensure that the water box of the simulated systems had reached the appropriate density . in the present study , the system threshold energy is set as e = vmax for all gamd simulations . the maximum , minimum , average , and standard deviation values of the system potential ( vmax , vmin , vav , and v ) were obtained from an initial 2 ns nvt simulation with no boost potential . for optimal acceleration , the greatest 0 and k0 were determined through short testing simulations ( e.g. , 2 ns ) : with increasing 0 , either k0 calculated using eq 9 reaches 1.0 ( highest acceleration level ) or v reaches 10kbt ( the upper limit of the v distribution width for accurate reweighting ) . each gamd simulation proceeds with a 2 ns equilibration run , followed by production simulations . dihedral potential boost only to identify the optimal 0 and k0 acceleration parameters ( supporting information tables s1s3 ) . long - time dual - boost gamd simulations were then obtained for analysis , including three independent 30 ns simulations of alanine dipeptide and three independent 300 ns simulations of chignolin . complete binding of benzene to the target ligand - binding site was observed in one of the five simulations . even when the simulation was extended to 1,800 ns , time courses of dihedral angles , rmsd , radius of gyration ( rg ) , and residue distances in the amber simulation trajectories were analyzed using the cpptraj tool . particularly , the backbone dihedral angles and were calculated for alanine dipeptide ( figure 2a ) . for chignolin , the rg and rmsd of simulation frames relative to the pdb native structure ( figure 3a ) were calculated for the protein c atoms with the terminal residues gly1 and gly10 excluded . for ligand binding to the t4-lysozyme ( figure 5a ) , the symmetry - corrected rmsd of benzene was obtained by generating six symmetrically imaged reference benzene configurations from the 181l crystal structure , calculating the rmsds for the diffusing benzene molecules in each frame after aligning the protein c - terminal domain ( residues 80160 ) and then extracting the minimum value of the calculated rmsds . moreover , the number of protein atoms found within 5 of benzene ( ncontact , only heavy atoms are considered ) was calculated using pbwithin in vmd that accounts for the periodic boundary conditions . the pyreweighting toolkit was used to reweight the gamd simulations for calculating the pmf profiles and to examine the boost potential distributions . two - dimensional ( 2d ) pmf profiles were computed for backbone dihedrals ( , ) in alanine dipeptide . a bin size of 6 is selected to balance between reducing the anharmonicity and increasing the bin resolution as discussed earlier . two - dimensional pmf profiles were also constructed using ( rmsd , rg ) for chignolin with a bin size of ( 1.0 , 1.0 ) . for benzene binding to the t4-lysozyme , 2d pmf was constructed using ( ligand rmsd , ncontact ) with a bin size of ( 1.0 , 5 ) . when the number of simulation frames within a bin is lower than a certain limit ( i.e. , cutoff ) , the bin is not sufficiently sampled and thus is excluded for reweighting . the cutoff can be determined by iteratively increasing it until the minimum position of the pmf profile does not change . the final cutoff was set as 10 , 50 , and 1000 for reweighting of gamd simulations on alanine dipeptide , chignolin , and t4-lysozyme , respectively . gamd provides enhanced sampling of conformational transitions in alanine dipeptide , chignolin folding , and ligand binding to the t4-lysozyme . furthermore , the boost potential applied in the present gamd simulations follows gaussian distribution , which allows accurate reweighting using cumulant expansion to the second order and recovery of the original biomolecular free energy landscapes , even for proteins as large as the t4-lysozyme . notably , hundreds - of - nanoseconds gamd simulations are able to capture complete folding of chignolin and ligand binding of benzene to the t4-lysozyme that take place on significantly longer time scales . in order to balance between achieving the highest acceleration ( large v ) and ensuring accurate reweighting ( small enough standard deviation , v ) , short gamd simulations ( 2 ns ) were tested on alanine dipeptide to search for optimal acceleration parameters . for the total potential boost , when 0p was adjusted from 1.0 to 2.0 , the resulting k0p calculated using eq 9 was increased from 0.21 to 1.0 , accompanied by increases of the vp standard deviation from 1.03 to 1.75 kcal / mol and the vp average from 2.36 to 3.85 kcal / mol ( see supporting information table s1a ) . after k0p reached its maximum 1.0 , slight changes were observed when 0p was further increased from 2.0 to 3.0 . for the dihedral potential boost , the calculated k0d reached 1.0 when 0d was increased to 3.0 ( supporting information table s1b ) . therefore , ( 0p,0d ) were set to ( 3.0 , 3.0 ) for the production dual - boost gamd simulations of alanine dipeptide , which enables the highest acceleration as well as accurate reweighting with both k0p and k0d equal to 1.0 ( supporting information table s1c ) . as shown in figure 2b , the boost potential v applied in gamd simulation of the alanine dipeptide follows gaussian distribution . the average and standard deviation of v are 10.9 and 2.9 kcal / mol , respectively ( table 1 ) . figure 2c plots the 2d pmf of backbone dihedrals ( , ) that are obtained by reweighting the three 30 ns gamd simulations combined using cumulant expansion to the second order . the reweighted pmf is able to recover five energy minimum wells that are centered around ( 162 , 12 ) and ( 72 , 12 ) for the right - handed helix ( r ) , ( 48 , 18 ) for the left - handed helix ( l ) , ( 156 , 162 ) for the -sheet , and ( 66 , 156 ) for the polyproline ii ( pii ) conformation . the corresponding minimum free energies are estimated as 0 , 1.40 , 1.07 , 2.94 , and 4.27 kcal / mol , respectively . this is in good agreement with the pmf results of an exceptionally long 1000 ns cmd simulation as presented earlier . additionally , the distribution anharmonicity of vof frames clustered in each bin of the 2d pmf exhibits values smaller than 0.10 in the low - energy regions ( figure 2d ) , which suggests sufficient sampling for reweighting using cumulant expansion to the second order . greater anharmonicity is found in high - energy regions ( especially the energy barriers ) , for which low boost potential is applied and less sampling is normally achieved . therefore , the anharmonicity of v distribution appears to be a good indicator for sufficiency of the enhanced sampling and accuracy of the reweighted free energy . ( a ) scheme representation of backbone dihedrals and in alanine dipeptide . ( c ) 2d potential of mean force ( pmf ) of backbone dihedrals ( , ) calculated from three 30 ns gamd simulations combined using cumulant expansion to the second order . the low - energy wells are labeled corresponding to the right - handed helix ( r ) , left - handed helix ( l ) , -sheet ( ) , and polyproline ii ( pii ) conformations . ( d ) distribution anharmonicity of v of frames found in each bin of the pmf profile . for chignolin , short 2 ns testing gamd simulations showed that k0p reached the 1.0 maximum for the highest total potential boost when 0p was increased to 3.0 ( supporting information table s2a ) . for the dihedral potential boost , the simulation crashed when 0d was increased to 0.9 and stable simulation was achieved only when 0d was increased to 0.6 ( supporting information table s2b ) . thus , ( 0p,0d ) were set to ( 3.0 , 0.6 ) for the production dual - boost gamd simulations of chignolin ( supporting information table s2c ) . the resulting boost potential follows gaussian distribution and the calculated = 9.22 10 ( figure 3b ) . the average and standard deviation of v are 9.8 and 2.4 kcal / mol , respectively ( table 1 ) . folding of chignolin captured in gamd simulations : ( a ) comparison of simulation - folded chignolin ( blue ) with the pdb ( 1uao ) native structure ( red ) that exhibits 0.2 rmsd , ( b ) distribution of the boost potential v , ( c ) 2d ( rmsd , rg ) pmf calculated by reweighting the three 300 ns gamd simulations combined , and ( d ) the distribution anharmonicity of v of frames found in each bin of the pmf profile . started from an extended conformation , gamd simulations were able to capture complete folding of chignolin into its native structure ( supporting information movie s1 ) . the rmsd obtained between the simulation - folded chignolin and nmr experimental native structure ( pdb , 1uao ) reaches a minimum of 0.2 ( figure 3a ) . using the protein rmsd relative to the pdb native structure and rg , a 2d pmf profile was calculated by reweighting the three 300 ns gamd simulations combined ( figure 3c ) . the reweighted pmf allows us to identify three distinct low - energy conformational states : the folded ( f ) , unfolded ( u ) , and an intermediate ( i ) . the folded state corresponds to the global energy minimum at ( 1.0 , 4.0 ) , the unfolded state is 3.68 kcal / mol higher in a local - energy well centered at ( 6.0 , 7.0 ) , and the intermediate in a 3.06 kcal / mol free energy well centered at ( 4.0 , 5.5 ) . the energy barrier for chignolin folding between the unfolded and folded states is 4.0 kcal / mol , and 3.5 kcal / mol for transitions between the intermediate and folded states ( figure 3c ) . in comparison , when the three 300 ns gamd simulations were analyzed separately , the reweighted pmf profiles exhibit significant differences ( supporting information figure s2 ) . whereas the three folded , intermediate , and unfolded low - energy states were captured in sim2 , the unfolded and intermediate states were not sufficiently sampled ( no converged low - energy wells ) in sim1 and sim3 , respectively . figure 3d plots the distribution anharmonicity of v for frames found in each bin of the 2d pmf as shown in figure 3c . the anharmonicity exhibits values smaller than 0.05 in the simulation sampled conformational space , suggesting that v achieves sufficient sampling for reweighting using cumulant expansion to the second order . compared with the native structure , the unfolded chignolin exhibits an extended conformation without proper secondary structure formed in the protein backbone as shown in figure 4a . the intermediate conformation observed during folding of chignolin is characterized by hydrophobic interactions between the pro4 and trp9 residue side chains and a turn at thr8 . such conformation was also observed in previous microsecond - time - scale cmd simulations . in the simulation - derived folded state , the protein residue side chains exhibit closely similar conformations as in the nmr native structure ( figure 4c ) . residues tyr2 and pro4 form hydrophobic interactions between their side chains . hydrophilic residues asp3 , glu5 , thr6 , and thr8 expose side chains to the solvent . structures of chignolin observed during gamd simulations corresponding to the ( a ) unfolded ( u ) , ( b ) intermediate ( i ) , and ( c ) folded ( f ) states ( blue ) aligned to the pdb native structure ( red ) . residues including tyr2 , asp3 , pro4 , glu5 , thr6 , thr8 , and trp9 are represented by sticks . finally , a 2d free energy profile of chignolin was calculated using the rmsd of the protein c atoms relative to the native pdb structure and the backbone dihedral of residue gly7 ( supporting information figure s3 ) . this allows for identification of a misfolded ( m ) low - energy state , in addition to the f , i , and u states as observed in figure 3c . the m conformation of chignolin shows 180 rotation of the c - terminal strand about its long axis relative to the native pdb structure . the free energy of the misfolded chignolin is found to be 1.89 kcal / mol greater than that of the folded state . this is consistent with previous findings that the misfolded chignolin was observed with higher probability in simulations with the amber ff99sb force field , which is used in the present study . short testing gamd simulations of the t4-lysozyme showed that k0p reached the 1.0 maximum for the highest total potential boost when 0p was increased to 3.0 ( supporting information table s3a ) . for the dihedral potential boost , the simulation crashed when 0d was increased to 5.0 ( supporting information table s3b ) . stable simulation was achieved when 0d was increased to 4.0 and the calculated k0d was 0.35 . with further testing for stable dual - boost gamd simulation , ( 0p,0d ) were set to ( 3.0 , 4.0 ) for the final production simulations of the t4-lysozyme ( supporting information table s3c ) . gamd captured complete binding of benzene to the deeply buried ligand - binding cavity in the leu99ala t4-lysozyme within 100 ns in one of the five independent 800 ns simulations ( supporting information movie s2 ) . benzene remained bound in the ligand - binding site even when the simulation was extended to 1,800 ns . by aligning the c - terminal domain ( residues 80160 ) of the t4-lysozyme , the rmsd of the diffusing benzene molecules relative to the bound pose in the 181l x - ray crystal structure reaches a minimum of 0.1 ( figure 5a ) . the boost potential applied during the 1,800 ns gamd simulation follows gaussian distribution , and its = 1.39 10 ( figure 5b ) . the average and standard deviation of v are 36.5 and 4.7 kcal / mol , respectively ( table 1 ) . although the v average values exhibit variations between five independent simulations , the v standard deviations are closely similar to each other provided that ( 0p,0d ) were set to ( 3.0 , 4.0 ) ( supporting information table s3c ) . using the rmsd of benzene relative to the bound pose and the number of protein heavy atoms that are within 5 of benzene ( ncontact ) , a 2d pmf profile was calculated by reweighting the 1,800 ns gamd simulation ( figure 5c ) . the reweighted pmf allows us to identify three distinct low - energy states : the unbound ( u ) , intermediate ( i ) , and bound ( b ) states . the bound state corresponds to the global energy minimum located at (0 , 30 ) , the unbound state in a local - energy well centered at (33 , 0 ) , and the intermediate centered at (11 , 20 ) . it is important to note that since the complete binding of benzene to the target ligand - binding site was observed only once , the calculated binding free energy between the bound and unbound states is subject to the error of limited sampling . nevertheless , benzene visits the intermediate site many times during the 1800 ns gamd simulation with the ligand rmsd decreased to 11 ( supporting information figure s2a ) . repeated sampling of the intermediate state was observed in the other four 800 ns gamd simulations as well ( supporting information figure s2 ) , for which a local - energy well appears around ( 11.0 , 20 ) in the 2d pmf profiles ( supporting information figure s3 ) . the relative free energy between the intermediate and unbound states is estimated to be 0.53 0.46 kcal / mol from pmf profiles of the five gamd simulations . furthermore , benzene was observed to bind another intermediate 2 ( i2 ) site that is located in the pocket formed by the hinge c helix and the b helix from the n - terminal domain ( supporting information figure s2 ) . a corresponding local energy well of the i2 state appears in the calculated 2d pmf profiles . figure 5d plots the v distribution anharmonicity , , for frames found in each bin of the 2d pmf . it exhibits relatively large values in the high - energy regions ( less sampling ) , notably the energy barrier between the intermediate and bound states . the ligand entry from the intermediate to the bound state is thus suggested to be the rate - limiting step for benzene binding . in comparison , exhibits values smaller than 0.01 in the energy well regions , suggesting that v achieves sufficient sampling for reweighting using cumulant expansion to the second order . binding of benzene to the leu99ala t4-lysozyme simulated via gamd : ( a ) comparison of simulation - derived complex structure that captures benzene binding ( blue ) with 0.1 ligand rmsd relative to the 181l pdb structure ( red ) , ( b ) distribution of the boost potential v , ( c ) 2d ( ligand rmsd , ncontact ) pmf calculated by reweighting the 1,800 ns gamd simulation , and ( d ) distribution anharmonicity of v of frames found in each bin of the free energy profile . a complete binding pathway of benzene that was observed in the gamd simulation is shown in figure 6a . benzene diffuses from the bulk solvent to the protein surface formed by the d and g helices and then to the target ligand - binding site in the protein c - terminal domain . figure 6b depicts the unbound pose of a benzene molecule that is located far away from the ligand - binding site in the 181l x - ray crystal structure . in the intermediate state , benzene interacts with residues lys83 , pro86 , and val87 from the d helix and the thr115 , thr119 , and gln122 residues from the g helix ( figure 6c ) . in the bound pose , benzene is superimposable with the ligand cocrystallized in the 181l crystal structure . it forms hydrophobic interactions with residues ile78 , leu84 , tyr88 , val87 , leu91 , val111 , leu118 , and leu121 in the deeply buried protein cavity ( figure 6d ) . supporting information figure s5 shows a transient snapshot observed during benzene binding between the intermediate and bound poses . benzene appears to slide into the ligand - binding cavity , interacting with residues gln81 , lys83 , leu84 , val111 , phe114 , thr115 , and leu118 from the d , f , and g helices . ( a ) pathway of benzene binding to the t4-lysozyme observed during the gamd simulation . ( b d ) the ( b ) unbound ( u ) , ( c ) intermediate ( i ) , and ( d ) bound ( b ) poses of the protein ligand complex ( blue ) with the protein c - terminal domain ( residues 80160 ) aligned to the pdb native structure ( red ) . the protein and benzene are represented by ribbons and spheres , respectively , and they are colored by blue for the simulation structure while red for the pdb native structure , except that in a the simulated benzene is represented by lines and colored by simulation time in a bwr color scale . residues with heavy atoms found within 3 of benzene are represented by sticks . by adaptively adding a harmonic boost potential to smoothen the system energy surface , gamd provides both unconstrained enhanced sampling and free energy calculation of biomolecules . important statistical properties of the system potential , such as the average , maximum , minimum , and standard deviation values , are used to calculate the simulation acceleration parameters , particularly the threshold energy , e , and force constant , k0 . for alanine dipeptide , both k0p and k0d can be increased to the 1.0 maximum for the greatest possible boost of the total and dihedral potential energies . the resulting standard deviation of v that follows gaussian distribution is 2.9 kcal / mol , which allows for accurate reweighting using cumulant expansion to the second order . notably , high - energy regions of the gamd sampled free energy surface ( especially the energy barriers ) exhibit increased anharmonicity compared with the low - energy wells ( figure 2d ) . thus , the free energy barriers appear unconverged and still suffer from insufficient sampling during the three short 30 ns gamd simulations . nevertheless , the gamd reweighted pmf profile is able to recover low - energy states of the system that are in good agreement with those observed in a 1000 ns cmd simulation . furthermore , gamd is able to fold chignolin from an extended conformation to the nmr native structure in three independent 300 ns simulations . while k0p can be increased to 1.0 for the greatest total potential boost , k0d can reach only 0.150.17 for stable simulation . nevertheless , this seems to provide sufficient sampling of the chignolin - folding process . among the three independent simulations , although the folded , intermediate , and unfolded low - energy states are captured in sim2 , the unfolded and intermediate states are not sufficiently sampled in sim1 and sim3 , respectively ( supporting information figure s2 ) . all three folded , intermediate , and unfolded states are identified from the resulting reweighted free energy profiles . particularly , the intermediate conformation was also observed in earlier microsecond - time - scale cmd simulations . gamd appears to achieve better convergence than the previous amd simulations that were not able to distinguish between the intermediate and unfolded states within the same simulation time . finally , gamd captured complete binding of benzene to the ligand - binding site of the t4-lysozyme . in one of the five independent simulations , benzene diffused into the deeply buried ligand - binding cavity within 100 ns and remained bound even when similar to chignolin , while k0p can be increased to the 1.0 maximum for the greatest total potential boost in the t4-lysozyme simulations , k0d can reach only 0.330.35 for the greatest dihedral boost . the resulting v standard deviation is 4.7 kcal / mol in the final dual - boost gamd simulations . such narrow distribution of v ensures accurate reweighting using cumulant expansion to the second order . distinct low - energy unbound , intermediate , and bound states were identified from the reweighted pmf profiles . the atomistic gamd simulation also elucidates a highly detailed binding pathway of benzene that diffuses from the bulk solvent to an intermediate site located on the protein surface formed by the d and g helices and then slides into the target ligand - binding cavity through a channel formed by the d , f , and g helices . the free energy difference between the intermediate and unbound states was found to be small at 0.53 0.46 kcal / mol as estimated from the five independent gamd simulations . benzene repeatedly visits the intermediate site on the protein surface . in comparison , the ligand entry from protein surface to the deeply buried protein cavity appears to be the rate - limiting step for complete benzene binding . it is important to note that the complete ligand binding was not observed in the four 800 ns gamd simulations , suggesting that the present gamd simulations still suffer from insufficient sampling of the ligand entry process and the reweighted free energy profiles remain unconverged ( figure 5c and supporting information figure s5 ) . this is also indicated by the increased anharmonicity corresponding to the free energy barrier between the intermediate and bound states as shown in figure 5d . nevertheless , our gamd simulation captured a binding pathway of benzene to the t4-lysozyme . the ligand entry site is indeed adjacent to the mobile f helix ( residues 108113 ) , which has been suggested earlier based on the finding that the f helix exhibits increased b - factors in the leu99ala complex structures compared to the apo structures . here , gamd is compared with the original amd , particularly on the performance of smoothening the potential energy surface and energetic reweighting . supporting information table s4 summarizes the statistical properties of the boost potential and the original and modified potential energies as obtained from amd and gamd simulations of the alanine dipeptide , chignolin , and t4-lysozyme , with reference to the cmd simulations . specifically , dual - boost amd simulations of the alanine dipeptide and chignolin obtained from our previous study were used for comparison . for the t4-lysozyme , restarting from the initial configuration of gamd simulations , a 200 ns dual - boost amd simulation was performed using the following acceleration parameters : edihed = vdihed_av + 4nres , dihed = 4nres/5 , etotal = vtotal_av + 0.2natoms , and total = 0.2natoms , where vdihed_av and vtotal_av are the average dihedral and total potential energies calculated from a short 10 ns cmd simulation . as shown in supporting information table s4 , although higher average boost potentials are applied in gamd simulations of the three systems than in the amd simulations , the boost potentials exhibit smaller standard deviations ( i.e. , narrower distribution ) in the gamd simulations , except for the alanine dipeptide . furthermore , anharmonicity of the boost potential distribution is significantly reduced in the gamd simulations . relative to cmd , both amd and gamd simulations mostly exhibit smaller standard deviations in the modified dihedral and total potential energies . notably for the t4-lysozyme , standard deviations of the modified potential energies are significantly smaller in the gamd simulations than in amd . thus , the potential energy surfaces appear more smoothened in gamd enhanced sampling of the t4-lysozyme . provided the narrower distribution and lower anharmonicity of the boost potential ( supporting information table s4 ) , gamd allows for more accurate approximation of exponential average reweighting factor using cumulant expansion to the second order and thus improved free energy calculation , especially for the t4-lysozyme . in the present simulations of ligand binding to the t4-lysozyme , complete binding of benzene was observed only once , largely because the target ligand - binding site is deeply buried in the protein . however , benzene binding to the intermediate site on the protein surface formed by the d and g helices was captured many times , which provides better statistics for free energy calculation . it has been suggested that a pmf - based approach is appropriate to calculate binding free energy for ligands ( especially the charged ) that bind to the protein surface . systems of this type for future applications of gamd might include binding of benzamidine to trypsin and allosteric modulators to the protein surface of the g - protein coupled receptors . in comparison with many enhanced sampling methods such as umbrella sampling , conformational flooding , metadynamics , abf calculations , and orthogonal space sampling , gamd has the advantage of no need to set predefined reaction coordinates . metadynamics , in particular , is another potential biasing technique that has been widely used to map the free energy landscapes of biomolecules such as protein conformational changes and protein ligand binding . by monitoring the energy surface of biomolecules during the simulation , metadynamics keeps adding small gaussians of potential energies to the low - energy regions . this will eventually fill the low - energy wells and achieve uniform sampling of the free energy surface along selected reaction coordinates . the usage of predefined coordinates greatly reduces the complexity of biomolecular simulation problems and facilitates the free energy calculations ( e.g. , significantly lower energetic noise compared with amd simulations ) . however , it is key to select proper reaction coordinates , which often requires expert knowledge of the studied systems . construction of biomolecular reaction coordinates or collective variables has thus been one of the main objectives in metadynamics studies . when important reaction coordinates are missed during the simulation setup , metadynamics simulations may suffer from slow convergence problems as discussed earlier . furthermore , the predefined reaction coordinates tend to place constraints on the sampled space and pathways . it seems difficult to identify certain intermediate states during the protein - folding and ligand - binding pathways , e.g. , intermediate 2 as observed during the binding of benzene to the t4-lysozyme . in comparison , amd simulations are not constrained by reaction coordinates , but this also leads to much higher energetic noise and presents grand challenge for accurate reweighting to recover the original free energy landscapes of biomolecules . although cumulant expansion to the second order was shown to improve amd reweighting when the boost potential follows near gaussian distribution , such improved reweighting is still limited to small systems such as protein with 35 residues . here , by constructing boost potential using a harmonic function that follows gaussian distribution , gamd enables rigorous energetic reweighting through cumulant expansion to the second order , even for simulations of larger proteins ( e.g. , t4-lysozyme ) . with this first , while the present gamd simulations seem to provide sufficient sampling of the low - energy regions , they appear to remain unconverged in sampling of the high - energy barriers . this is particularly true for the ligand entry step in the gamd simulation of benzene binding to the t4-lysozyme . it is worth recalling that the threshold energy for adding the boost potential is set to its lower bound in the present gamd simulations . it is subject to future investigation into whether using the upper bound of the threshold energy will facilitate sampling of the high - energy barriers in gamd simulations . second , based on a potential biasing approach , gamd mainly accelerates transitions across enthalpic energy barriers . improvement for its application to systems with high entropic barriers is still in need . on this regard , gamd can be potentially combined with the parallel tempering and replica exchange algorithms for further enhanced sampling . particularly , the combination of parallel tempering and metadynamics ( pt - metad ) has been shown to facilitate enhanced sampling of biomolecules over entropic barriers . in summary , without the need to set predefined reaction coordinates , gamd is generally applicable to a wide range of biomolecular systems as demonstrated on protein folding and ligand binding in this study . for systems of increasing size , the upper limit of the v standard deviation , 0 can be adjusted dynamically to ensure that the distribution width of the applied boost potential is narrow enough for accurate energetic reweighting using cumulant expansion to the second order . therefore , gamd provides both unconstrained enhanced sampling and free energy calculation for biomolecular simulations .

a gaussian accelerated molecular dynamics ( gamd ) approach for simultaneous enhanced sampling and free energy calculation of biomolecules is presented . by constructing a boost potential that follows gaussian distribution , accurate reweighting of the gamd simulations is achieved using cumulant expansion to the second order . here , gamd is demonstrated on three biomolecular model systems : alanine dipeptide , chignolin folding , and ligand binding to the t4-lysozyme . without the need to set predefined reaction coordinates , gamd enables unconstrained enhanced sampling of these biomolecules . furthermore , the free energy profiles obtained from reweighting of the gamd simulations allow us to identify distinct low - energy states of the biomolecules and characterize the protein - folding and ligand - binding pathways quantitatively .

Introduction Methods Results Discussion

a 40-year - old , previously healthy man presented with left hemiparesis and left facial weakness which had developed four hours before admission after being struck on the right side of the neck by the edge of a door . ct and mri of the brain revealed acute infarction in the vascular territory of the right middle cerebral artery . intravenous heparinization was initiated , thus maintaining a twice - normal activated partial thromboplastin time ( aptt ) . angiography performed on admission revealed near - complete occlusion of the suprabulbar portion of the right internal carotid artery and complete occlusion of the ipsilateral proximal middle cerebral artery , suggesting dissection of the vessel and subsequent arterial thromboembolism ( figs . following admission the patient 's mental status deteriorated suddenly , and ct revealed a large intracerebral hemorrhage within the territory of the infarcted right middle cerebral artery . follow - up angiography at day 20 revealed partial restoration of luminal patency ; the lumen , however , was largely compromised by the presence of a thick intimal flap ( fig . because of the risk of continuing anticoagulation therapy , endovascular treatment of the dissection was planned . two days before angioplasty , performed at day 35 , daily doses of aspirin ( 100 mg ) and ticlopidine ( 250 mg ) were initiated and continued thereafter . a heparin bolus of 5000 iu was injected intravenously , and in order to maintain aptt at 1.5 to 2 times normal , was continued for one day . atropine ( 1 mg ) was injected intravenously just before stent placement ; for primary stenting of the dissected segment , a self - expandable uncovered metallic stent 30 mm in length and 8 mm in diameter ( easy wallstent ; boston scientific corporation , watertown , mass . , u.s.a . ) was used ( fig . 1d ) , and for postdilatation , a 6-mm diameter angioplasty catheter ( ultra - thin ; boston scientific corporation , watertown , mass . , u.s.a . ) at a low inflation pressure was employed . intermittent neurological examinations were conducted throughout the procedure , and after stenting , focal residual dissected false lumen at the posterior portion of the artery was observed ( fig . interestingly , the symptom of left side weakness showed slight improvement two days after stent placement , motor power having increased from grade 2 to 3 . thirty - six days after the procedure , follow - up axial ct scanning and 2d reconstruction images showed complete reconstitution of the arterial lumen and disappearance of the false lumen . ticlopidine was discontinued . a 39-year - old man presented with multiple fractures of the lower extremities and a fractured left zygoma and skull base after a motor vehicle accident . brain ct performed immediately after arrival in the emergency room showed no significant abnormality and orthopedic management was initiated . on the fourth day following admission , however , the patient showed mild weakness of the right extremities , and brain ct revealed cortical areas of low density at the left frontal and occipital lobes . angiography was performed the following day , and focal but significant stenosis associated with a dissecting aneurysm was observed at the subpetrosal portion of the left internal carotid artery ( fig . because the patient was not in a fit condition for sustained anticoagulation therapy , percutaneous angioplasty aimed at reestablishing the vessel lumen was performed during the same session . a 7-fr guiding catheter was introduced into the proximal portion of the internal carotid artery , and to prevent thromboembolic complications a heparin bolus of 5000 iu was administered intravenously . for primary stenting of the affected portion of the vessel , a premounted balloon - expandable uncovered stent ( nir primo ; boston scientific scimed , inc . , maple grove , minn . this was 16 mm in length and was dilated to 4.2 mm with a balloon at a pressure of 10 atm ( fig . a postangioplastic angiogram showed improved luminal patency , with reduction of the aneurysmal sac ( fig . 2c ) . aspirin ( 100 mg / day ) and ticlopidine ( 250 mg / day ) were subsequently administered , and to maintain aptt at 1.5 to 2 times normal , [ jw2]heparinization was continued for five days after stenting . although the filled aneurysmal sac was significantly smaller , focal filling of a part of it was noted ( fig . the patient 's symptom was improved gradually after the procedure , and the motor power of the affected extremities returned to normal at the time of follow - up angiography . a 40-year - old , previously healthy man presented with left hemiparesis and left facial weakness which had developed four hours before admission after being struck on the right side of the neck by the edge of a door . ct and mri of the brain revealed acute infarction in the vascular territory of the right middle cerebral artery . intravenous heparinization was initiated , thus maintaining a twice - normal activated partial thromboplastin time ( aptt ) . angiography performed on admission revealed near - complete occlusion of the suprabulbar portion of the right internal carotid artery and complete occlusion of the ipsilateral proximal middle cerebral artery , suggesting dissection of the vessel and subsequent arterial thromboembolism ( figs . following admission the patient 's mental status deteriorated suddenly , and ct revealed a large intracerebral hemorrhage within the territory of the infarcted right middle cerebral artery . follow - up angiography at day 20 revealed partial restoration of luminal patency ; the lumen , however , was largely compromised by the presence of a thick intimal flap ( fig . because of the risk of continuing anticoagulation therapy , endovascular treatment of the dissection was planned . two days before angioplasty , performed at day 35 , daily doses of aspirin ( 100 mg ) and ticlopidine ( 250 mg ) were initiated and continued thereafter . a heparin bolus of 5000 iu was injected intravenously , and in order to maintain aptt at 1.5 to 2 times normal , was continued for one day . atropine ( 1 mg ) was injected intravenously just before stent placement ; for primary stenting of the dissected segment , a self - expandable uncovered metallic stent 30 mm in length and 8 mm in diameter ( easy wallstent ; boston scientific corporation , watertown , mass . , u.s.a . ) was used ( fig . 1d ) , and for postdilatation , a 6-mm diameter angioplasty catheter ( ultra - thin ; boston scientific corporation , watertown , mass . , u.s.a . ) at a low inflation pressure was employed . intermittent neurological examinations were conducted throughout the procedure , and after stenting , focal residual dissected false lumen at the posterior portion of the artery was observed ( fig . interestingly , the symptom of left side weakness showed slight improvement two days after stent placement , motor power having increased from grade 2 to 3 . thirty - six days after the procedure , follow - up axial ct scanning and 2d reconstruction images showed complete reconstitution of the arterial lumen and disappearance of the false lumen . a 39-year - old man presented with multiple fractures of the lower extremities and a fractured left zygoma and skull base after a motor vehicle accident . brain ct performed immediately after arrival in the emergency room showed no significant abnormality and orthopedic management was initiated . on the fourth day following admission , however , the patient showed mild weakness of the right extremities , and brain ct revealed cortical areas of low density at the left frontal and occipital lobes . angiography was performed the following day , and focal but significant stenosis associated with a dissecting aneurysm was observed at the subpetrosal portion of the left internal carotid artery ( fig . because the patient was not in a fit condition for sustained anticoagulation therapy , percutaneous angioplasty aimed at reestablishing the vessel lumen was performed during the same session . a 7-fr guiding catheter was introduced into the proximal portion of the internal carotid artery , and to prevent thromboembolic complications a heparin bolus of 5000 iu was administered intravenously . for primary stenting of the affected portion of the vessel , a premounted balloon - expandable uncovered stent ( nir primo ; boston scientific scimed , inc . , maple grove , minn . this was 16 mm in length and was dilated to 4.2 mm with a balloon at a pressure of 10 atm ( fig . a postangioplastic angiogram showed improved luminal patency , with reduction of the aneurysmal sac ( fig . 2c ) . aspirin ( 100 mg / day ) and ticlopidine ( 250 mg / day ) were subsequently administered , and to maintain aptt at 1.5 to 2 times normal , [ jw2]heparinization was continued for five days after stenting . follow - up angiography performed 65 days after stent placement showed good luminal patency . although the filled aneurysmal sac was significantly smaller , focal filling of a part of it was noted ( fig . the patient 's symptom was improved gradually after the procedure , and the motor power of the affected extremities returned to normal at the time of follow - up angiography . carotid dissection is initiated by hemorrhage into the medial layer of the artery due to various causes . where the dissection primarily involves the subintima , stenosis or occlusion of the artery usually results , and angiography reveals the so - called ' string sign ' . if the subadventitia is principally involved , an out - pouching sac , a dissecting aneurysm in the form of an outponihing sae usually arises . luminal stenosis can cause distal ischemia leading to stroke or transient ischemic attacks . in both subintimal and subadventitial dissections the resulting exposure of the basement membrane leads to platelet aggregation , and this serves as an embolic source ( 1 ) . it has been suggested that ischemic symptoms are more often secondary to embolic phenomena than to hypoperfusion caused by stenosis ( 2 ) . because many cases escape diagnosis or patients receive some form of therapy , the natural history of carotid dissection remains somewhat unclear . although its clinical course may be benign ( 1 ) , overall mortality rates have been consistently reported at 20 - 40% ( 3 ) . in addition , in a recent study of the time course of symptoms in extracranial carotid artery dissection , the authors observed ischemic events in 82.5% of afflicted patients and complete stroke in two - thirds ( 12 ) . these results imply that the time course of carotid dissection is not at all benign , and the same authors emphasized the need for early preventive measures to avoid ischemic complications ( 12 ) . the management goal in cases of extracranial carotid dissection is to prevent the development or worsening of neurologic deficits by preventing thrombosis and embolization ( 3 ) : options include observation , anticoagulation therapy , surgery , and endovascular treatment . fabian et al . suggested that anticoagulative measures should be used in all non - occlusive arterial dissections without contraindications , regardless of the degree of luminal narrowing ( 13 ) . anticoagulation is often initially achieved with heparin and then , after 1 - 3 weeks , maintained with warfarin , which is continued for up to 6 months . surgical repair is reserved for the management of symptomatic patients with lesions in an accessible location ( 3 ) , but the technique for the preservation of the artery is technically demanding and time consuming . for many years , endovascular occlusion of the affected artery has involved the use of detachable balloons or coils as an alternative to surgery . the advent of stenting techniques in carotid artery angioplasty has , however , led to their exploitation in the preservation of arterial patency . the techniques are much easier and safer than surgical repair , and published case reports have described their successful application ( 4 - 11 ) . various stents can be used for the restoration of luminal patency and occlusion of the sac of a dissecting aneurysm : some authors have insisted that a covered stent is especially useful ( 11 ) , though stents without fabric covering have also been used for the same purpose with successful results , as in our cases . to obliterate dissecting aneurysms , guglielmi detachable coils along with stents have also been used ( 10 ) . for suprabulbar internal carotid artery dissection we applied a self - expandable stent , and for subpetrosal internal carotid artery dissection , the balloon - expandable type : in both cases the results were successful . emphasized the superiority of self - expandable stents over the balloon - expandable type in terms of their radial expansile force and longitudinal flexibility ( 5 ) . they surmised that the first of these two factors was helpful for effectively sealing off the dissecting aneurysm , and in a case involving the use of a balloon - expandable stent the aneurysm sac also effectively disappeared . the balloon - expandable stent we used was also flexible , a feature which allowed us to place it in an angled portion of the vessel . follow - up showed that the softness of the stent did not compromise luminal patency of the vessel . selection of a stent must be individualized on the basis of factors which include the characteristics and location of the lesion and the diameter and geometry of the vessel . during the management of case 1 , we delayed stent placement because of an infarction in the patient 's right middle cerebral artery territory due to embolic occlusion . where there is overt parenchymal infarction , as in that patient for the stabilization of already - infarcted parenchyma to which anticoagulative measures have been applied , a wait of three to four weeks is recommended , though in case 1 there was unexpected hemorrhagic transformation of the infarction . because flow through the patent anterior communication artery was good , management could have involved occlusion of the dissected ica : because of the relative ease and safety of the endovascular recanalization methods now available , however , a destructive method of this kind was not considered . in case 2 , the cause of the left cerebral cortical infarction was not clear . because a dissecting aneurysm is already known to be a potential embolic source , the dissected segment was recanalized after initial angiography without evaluating the significance of the stenosis . angiography appeared to indicate that this was narrow enough to cause a watershed infarction due to hemodynamic insufficiency , and that stenting of the stenotic segment could recanalize the stenosis as well as seal the aneurysmal sac . follow - up angiography , however , revealed a focal residual sac . suggesting that the dissection had opened : in such cases endovascular stent placement is a safe and effective method for the management of patients with extracranial carotid artery dissections with or without associated aneurysm .

extracranial carotid artery dissection may manifest as arterial stenosis or occlusion , or as dissecting aneurysm formation . anticoagulation and/or antiplatelet therapy is the first - line treatment , but because it is effective and less invasive than other procedures , endovascular treatment of carotid artery dissection has recently attracted interest . we encountered two consecutive cases of trauma - related extracranial internal carotid artery dissection , one in the suprabulbar portion and one in the subpetrosal portion . we managed the patient with suprabulbar dissection using a self - expandable metallic stent and managed the patient with subpetrosal dissection using a balloon - expandable metallic stent . in both patients the dissecting aneurysm disappeared , and at follow - up improved luminal patency was observed .

CASE REPORTS Case 1 Case 2 DISCUSSION

techniques for isolation and culture of fetal type ii alveolar epithelial cells , as well as the morphologic and biochemical characteristics of these histotypic cultures , are described . type ii alveolar epithelial cells can be isolated from fetal rat lungs and grown in an organotypic culture system as described in this review . the fetal type ii cells resemble differentiated rat type ii cells in morphology , biochemistry , and karyotype as they grow in culture for up to 5 weeks . the cells of the mature organotypic cultures form alveolarlike structures while growing on a gelatin sponge matrix . the type ii cells also synthesize and secrete pulmonary surfactant similar in biochemical composition to that produced in vivo . this system has been used to study the effects of hormones on surfactant production and composition . the organotypic model has many potential applications to the study of pulmonary toxicology.imagesfigure 1.figure 2 .

Images

defining alterations in signalling pathways in normal and malignant cells is becoming a major field in proteomics . a number of different approaches have been established to isolate , identify and quantify phosphorylated proteins and peptides . in the current report , a comparison between scx prefractionation versus an antibody based approach , both coupled to tio2 enrichment and applied to tmt labelled cellular lysates , is described . the antibody strategy was more complete for enriching phosphopeptides and allowed the identification of a large set of proteins known to be phosphorylated ( 715 protein groups ) with a minimum number of not previously known phosphorylated proteins ( 2 ) .

Supplementary Material

allogeneic bone marrow transplantation ( bmt ) is widely used in the treatment of various hematologic disorders , such as leukemia and aplastic anemia , and the major complication of bmt is graft - versus - host disease ( gvhd ) . we discuss whether autologous melanocyte transplantation is an appropriate way as a treatment of the vitiligo after allogeneic bmt . in this report we describe a patient with hodgkin 's lymphoma who developed universal vitiligo after allogeneic bmt from his sister . he underwent four times melanocyte - keratinocyte transplantation ( mkt ) for the treatment of vitiligo ; all treated depigmented patches had a significant repigmentation . an 18-year - old patient was diagnosed with epstein - barr virus - related hodgkin - like lymphoma in 2003 , and antivirotic had been used to control his lymphoma , but the treatment was ineffective . then in march 2009 , he received the histocompatibility leukocyte antigen ( hla)-matched allogeneic bmt from his sister . cyclosporine and methotrexate were used as a prophylaxis strategy to prevent gvhd for 6 months after the allogeneic bmt . during the 6-month recovering period , the patient developed a small erythematous rash , which was consistent with the clinical features of graft - versus - host disease ( gvhd ) of the skin . no external agents were used and the rash dissipated in a short period of time . small depigmented macules started to appear on his cheek 11 months after allogeneic bmt in february 2010 , and followed by rapid progression to the whole body from the face to hands within 1 month . we indicated it to be a typical universal vitiligo by the reason that the depigmentation macules had a chalky white appearance under wood 's light . moreover , leukotrichia appeared at the same time associated with his vitiligo , affecting back and arms [ figure 1 ] . the patient with extensive vitiligo of the back and arms the depigmentation macules did not expanded any more after he received whole - body nb - uvb therapy twice a week for 2 months . simultaneously , following the doctor 's advice , 0.1% tacrolimus was used and traditional chinese medicine was orally taken . the patient underwent the therapies mentioned above for 8 months in total , but with poor repigmentation . then he discontinued medical therapies and performed non - cultured mkt on january 17 , 2011 , including left forehead and temple [ figure 2 ] . almost complete repigmentation was shown after 8 months since the first transplantation [ figure 3a ] . then he received mkt on his right face on september 29 , 2011 . and 8 months after the second mkt , 28 months after the first - time transplantation , all of the transplanted areas had almost completely repigmented and the color of the repigmented area matched with the normal surrounding skin excellently [ figure 3c ] . then the patient underwent the third and the fourth mkt procedure on the right and the left side of his neck respectively on may 28 , 2012 and january 22 , 2013 [ figure 4a ] . significant improvement was shown in the area around the left side of his neck while there 's also a majority repigmentation on the right side [ figure 4b and c ] . no hyperpigmentation , scar , infection and any other adverse effects on both recipient and donor sites by now were noticed . leukasmus involving the face symmetrical , around the nose , eyes and mouth repigmentation of vitiligo by mkt on his face . ( c ) 28 months after the first time transplantation , significant repigmentation obtained and normal skin achieved mkt performed on his neck . ( b ) 12 months after the surgery of right neck showing nearly 100% repigmentation . vitiligo is a most common depigmentation disorder and affects 0.5 - 2.0% of the world population . the etiology of vitiligo remains obscure , the most prevalent hypothesis today considers vitiligo as an autoimmune disease and focuses on a melanocyte - specific cytotoxic t - cell immune reaction in the destruction of melanocytes . supported that there perhaps be four explanations for the development of vitiligo after bmt , such as the destruction of melanocytes stimulated by pretransplantation chemotherapy and radiotherapy , chronic gvhd , infusion of a larger number of lymphocytes and adoptive passive transfer from the donor to the recipient . in the case of our patient , vitiligo might be a result of an immune response directed against the melanocyte destruction initiated by gvhd . there are reported cases about allogeneic posted - bmt vitiligo . but most of the pre - bmt diagnosis were chronic myelogenous leukemia , none of them were diagnosed with hodgkin 's lymphoma as our report . in terms of treatment during those cases , just partial of them accepted therapies , but none of them performed with transplantation . in the bmt context , the extensive and relentless vitiligo progression indicated an aggressive alloimmune process , so the destruction of melanocyte is alloimmune in nature . there was no report to make discriminate of melanocyte destruction between alloimmune and autoimmune , but au et al . proposed the success of melanocyte autografts for autoimmune vitiligo could be used in an alloimmune setting . therefore , we came up with the idea of vitiligo melanocyte transplantation while other treatment was invalid for the patient . melanocyte grafting technique include cultured autologous melanocyte suspension transplantation and autologous noncultured melanocyte - keratinocyte . in our report , the patient obtained universal vitiligo after allogeneic bone marrow transplantation for hodgkin 's lymphoma . in the condition of vitiligo failed to respond to non - surgical treatment , the patient underwent four times of mkt during 2 years . the largest area of treated lesions was 50 cm ; repigmentation rate achieved more than 95% after each transplantation without any adverse effects or koebner 's phenomenon . the pattern of the repigmentation was uniform in the first three times mkt but diffuse in the last one ; whatever , more follow - up time is needed in the future . after 2 years of follow - up , graft sites still repigmented obviously , and the color was similar to the surrounding normal skin which illustrated mkt can treat allogeneic vitiligo successfully . this is the first time to report about treating vitiligo after allogeneic bone marrow transplantation by mkt . we suggest that autotransplantation especially autologous grafting with noncultured melanocyte is simple , safe , and inexpensive for remedying the loss of melanocyte after bmt in the case of depigmented macules is stable . but whether the melanocyte transplantation used in alloimmune vitiligo is effective as in autoimmune , we need more follow - ups . this is the first time to report about treating vitiligo after allogeneic bone marrow transplantation for hodgkin 's lymphoma by non - cultured melanocyte - keratinocyte transplantation without koebner 's phenomenon and adverse effects but all successful . it probably suggests that therapy of transplantation can be used in the treatment of vitiligo after allogeneic bone marrow transplantation .

the treatment of vitiligo is derisory since the pathogenesis of vitiligo is not clear at present . most conservative treatments are difficult to approach satisfactory therapy . so transplantation is the only way left when the disease becomes insensitive to those conservative treatments . here we describe an 18-year - old patient who developed vitiligo , which was triggered by graft - versus - host disease after a allogeneic bone marrow transplantation for the treatment of hodgkin 's lymphoma from his sister . in the following treatment to vitiligo , the patient successfully performed the transplantation of autologous uncultured melanocyte on the premise of poor reaction to other conservative methods . we infer that transplantation can be a treatment of the vitiligo after allogeneic bone marrow transplantation .

Introduction Case Report Discussion

spondylolisthesis is defined as an anterior migration of a vertebral body in relation to the vertebra located immediately caudal . in 1930 junghanns was the first to describe anterior translation of a lumbar vertebra without any defect in the neural arch following this the term degenerative spondylolisthesis ( ds ) was introduced by newman in 1955 . five types of spondylolisthesis have been described including dysplastic , isthmic , traumatic , pathologic , and degenerative . there are many predisposing factors like sagittally - placed facet joint , a high iliac crest [ 4 - 6 ] , etc . ds which is characterized by an intact vertebral ring is presumed to result from degeneration of facet joints and intervertebral discs with aging and thus has traditionally been considered to represent instability of the vertebral segment . a 66-year - old gentleman , farmer by occupation , came with complaints of lower backache for 2 years which was insidious in onset , gradually progressive and non - radiating . he also complained of acute retention of urine since 15 days for which he was catheterized . he also had a history of neurogenic claudication at a distance of 100 m. there was no history of any trauma . a detailed systemic and neurological examination revealed power of flexor hallucis longus(fhl ) and flexor digitalis longus(fdl ) as 4/5 , ankle jerks were absent , sensory deficits in s1-s2 dermatome and per - rectal examination revealed decreased perianal sensations and anal tone with absent anal wink . plain radiographs of lumbo - sacral spine revealed spondylolisthesis of s1-s2 ( meyerding s grade 1 ) ( fig.1 ) . magnetic resonance imaging and computed tomography scan of the spine revealed lumbarization of s1 with spondylolisthesis of s1 over s2 , facetal hypertrophy at l5-s1 and canal stenosis at s1-s2 ( figs . 2 , and 3 ) . anteroposterior ( a ) and lateral ( b ) radiographs showing grade 1 spondylolisthesis ats1-s2b . the patient underwent posterior spine surgery where decompression was done with laminotomy of s1 bilaterally and then pedicular screw fixation was done bilaterally at l5 , s1 , and s2 ( fig.4 ) . s1 and post - operative radiographs - anteroposterior ( a ) and lateral ( b ) view . the bladder symptoms disappeared after 3 weeks and the power of fhl / fdl improved from 4/5 to 5/5 . the patient underwent posterior spine surgery where decompression was done with laminotomy of s1 bilaterally and then pedicular screw fixation was done bilaterally at l5 , s1 , and s2 ( fig.4 ) . s1 and post - operative radiographs - anteroposterior ( a ) and lateral ( b ) view . the bladder symptoms disappeared after 3 weeks and the power of fhl / fdl improved from 4/5 to 5/5 . the patient underwent posterior spine surgery where decompression was done with laminotomy of s1 bilaterally and then pedicular screw fixation was done bilaterally at l5 , s1 , and s2 ( fig.4 ) . s1 and post - operative radiographs - anteroposterior ( a ) and lateral ( b ) view . the bladder symptoms disappeared after 3 weeks and the power of fhl / fdl improved from 4/5 to 5/5 . the deformity occurs at l4 - 5 6 times more often than at other lumbar levels and four times more often above a sacralized l5 . the lumbosacral junction and middle lumbar spine are most often involved , but the lesion is also found in cervical or rarely the thoracic vertebra . to the best of our knowledge , ds of sacral vertebrae has not been reported in the available english literature till now . the prevalence of complete lumbarization is 1.8% and to get a spondylolisthesis is even rarer . there have been many publications in the literature mentioning incidence of spondylolisthesis with sacralization but hardly any on spondylolisthesis with lumbarization . further case series or longitudinal studies of such cases may help understand better the pathomechanics related to spondylolisthesis at this level . ds of s1-s2 is a very rare entity and further case reports will help us to explore the biomechanics at this level .

introduction : degenerative spondylolisthesis ( ds ) is usually seen at l4-l5 level and less frequently at l5-s1 level . this is a rare case report of spondylolisthesis of s1 over s2 with lumbarization of s1 . lumbarization of s1 is seen in just 1 - 2% of the population and to have spondylolisthesis in this segment is even rarer . the purpose is to report a rare case of ds at s1-s2 level.case report : this is a single case report of a 66-year - old gentleman who presented with complains of lower backache for 2 years and acute retention of urine to the emergency department . detailed clinical and radiological evaluation of the spine was done which revealed lumbarization of s1 with spondylolisthesis at s1-s2 and facetal hypertrophy at l5 , s1 , and s2 . he underwent decompression and stabilization at l5 , s1 , and s2 along with placement of autologous bone graft . the bladder symptoms disappeared after 3 weeks . at 1-year follow - up , patient s clinical symptoms were relieved , and he improved clinically.conclusion:to the best of our knowledge , this is probably the first case of ds of sacral vertebrae to be reported in english literature . the prevalence of complete lumbarization is around 1.8% and to get spondylolisthesis in this segment is even rarer , hence the lack of literature in this regard . since this is the first of its kind of case , further case series or longitudinal studies of such cases may help understand better the pathomechanics related to spondylolisthesis at this level .

Introduction Case report None Operative procedure Results Discussion Conclusion

the research protocol was approved by the institutional review board at the oregon health & science university ( ohsu ) and performed in accordance with the tenets of the declaration of helsinki . written informed consent was obtained from each participant after explanation of the nature and possible consequences of the study . participants were recruited at the casey eye institute / ohsu according to the aig study protocol . the ohsu ancillary site followed the same eligibility and endpoint protocol as the aig study , but used an advanced swept - source oct prototype system instead of commercially available oct instruments . briefly , normal control participants met the following criteria in both eyes : iop of less than 21 mm hg in both eyes , and a normal humphrey visual field ( hvf ) on achromatic standard automated perimetry by swedish interactive threshold algorithm 24 - 2 testing ( hfa ii ; carl zeiss meditec , inc . , dublin , ca , usa ) with mean deviation ( md ) , glaucoma hemifield test ( ght ) , and pattern standard deviation ( psd ) within normal limits . in addition , normal subjects had a normal - appearing optic nerve head ( onh ) and nfl on ophthalmoscopic examination , and an open angle by gonioscopy . glaucoma participants were required to have a glaucomatous onh rim or nfl thinning on ophthalmoscopic examination . glaucomatous eyes were classified in the pg subgroup if they have visual field ( vf ) psd or ght outside normal limits ( p < 0.05 and p < 1% , respectively ) in a consistent pattern on two qualifying vf exams . exclusion criteria for all groups included visual acuity less than 20/40 , age < 40 or > 79 years at enrollment , any ocular surgery other than uncomplicated cataract extraction , other diseases that might cause vf or onh abnormality , and factors that might preclude the participant from performing study procedures or completing the study . a prototype high speed swept - source fourier - domain oct system was used in this study . the device operated at an axial scan speed of 100 khz using a swept - source cavity laser operating at 1050 nm with a tuning range of 100 nm . a resolution of 5.3 m axially and 18 m laterally at an imaging depth of 2.9 mm in tissue was achieved . the ocular light power exposure was 1.9 mw , within the american national standards institute ( ansi ) safety limit . participants were scanned using a high density 8 8 mm raster scan pattern , centered at the onh . in the fast transverse scan direction , the time to acquire each 3d volumetric scan was 4.3 seconds . in the scan protocol , one eye of each participant was scanned with 4 scans consisting of 2 horizontal and 2 vertical scans . an orthogonal registration algorithm was applied to register and merge all 4 scans to reduce eye motion . concentric , cylindrical , cross - sectional oct images of varying radii were resampled from the volume scan ( fig . we selected rings with radii of 1.5 , 1.7 , 2 , 2.5 , 3 , and 3.5 mm for analysis . the disc center was decided manually by one of us ( ll ) by matching the disc boundary with an ellipse on the en face view ( fig . ( a ) en face view ( average projection of log reflectance intensity ) of the 8 8 mm volumetric cube scan of the onh region in a glaucomatous eye . the 3-mm radius circle centered on the onh ( b ) the 3-mm radius cylindrical cross section is resampled from the cube scan . the ilm contour ( white dashed line ) is measured from this circular cross section . ( c ) en face oct showing resampling circles at radii of 1.5 , 1.7 , 2.0 , 2.5 , 3.0 , and 3.5 mm . the ilm was detected as the most inner boundary of retina in the cylindrical cross - section images . an automated algorithm was developed to follow the vessel bumps and avoid artifacts , such as points of vitreous macular adhesion . the ilm elevation profile ( contour ) one of us ( ll ) inspected the cross - sectional images with ilm contour overlay and performed manual correction of the contour if necessary . the ilm elevation profile was transformed to spatial frequency domain using the fast fourier transform ( fig . the spatial frequency components at lowest frequencies were removed because they simply relate to the average axial position and the tilt of the oct scan beam relative to the onh plane . the highest frequency components also were nondiagnostic because of high noise and little anatomic information . the middle frequency band that optimized the detection of retinal vessel relief and nfl bundle defects was determined empirically based on the difference between the average normal and glaucoma ilm spatial frequency spectrum ( fig . the rscv is defined as the average log value within a middle spatial frequency band of the fourier transform to the elevation profile of the inner retinal surface . ( a ) spatial frequency spectrum of ilm elevation showing the averages of all glaucoma eyes and all normal eyes derived from the 3.0-mm oct cylindrical sections . ( b ) shows the difference spectrum ( glaucoma normal ) and the optimize frequency band ( between dotted lines ) used to compute the rscv . optimized passbands for rscv calculation at different radii . on the cylindrical cross - sectional oct image with radius of 1.7 mm the wilcoxon rank sum test was performed to determine statistical significance between the study groups . the area under receiver operating characteristic curve ( aroc ) was calculated for the diagnostic accuracy . pearson correlation was used to determine correlations between rscv , axial length , nflt , and visual field test md . a coefficient of variance ( cv ) the first one compares rscv value at r = 2.8 mm and r = 3.0 mm . the second test compares results graded independently by two of the authors ( ot and ll ) . all image processes and statistical analyses were done using matlab ( mathworks , natick , ma , usa ) . the research protocol was approved by the institutional review board at the oregon health & science university ( ohsu ) and performed in accordance with the tenets of the declaration of helsinki . written informed consent was obtained from each participant after explanation of the nature and possible consequences of the study . participants were recruited at the casey eye institute / ohsu according to the aig study protocol . the ohsu ancillary site followed the same eligibility and endpoint protocol as the aig study , but used an advanced swept - source oct prototype system instead of commercially available oct instruments . briefly , normal control participants met the following criteria in both eyes : iop of less than 21 mm hg in both eyes , and a normal humphrey visual field ( hvf ) on achromatic standard automated perimetry by swedish interactive threshold algorithm 24 - 2 testing ( hfa ii ; carl zeiss meditec , inc . , dublin , ca , usa ) with mean deviation ( md ) , glaucoma hemifield test ( ght ) , and pattern standard deviation ( psd ) within normal limits . in addition , normal subjects had a normal - appearing optic nerve head ( onh ) and nfl on ophthalmoscopic examination , and an open angle by gonioscopy . glaucoma participants were required to have a glaucomatous onh rim or nfl thinning on ophthalmoscopic examination . glaucomatous eyes were classified in the pg subgroup if they have visual field ( vf ) psd or ght outside normal limits ( p < 0.05 and p < 1% , respectively ) in a consistent pattern on two qualifying vf exams . exclusion criteria for all groups included visual acuity less than 20/40 , age < 40 or > 79 years at enrollment , any ocular surgery other than uncomplicated cataract extraction , other diseases that might cause vf or onh abnormality , and factors that might preclude the participant from performing study procedures or completing the study . a prototype high speed swept - source fourier - domain oct system was used in this study . the device operated at an axial scan speed of 100 khz using a swept - source cavity laser operating at 1050 nm with a tuning range of 100 nm . a resolution of 5.3 m axially and 18 m laterally at an imaging depth of 2.9 mm in tissue was achieved . the ocular light power exposure was 1.9 mw , within the american national standards institute ( ansi ) safety limit . participants were scanned using a high density 8 8 mm raster scan pattern , centered at the onh . in the fast transverse scan direction , the time to acquire each 3d volumetric scan was 4.3 seconds . in the scan protocol , one eye of each participant was scanned with 4 scans consisting of 2 horizontal and 2 vertical scans . an orthogonal registration algorithm was applied to register and merge all 4 scans to reduce eye motion . concentric , cylindrical , cross - sectional oct images of varying radii were resampled from the volume scan ( fig . we selected rings with radii of 1.5 , 1.7 , 2 , 2.5 , 3 , and 3.5 mm for analysis . the disc center was decided manually by one of us ( ll ) by matching the disc boundary with an ellipse on the en face view ( fig . ( a ) en face view ( average projection of log reflectance intensity ) of the 8 8 mm volumetric cube scan of the onh region in a glaucomatous eye . the 3-mm radius circle centered on the onh ( b ) the 3-mm radius cylindrical cross section is resampled from the cube scan . the ilm contour ( white dashed line ) is measured from this circular cross section . ( c ) en face oct showing resampling circles at radii of 1.5 , 1.7 , 2.0 , 2.5 , 3.0 , and 3.5 mm . the ilm was detected as the most inner boundary of retina in the cylindrical cross - section images . an automated algorithm was developed to follow the vessel bumps and avoid artifacts , such as points of vitreous macular adhesion . the ilm elevation profile ( contour ) one of us ( ll ) inspected the cross - sectional images with ilm contour overlay and performed manual correction of the contour if necessary . the ilm elevation profile was transformed to spatial frequency domain using the fast fourier transform ( fig . 3a ) . the spatial frequency components at lowest frequencies were removed because they simply relate to the average axial position and the tilt of the oct scan beam relative to the onh plane . the highest frequency components also were nondiagnostic because of high noise and little anatomic information . the middle frequency band that optimized the detection of retinal vessel relief and nfl bundle defects was determined empirically based on the difference between the average normal and glaucoma ilm spatial frequency spectrum ( fig . the rscv is defined as the average log value within a middle spatial frequency band of the fourier transform to the elevation profile of the inner retinal surface . ( a ) spatial frequency spectrum of ilm elevation showing the averages of all glaucoma eyes and all normal eyes derived from the 3.0-mm oct cylindrical sections . ( b ) shows the difference spectrum ( glaucoma normal ) and the optimize frequency band ( between dotted lines ) used to compute the rscv . on the cylindrical cross - sectional oct image with radius of 1.7 mm , the lower nfl boundary also was detected using a method described previously . the wilcoxon rank sum test was performed to determine statistical significance between the study groups . the area under receiver operating characteristic curve ( aroc ) was calculated for the diagnostic accuracy . pearson correlation was used to determine correlations between rscv , axial length , nflt , and visual field test md . a coefficient of variance ( cv ) the first one compares rscv value at r = 2.8 mm and r = 3.0 mm . the second test compares results graded independently by two of the authors ( ot and ll ) . all image processes and statistical analyses were done using matlab ( mathworks , natick , ma , usa ) . of 17 participants in the glaucoma group ( 8 in the pg and 9 in the ppg subgroups ) , 15 had early vf damage ( md > 6 db ) and 2 had more severe vf damage . we enrolled 25 participants in the normal control group , but due to their younger average age , only the 17 oldest control participants were used in the present analysis . the age matching was not exact , as the control participants still averaged 9 years younger ( table 1 ) . as expected , the glaucoma group had significantly worse vf md and thinner nfl in the overall area , superior and inferior quadrants . the image quality of the normal group is significantly higher than that of the glaucoma group based on signal strength index . participant characteristics the spatial frequency transforms of the ilm profile were averaged for the normal and glaucoma groups ( fig . optimized bands were detected at frequency components where the glaucoma group was significantly larger than the normal group ( fig . we averaged rscvs in the optimized bands for each circle and the rscv distribution for normal and glaucoma groups are illustrated in figure 5 . the rscv was significantly higher at all sampling radii in the glaucoma group compared to the normal control group ( table 2 ) . distribution of rscv values for normal ( n ) and glaucoma ( g ) groups at sampling radii between 1.5 and 3.5 mm . retinal surface contour variability the diagnostic accuracy of nfl and rscv were assessed using aroc values at each sampling radius ( table 3 ) . the diagnostic accuracy of rscv improved with larger radii and the highest aroc value ( 0.90 ) was found at a radius of 3.5 mm . this was higher than that for nflt parameters , but only significantly in the temporal and nasal quadrants ( < 0.01 ) . we examined averages of rscv over various ranges of radii and found that the average over 2.5 to 3.5 mm had the highest aroc ( 0.91 ) . the combination had marginally higher aroc ( 0.91 ) than nfl only ( p = 0.08 ) . diagnostic accuracy comparison venn diagram analysis ( fig . 6 ) showed that rscv had higher sensitivity for glaucoma diagnosis ( 53% ) than nflt ( 29% ) . it also was notable that all abnormal eyes detected by nflt also were detected by rscv . however , the difference in sensitivity was not statistically significant ( p = 0.13 ) . the threshold for rscv was set 2.33 sd above the mean of the normal group ( 99 percentile cutoff assuming normal distribution ) and the threshold of nfl was 2.33 sd below the mean of normal group . in the normal group , all eyes were within the cutoff for both parameters , indicating 100% specificity . in the glaucoma groups , rscv detected abnormality in nine participants , including the five detected by nfl . the correlations between averaged rscv and nflt , or between averaged rscv and vf md , were estimated for glaucomatous participants . significant correlations were found between rscv and nfl ( r = 0.54 , p = 0.03 ) but not md ( r = 0.32 , p = 0.21 ) . a significant correlation also was found between nfl and md ( r = 0.68 , p = 0.002 ) . we also evaluated the correlation between rscv and nfl for normal participants , which was not significant ( r = 0.10 , p = 0.70 ) . we tested whether the rscv was sensitive to transverse magnification changes ( i.e. , axial eye length variation ) by comparing the values evaluated at the sampling radii of r = 2.8 and 3 mm . no significant difference was found between the two radii in either the glaucoma or normal group ( p > 0.26 ) . 1.3% for glaucoma eyes . we also tested the between - grader ( ot and ll ) reproducibility of rscv because the onh boundary and center were defined manually . the reproducibility ( cv ) was 1.6% for normal eyes and 1.1% for glaucoma eyes . we did not find a significant correlation between axial length and rscv parameter ( r = 3.5 mm ) for the normal ( r = 0.06 , p = 0.83 ) and glaucoma ( r = 0.42 , p = 0.09 ) groups . we also did not find a significant correlation between axial length and rscv parameters at any radius for the normal group ( p > 0.32 ) . in this pilot study , we reported , to our knowledge , the first use of spatial frequency analysis to detect small scale retinal surface contour change in glaucoma . the rscv , measured in the peripapillary area , was significantly larger in glaucoma eyes . the diagnostic accuracy of rscv was at least as good as nflt , although a larger study would be needed to see if there is a significant advantage . in early glaucoma , nflt has limited diagnostic sensitivity , with literature values of 33.3% to 67.4% at a fixed specificity of 99% . this is not surprising as there is a relatively wide range of population variation in the average nflt , which has been reported as between 9.0% and 10.0% sd . because the rscv measures small scale , focal changes that are unlikely to be due to the inborn variation of nflt , it may be able to better detect glaucoma in the earlier stages , when nfl damage is small and variable in the location . in this study , nflt was abnormally ( 99% specificity cutoff ) thin in only 5 of 17 participants in the glaucoma group , while rscv was abnormally elevated in 9 of 17 glaucoma participants . while this advantage is not statistically significant , it shows a promising trend that deserves further study . because the nflt and the rscv measure different aspects of nfl damage while we previously have developed another parameter to measure focal peripapillary nfl and macular ganglion cell complex ( gcc ) thinning called focal loss volume ( flv ) , that algorithm was optimized to detect relatively larger areas ( 500 m superpixels ) of focal damage . by contrast the rscv spatial frequency analysis detects changes in the 2 to 100 radians / mm , which corresponds to spatial features as small as 60 m ( the diameter of a small arteriole ) . thus , we believe all three parameters will be synergistic based on the fact they measure glaucoma damage on different spatial scales . we found that the rscv appeared to have higher diagnostic accuracy in circles with large radii ( 2.53.5 mm ) than small radii ( 1.52 mm ) . firstly , we observed that , in the normal eye , larger retinal vessels submerge into the retina further from the disc margin . thus , the inner retinal surface is smoother and rscv is lower further from the disc . this observation also might be due to the fact that glaucoma damages axon bundles nonuniformly within the onh ( laminar pores , and so forth ) and these bundle losses may be more detectable within the nfl where all bundles are more spread out . another assumption is that the thinner nfl away from the disc is going to be less likely to mask blood vessels and loss of axon bundles may reveal blood vessels more readily in these regions . the second observation is that the population variances were tighter with larger radii ( table 2 ) . we suspected this was simply due to the larger number of a - scans in sampling circles of larger radii . thus , maximizing the oct scan density in the 2.5- to 3.5-mm radius annulus around the optic disc may improve rscv evaluation . an additional advantage of rscv over nflt is that the rscv value is relatively insensitive to magnification variation or decentration relative to the optic disc center . between the scan radii of 2.5 to 3.5 mm , the mean rscv values varied less than 2% in the normal and glaucoma groups . intergrader variation in disc boundary identification also affected rscv by less than 2% . by comparison furthermore , nfl quadrant thickness averages are known to be exquisitely sensitive to decentration of the sampling circle relative to the optic disc . therefore , rscv may be a more robust diagnostic parameter that is relatively resistant to the introduction of bias due to patients , oct operators , or graders . in previous studies , contour variability was accessed by heidelberg retinal tomography ( hrt ) but was considerably inferior in its ability to diagnose glaucoma or progression compared to nfl measured by oct . we believe there are two reasons why contour variability by hrt was considerably inferior in diagnostic power . thus , it is difficult for hrt to catch small changes in the contour variability caused by protrusions of retinal vessels . , we found that the contour variability is less sensitive in location near the disc edge . one of the limitations of this study is the relatively small number of participants , especially for advanced glaucoma ( vf < 6db ) . a power calculation shows that our sample size of 17 normal and 17 glaucoma subjects should be able to detect a difference in aroc of 0.67 compared to the aroc of rscv of 0.90 with a probability of 0.80 at a significance threshold of p < 0.05 . however , rscv was not significantly affected by sex in our control group ( p = 0.62 ) . second , the glaucoma group also is 9 years older than the normal group . as a consequence , the oct image quality in normal however , the ssi is good for the normal ( 60 9 ) and glaucoma ( 50 12 ) groups . the rscv parameters are not sensitive to ssi when the image quality is good enough for accurate ilm boundary detection . we also tested the correlation between ssi and rscv but did not find any significance for the normal and glaucoma groups ( p > 0.38 ) . furthermore , rscv was not correlated with age in the normal group ( p = 0.43 ) . retinal surface contour variability also may be sensitive to focal nfl loss caused by other optic neuropathies , such as anterior ischemic optic neuropathy ( aion ) and optic neuritis . since rscv is a nonspecific detector of nfl loss on a small spatial scale , specific diagnosis of particular conditions would require the physician to consider the clinical presentation as well as patterns of nfl , gcc , optic disc topography , and vf changes . in summary , since the rscv detects small - scale focal damage and the average nflt measures global damage , they provide different diagnostic information that may be synergistic .

purposewe investigated the feasibility of glaucoma detection by measuring retinal surface contour variability ( rscv ) using optical coherence tomography ( oct).methodsthe peripapillary region in one eye of each participant was scanned over an 8 8 mm area with a swept source oct prototype . the retinal surface contour was sampled at approximately 1.5- to 3.5-mm radius circles centered on the optic nerve head . the rscv is defined as the average log value within a middle spatial frequency band of the fourier transform to the elevation profile of the inner retinal surface . the spatial frequency band was optimized to distinguish glaucoma from normal . nerve fiber layer thickness ( nflt ) was sampled around a 1.7-mm radius circle . glaucoma severity was assessed by automated static perimetry.resultswe enrolled 17 glaucomatous eyes and 17 healthy eyes . a great majority of the glaucoma group were in the early stage ( visual field mean deviation average 2.48 3.73 db ) . significant differences were found for rscv between glaucoma and control eyes ( p < 0.003 ) at all radii . the area under the receiver operating characteristic curve ( aroc = 0.90 ) of rscv was best at the 3.5-mm radius . this was not significantly better than nflt ( aroc = 0.84 ) . with the 99% specificity , the glaucoma detection sensitivity was 53% for rscv and 29% for nflt ( p = 0.13).conclusionsretinal surface contour variability was significantly increased in glaucoma patients . the diagnostic accuracy of rscv was equal to nflt in early glaucoma . since the rscv detects small - scale focal damage and the average nflt measures global damage , they provide different diagnostic information that may be synergistic .

Method Participants OCT and Scanning ILM Detection Retinal Surface Contour Variability (RSCV) Nerve Fiber Layer Thickness (NFLT) Statistical Analysis Results Discussion

organic foreign bodies are generally associated with severe inflammatory reaction and infection , while the nature of reaction elicited by inorganic foreign bodies depends on the material of the foreign body . graphite , which is the major constituent of pencil lead , has been reported to remain inert in the eye for a long time . however , it has also been reported to cause severe endophthalmitis - like reaction in the eye . we report a rare case of retained graphite pencil tip in the anterior chamber of a six - year - old girl . a six - year - old girl presented to us with history of mild pain in the left eye of two days duration . the child s mother gave a history of trauma with a graphite lead pencil about four months ago at school when she was accidentally poked in the left eye by another child . the child had not been examined by an ophthalmologist after the incident as she was apparently asymptomatic at the time . on examination , the best corrected visual acuity was 20/20 in the right eye and 20/40 in the left eye . there was a full thickness corneal scar ( figure 1 ) in the left eye . a small area of iris atrophy with a sphincter tear was noted at the edge of the pupil at the 6 o clock position . there was a black foreign body resembling a graphite pencil lead tip , measuring about 1.5 mm in size , on the iris at the 7 o clock position ( figure 1 ) . ocular ultrasonography of the left eye did not reveal any abnormality in the posterior segment . figure 1slit lamp photograph of the left eye showing the corneal scar and the graphite pencil lead tip on the iris . slit lamp photograph of the left eye showing the corneal scar and the graphite pencil lead tip on the iris . a corneal incision was made at 5 o clock position with a 2.8 mm keratome . the anterior chamber was filled with 2% methyl cellulose and the foreign body ( figure 2 ) was removed in toto with a bechert - mcpherson forceps . the remaining graphite particles were aspirated out with a simcoe cortex aspiration cannula using an anterior chamber maintainer . post - operatively , the patient was put on tapering doses of topical steroids and cycloplegics . on follow up , one month later , the best corrected visual acuity in the left eye was 20/40 . there was no inflammation ; the lens was clear and the fundus was normal . the reaction of the eye to a retained intraocular foreign body varies depending on its composition . foreign bodies comprised of materials like gold , silver and platinum have been reported to remain inert in the intraocular environment . there are only a few reports of ocular trauma with retention of graphite pencil lead in the eye . retained graphite has been described in the conjunctiva , cornea , angle of the anterior chamber and the posterior segment . a case of pencil - tip injury to the orbit with retained graphite foreign body associated with delayed orbital infection has also been described . however , the potential toxicity of the other constituents of pencil lead like animal fats and clay is not clearly known . there has been a report of severe endophthalmitis - like reaction incited by retained graphite foreign bodies in the vitreous . in this case , it is unclear whether the reaction was induced by the other constituents of pencil lead like the aluminium in the kaolinite or if there was an associated infection . in our case , we decided to surgically remove the intraocular foreign body in spite of the fact that it had obviously remained in the eye for some time ( as evidenced by the healed corneal scar and presence of iris atrophy ) without inciting an inflammatory response or causing much damage to the intraocular structures . there was a distinct possibility of causing damage to the lens and inciting an inflammatory reaction during surgical removal of the foreign body . this risk had to be weighed against that of the damage the foreign body might cause if it was left in the eye . there was a chance that the foreign body would get dislodged into the angle at a later stage and cause progressive damage to the angle structures and the cornea as in the case reported by han et al . honda et al . have reported a case of a five - year - old child with a graphite foreign body lodged in the peripheral retina , whom they followed up for six years with serial electroretinograms , fundus photographs and fundus fluorescein angiography . there was no evidence of any damage to the eye caused by the foreign body at the end of their follow up . however , in our case , such meticulous follow - up would have been quite impossible as the child s family belonged to a poor socio - economic background and the parents would not have been able to afford the cost of repeated hospital visits and investigations . our case also differed from honda et al.s case in that the foreign body was in the anterior segment and therefore , was at a more accessible site . there was a higher chance of removal of the foreign body without causing damage to the intraocular structures in our case . in conclusion , graphite foreign bodies may be retained in the eye without causing any inflammation or damage to the intraocular structures . on the other hand , there is also the possibility of progressive damage to intraocular structures by these foreign bodies due to various mechanisms . therefore , the decision about surgical removal of the foreign body has to be made on an individual basis after taking multiple factors into consideration and estimating the risk - benefit ratio in each patient .

retained intraocular graphite foreign bodies are uncommon . although they are generally inert , they have been reported to cause severe inflammatory reaction and progressive damage to intraocular structures . we report a case of a six - year - old girl with a retained intraocular graphite pencil lead foreign body in the anterior chamber of the eye and discuss the various considerations in the management of such cases .

Introduction Case Report Discussion

polyacrylonitrile ( pan ) are commercially important and used for the precursor for high - performance carbon fiber . in the fiber applications , pan is dissolved in a suitable solvent and spin to fibers by a wet or dry spinning process . the extensively used solvents are aqueous sodium sulfocyanate ( nascn ) , n , ndimethylformamide ( dmf ) , n , n - dimethylacetamide ( dmac ) , and n , n - dimethyl sulfone ( dmso ) . unfortunately , all the solvents mentioned above are unfriendly to the environment . to meet the requirements of green chemistry , scientists have been searching for a green solvent to be an alternative to these solvents . in recent years , room temperature ionic liquids ( rtils ) have received a lot of attention as the potential green and designable solvents . although the ionic liquids have been studied since the 1950s , but they can not to be handled under an inert atmosphere . these are not part of our studies until some kinds of water - stable and air - stable ionic liquids are found such as [ bmim]cl . because of its extremely low volatility , some of the rtils are promising environment - friendly solvents instead of the volatile organic solvents in a range of science and technology applications such as media for organic and inorganic reactions , materials processing electrochemistry and chemical separation . during the copolymerization of an and mma in rtils our research group find that rtils are good solvents for pan copolymer , and pan copolymer could be precipitated from the rtils solutions by the addition of water . therefore rtils are promising to be green solvents for pan . understanding the rheological properties of a solution is a convenient and effective way to gain a fundamental knowledge of the spinnability and structure - property relationships for the spinning solution . thus , this paper investigates the rheological properties of pan copolymer / bmimcl concentrated solutions in some detail . the pan copolymer , chlorobutane , 1-methylimidazole , ethyl acetate and acetone were used as supplied . firstly , pan powder was swollen by [ bmim]cl at room temperature to give a white , little viscous pulp . secondly the pulp was heated to 90c until a homogeneous , transparent solution was formed . the pan/[bmim]cl solution of 5 wt% to 22 wt% concentration was obtained within 5 hours at 90c . figure 1 shows the process of the dissolution of pan with ionic liquids [ bmim]cl at 90c at the concentration of 10 wt% . steady state rheological measurements were performed on a haake rs150l rotational rheometer using a 35 mm , 1 cone and plate . the apparent viscosity a is shown as a function of shear rate for the pan in two different solvents , [ bmim]cl and dmf ( see figure 2 ) . over the experimental range , the pan / dmf solution is newtonian fluid , while the pan/[bmim]cl solution is pseudoplastic , that is , the apparent viscosity decreases with increasing shear rate . according to the so - called power law equation , where k and n are constants , for pseudoplastic liquids the viscosity should decrease nearly linearly with shear rate on the log - log plot and the value of n is less than one . however , it is surprising to find that the data of pan/[bmim]cl solution should be considered having two linear stages ( see figure 2 ) . the value of n of the first stage is less than 1 , according with the shear thinning rheological behavior . while at the second stage the apparent viscosity decreases so dramatically that even the value of n is negative . it is known that n is used to indicate the non - newtonian property of fluids . the more the value of n deviates from 1 , the more non - newtonian the fluids would be . usually , the value of n of pan / traditional organic solvents solutions is between 0 and 1 . therefore , it could be postulated that there would be some much stronger interaction between pan and the new solvent [ bmim]cl which will be discussed in the following text . the data in figure 3 shows that the viscosity - shear rate curves for the pan/[bmim]cl solutions of different concentrations . these curves show that solutions of lower concentration remain newtonian in behavior at high shear rates than concentrated solutions . at high concentration , the rheological behavior of pan/[bmim]cl solutions acts like that of liquid crystalline polymers ( lcp ) . the solvent [ bmim]cl reduces the number of entanglements . reducing the number of entanglements at a given shear rate since the orientation of macromolecular is the major cause of non - newtonian behavior , increasing the shear rate would make the non - newtonian behavior more noticeable . besides , it could be found in figure 3 that when at high shear rate , the viscosity of high concentration solutions is lower than that of low concentration , for example , the viscosity of 22wt% solution is the lowest among all the solutions when the shear rate is close to 1000 1/s . as we known , liquid crystalline polymers usually have rigid chain segment such as aromatic polyamide and aromatic polyester . the flexibility of the c - c bond of the pan main chain is smaller than the c - o bond , c - n bond and decrease because of the strong polarity of the cn , but comparing to the liquid crystalline polymers , the pan chain segment can not be oriented because of lack of strong rigid chain segment . in this case , high concentration means more entanglements , which indicates that only low shear rates would be needed to orient the macromolecules . and the amount of entanglements is so large that there is no enough time and much more difficulties for most of them to slip and disengage . with the increasing of the shear rate the number of the oriented segments increases , so that the viscosity of the high concentration pan/[bmim]cl solution decreases greatly . commonly , the viscosity follows the andrade or arrhenius equation to a good approximation : in this equation , a is a constant , e is the activation energy . the slope of the straight line plotted by ln versus 1/t is e / r . the value of the energy of activation depends strongly on whether the viscosities at various temperatures are evaluated at constant shear stress or at constant shear rate . here since low molecular weight polymers has fewer entanglements than high molecular weight ones , it is not surprising that deviations from newtonian behavior start at higher shear rates for the low molecular weight solutions . however , in figure 5 , when the shear rate up to 210 1/s the viscosity of the high molecular weight pan/[bmim]cl solution is lower than the other one . the viscoelasticity of polymer is usually characterized by dynamic rheological experiment in which the sample is subjected to a sinusoidal strain of infinitesimal amplitude and fixed angular frequency . the term g , called the storage modulus , is the in - phase component of the modulus and represents the energy stored and recovered per cycle . correspondingly , the term g , called the loss modulus , is the out - of - phase component of the modulus and represents the energy dissipated as heat per cycle of deformation . figure 6 shows the modulus - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . the data shows that the loss modulus g is much higher than the storage modulus g. either module increases when the concentration increases or the temperature decreases . the storage modulus g increases faster than the loss modulus g because the elasticity of the solution increases . the data in fig . 7 shows that the complex viscosity - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . . the complex viscosity of the solution increases when the concentration increases or the temperature decreases . this non - newtonian behavior is of tremendous practical importance in the processing and fabrication of polymer material . first , the decreased viscosity makes the polymer solution easier to process or squirt through small channels , such as spinning . second , the decrease in viscosity is associated with the development of elasticity in the polymer solution . this elasticity produces such phenomena as die swell , or the puff - up of extruded strands . the possible dissolution mechanism of pan in ionic liquid [ bmim]cl is shown in figure 8 . it has been proved that the miscibility of ionic liquid with polymer is the function of their structural characteristic . we here focus on the polar group of pan which has strong interaction with the ionic liquid . because the electronegativity of the nitrogen atom on the nitrile grouping is very strong when the electron density is very large , the nitrogen atom on the nitrile grouping donate electrons and the carbon atom on the nitrile grouping attract electrons . as a result , there is strong interaction between the cation and the nitrogen atom on the nitrile grouping . similarly , there is strong interaction between the anion and the carbon atom on the nitrile grouping . the pan copolymer , chlorobutane , 1-methylimidazole , ethyl acetate and acetone were used as supplied . firstly , pan powder was swollen by [ bmim]cl at room temperature to give a white , little viscous pulp . secondly the pulp was heated to 90c until a homogeneous , transparent solution was formed . the pan/[bmim]cl solution of 5 wt% to 22 wt% concentration was obtained within 5 hours at 90c . figure 1 shows the process of the dissolution of pan with ionic liquids [ bmim]cl at 90c at the concentration of 10 wt% . steady state rheological measurements were performed on a haake rs150l rotational rheometer using a 35 mm , 1 cone and plate . the apparent viscosity a is shown as a function of shear rate for the pan in two different solvents , [ bmim]cl and dmf ( see figure 2 ) . over the experimental range , the pan / dmf solution is newtonian fluid , while the pan/[bmim]cl solution is pseudoplastic , that is , the apparent viscosity decreases with increasing shear rate . according to the so - called power law equation , where k and n are constants , for pseudoplastic liquids the viscosity should decrease nearly linearly with shear rate on the log - log plot and the value of n is less than one . however , it is surprising to find that the data of pan/[bmim]cl solution should be considered having two linear stages ( see figure 2 ) . the value of n of the first stage is less than 1 , according with the shear thinning rheological behavior . while at the second stage the apparent viscosity decreases so dramatically that even the value of n is negative . it is known that n is used to indicate the non - newtonian property of fluids . the more the value of n deviates from 1 , the more non - newtonian the fluids would be . usually , the value of n of pan / traditional organic solvents solutions is between 0 and 1 . therefore , it could be postulated that there would be some much stronger interaction between pan and the new solvent [ bmim]cl which will be discussed in the following text . the data in figure 3 shows that the viscosity - shear rate curves for the pan/[bmim]cl solutions of different concentrations . these curves show that solutions of lower concentration remain newtonian in behavior at high shear rates than concentrated solutions . at high concentration , the rheological behavior of pan/[bmim]cl solutions acts like that of liquid crystalline polymers ( lcp ) . the solvent [ bmim]cl reduces the number of entanglements . reducing the number of entanglements at a given shear rate reduces the amount of orientation of macromolecular . since the orientation of macromolecular is the major cause of non - newtonian behavior , increasing the shear rate would make the non - newtonian behavior more noticeable . besides , it could be found in figure 3 that when at high shear rate , the viscosity of high concentration solutions is lower than that of low concentration , for example , the viscosity of 22wt% solution is the lowest among all the solutions when the shear rate is close to 1000 1/s . as we known , liquid crystalline polymers usually have rigid chain segment such as aromatic polyamide and aromatic polyester . the flexibility of the c - c bond of the pan main chain is smaller than the c - o bond , c - n bond and decrease because of the strong polarity of the cn , but comparing to the liquid crystalline polymers , the pan chain segment can not be oriented because of lack of strong rigid chain segment . in this case , high concentration means more entanglements , which indicates that only low shear rates would be needed to orient the macromolecules . and the amount of entanglements is so large that there is no enough time and much more difficulties for most of them to slip and disengage . with the increasing of the shear rate the number of the oriented segments increases , so that the viscosity of the high concentration pan/[bmim]cl solution decreases greatly . commonly , the viscosity follows the andrade or arrhenius equation to a good approximation : in this equation , a is a constant , e is the activation energy . the slope of the straight line plotted by ln versus 1/t is e / r . the value of the energy of activation depends strongly on whether the viscosities at various temperatures are evaluated at constant shear stress or at constant shear rate . here the two molecular weights are high enough to become entangled . since low molecular weight polymers has fewer entanglements than high molecular weight ones , it is not surprising that deviations from newtonian behavior start at higher shear rates for the low molecular weight solutions . however , in figure 5 , when the shear rate up to 210 1/s the viscosity of the high molecular weight pan/[bmim]cl solution is lower than the other one . the viscoelasticity of polymer is usually characterized by dynamic rheological experiment in which the sample is subjected to a sinusoidal strain of infinitesimal amplitude and fixed angular frequency . the term g , called the storage modulus , is the in - phase component of the modulus and represents the energy stored and recovered per cycle . correspondingly , the term g , called the loss modulus , is the out - of - phase component of the modulus and represents the energy dissipated as heat per cycle of deformation . figure 6 shows the modulus - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . the data shows that the loss modulus g is much higher than the storage modulus g. either module increases when the concentration increases or the temperature decreases . the storage modulus g increases faster than the loss modulus g because the elasticity of the solution increases . the data in fig . 7 shows that the complex viscosity - angular frequency curves for the pan/[bmim]cl solutions of different concentrations(90c)(a ) and temperature(14wt%)(b ) . the complex viscosity of the solution increases when the concentration increases or the temperature decreases . this non - newtonian behavior is of tremendous practical importance in the processing and fabrication of polymer material . first , the decreased viscosity makes the polymer solution easier to process or squirt through small channels , such as spinning . second , the decrease in viscosity is associated with the development of elasticity in the polymer solution . this elasticity produces such phenomena as die swell , or the puff - up of extruded strands . the possible dissolution mechanism of pan in ionic liquid [ bmim]cl is shown in figure 8 . it has been proved that the miscibility of ionic liquid with polymer is the function of their structural characteristic . we here focus on the polar group of pan which has strong interaction with the ionic liquid . because the electronegativity of the nitrogen atom on the nitrile grouping is very strong when the electron density is very large , the nitrogen atom on the nitrile grouping donate electrons and the carbon atom on the nitrile grouping attract electrons . as a result , there is strong interaction between the cation and the nitrogen atom on the nitrile grouping . similarly , there is strong interaction between the anion and the carbon atom on the nitrile grouping . within the limits of the experimental techniques accessible to us , the results obtained for pan/[bmim]cl solutions show that they are pseudoplastic , similar to the pan / traditional organic solvents solutions . the dependence on concentration and molecular weight shows that entanglements play an important role in the rheological behaviors of pan/[bmim]cl concentrated solutions . the dynamic rheological measurement indicates the elasticity of the solution increases when the concentration is increasing or the temperature decreasing . the rheological behaviors of the concentrated solution at high share rate are directive to the spinning of pan fiber .

one of the room temperature ionic liquids ( rtils ) , 1-butyl-3-methylimidazolium chloride ( [ bmim]cl ) was chosen to prepare the concentrated solutions of polyacrylonitrile ( pan ) . the rheological behaviors of the solutions were measured with rotational rheometry under different conditions , including temperatures , concentration , and molecular weight of pan . the solutions exhibited shear - thinning behaviors , similar to that of pan / dmf solutions . the viscosities decreased with the increasing of shear rates . however , the viscosity decreased sharply at high shear rates when the concentration was up to 16wt% . the dependence of the viscosity on temperature was analyzed through the determination of the apparent activation energy . unusually , the viscosity of solutions of higher concentration is lower than that of lower concentration . similarly , the viscosity of low molecular weight pan was higher than high molecular weight pan at high shear rates . the dynamic rheological measurement indicates the loss modulus is much higher than storage modulus . the trend of complex viscosity is similar with the result of static rheological measurement . the interaction between pan and ionic liquid [ bmim]cl was discussed .

1. Introduction 2. Experimental 2.1 Materials 2.2 Preparation of concentrated solutions 2.3 Rheometry 2.4 Results and discussion Conclusions

fistulae between the upper respiratory and gastrointestinal tracts are uncommon in adults . whereas developmental anomaly is the commonest cause in infancy and childhood , the etiology in adults is most frequently secondary to an esophageal malignancy . we report two cases of esophagobronchial fistulae one secondary to chronic chest tuberculosis and the other secondary to a squamous cell carcinoma of the upper esophagus diagnosed by multi - detector computed tomography ( mdct ) . traditionally , fluoroscopy with oral contrast swallow examination has been the mainstay radiological investigation for the diagnosis of these fistulae . however , it can be an inconvenient study with need for multiple projections to adequately demonstrate the fistula and its course . also , being a luminal study , the cause of the fistula is not adequately evaluated . use of the various post - processing features like thick maximum intensity projections ( mips ) and volume rendering techniques ( vrts ) enables better detection and depiction of these fistulae . use of virtual endoscopy also guides the clinician for the conventional endoscopy which would be needed for biopsy and treatment . a 15-year - old male presented with cough , mucopurulent expectoration , and dyspnea since 3 months . this finding of swallow cough sequence has been referred to as ono 's sign . a chest radiograph revealed complete opacification of the right hemithorax with volume loss suggesting complete collapse of the right lung with mediastinal shift to the right [ figure 1 ] . a plain and intravenous contrast - enhanced ct study with oral contrast swallow was performed on a 128-slice mdct scanner ( siemens somatom definition as , erlangen , germany ) . the study revealed complete collapse of the right lung with irregular dilated ectatic bronchi in the right lower lobe [ figure 2 ] . there was stenosis and diffuse narrowing of the right mainstem bronchus with nodularity of the mucosa seen best on the virtual bronchoscopy [ figure 3 ] . an air - filled tract was noted extending from one of the right lower lobar bronchi toward the posterior mediastinum with ill - defined soft tissue around it . the possibility of an esophagobronchial fistula was suspected , and we did a ct oral contrast swallow study to detect it . oral contrast swallow study performed with the patient in right decubitus position using diluted non - ionic iodinated contrast medium ( 1:20 dilution of iohexol with normal saline ) . it depicted the site and the fistula tract between the right lateral wall of the esophagus and one of the ectactic bronchi in the right lower lobe of the lung . thick mip images and vrt processing demonstrated the fistula site and tract [ figures 4 and 5 ] . these imaging findings led us to conclude that these changes were most likely the sequelae of chronic tuberculosis . histology of the lung specimen revealed distorted bronchioles with diffuse and focal dense infiltration by mononuclear cells and giant cells . frontal chest radiograph reveals severe volume loss and complete opacifi cation of the right hemithorax with ipsilateral tracheal and mediastinal shift . branching tubular radiolucencies are noted in the right lower zone representing bronchiectasis ( encircled area ) axial ( a ) and coronal ( b ) ct of the chest in lung window shows complete collapse of the right lung with shift of of the heart and the mediastinum to the right . dilated irregular fluid bronchi are seen in the right lower lobe ( thin black arrows ) with few having air - fluid levels within ( thick black arrow ) coronal ct of the chest in lung window ( a ) demonstrates narrowing of the right mainstem bronchus ( thick black arrow ) . a virtual bronchoscopy images ( b ) revealing stenotic orifice of the right mainstem bronchus with mucosal nodularity near the carina thick maximum intensity projection ( mip ) image of the ct contrast swallow study demonstrating the esophagus pulled to the right and opacification by oral contrast of the fistula tract between the esophagus and the dilated right lower lobe bronchi volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination demonstrating the fistula ( arrowheads ) between the right lateral aspect of the pulled esophagus and the right lower lobar bronchus with opacifi cation of the bronchial tree a 65-year - old male presented with progressive dysphagia to solids over the past 2 months . an ultrasound of the neck revealed few enlarged lymph nodes which on fine needle aspiration ( fna ) revealed neoplastic squamous cells . a plain and intravenous contrast - enhanced ct study with ct oral contrast swallow was performed . there was a loss of fat plane between the esophagus and the carina , left mainstem bronchus , and aorta . a defect was seen in the posterior wall of the proximal left mainstem bronchus suggesting formation of a fistula with the esophagus [ figure 6 ] . a ct oral contrast swallow study performed in prone position with diluted non - ionic contrast ( 1:20 dilution of iohexol in normal saline ) revealed a small fistula tract between the esophagus and the proximal left mainstem bronchus just distal to the carina [ figure 7 ] . post - processing of this contrast swallow study with volume rendering depicted the fistulous communication in 3d [ figure 8 ] . axial contrast enhanced ct ( a ) at a level just beyond the carina demonstrates defect in the posterior wall of the proximal left mainstem bronchus ( thick white arrow ) . a virtual bronchoscopy image ( b ) shows the site of the fistula opening ( thin white arrow ) oral contrast ct swallow- axial ct performed with patient in prone position shows the fistula tract ( white arrow ) opacifi ed by the diluted oral contrast volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination show the fistula tract between the esophagus and the proximal left mainstem bronchus a 15-year - old male presented with cough , mucopurulent expectoration , and dyspnea since 3 months . this finding of swallow cough sequence has been referred to as ono 's sign . a chest radiograph revealed complete opacification of the right hemithorax with volume loss suggesting complete collapse of the right lung with mediastinal shift to the right [ figure 1 ] . a plain and intravenous contrast - enhanced ct study with oral contrast swallow was performed on a 128-slice mdct scanner ( siemens somatom definition as , erlangen , germany ) . the study revealed complete collapse of the right lung with irregular dilated ectatic bronchi in the right lower lobe [ figure 2 ] . there was stenosis and diffuse narrowing of the right mainstem bronchus with nodularity of the mucosa seen best on the virtual bronchoscopy [ figure 3 ] . an air - filled tract was noted extending from one of the right lower lobar bronchi toward the posterior mediastinum with ill - defined soft tissue around it . the possibility of an esophagobronchial fistula was suspected , and we did a ct oral contrast swallow study to detect it . oral contrast swallow study performed with the patient in right decubitus position using diluted non - ionic iodinated contrast medium ( 1:20 dilution of iohexol with normal saline ) . it depicted the site and the fistula tract between the right lateral wall of the esophagus and one of the ectactic bronchi in the right lower lobe of the lung . thick mip images and vrt processing demonstrated the fistula site and tract [ figures 4 and 5 ] . these imaging findings led us to conclude that these changes were most likely the sequelae of chronic tuberculosis . histology of the lung specimen revealed distorted bronchioles with diffuse and focal dense infiltration by mononuclear cells and giant cells . frontal chest radiograph reveals severe volume loss and complete opacifi cation of the right hemithorax with ipsilateral tracheal and mediastinal shift . branching tubular radiolucencies are noted in the right lower zone representing bronchiectasis ( encircled area ) axial ( a ) and coronal ( b ) ct of the chest in lung window shows complete collapse of the right lung with shift of of the heart and the mediastinum to the right . dilated irregular fluid bronchi are seen in the right lower lobe ( thin black arrows ) with few having air - fluid levels within ( thick black arrow ) coronal ct of the chest in lung window ( a ) demonstrates narrowing of the right mainstem bronchus ( thick black arrow ) . a virtual bronchoscopy images ( b ) revealing stenotic orifice of the right mainstem bronchus with mucosal nodularity near the carina thick maximum intensity projection ( mip ) image of the ct contrast swallow study demonstrating the esophagus pulled to the right and opacification by oral contrast of the fistula tract between the esophagus and the dilated right lower lobe bronchi volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination demonstrating the fistula ( arrowheads ) between the right lateral aspect of the pulled esophagus and the right lower lobar bronchus with opacifi cation of the bronchial tree a 65-year - old male presented with progressive dysphagia to solids over the past 2 months . an ultrasound of the neck revealed few enlarged lymph nodes which on fine needle aspiration ( fna ) revealed neoplastic squamous cells . a plain and intravenous contrast - enhanced ct study with ct oral contrast swallow was performed . there was a loss of fat plane between the esophagus and the carina , left mainstem bronchus , and aorta . a defect was seen in the posterior wall of the proximal left mainstem bronchus suggesting formation of a fistula with the esophagus [ figure 6 ] . a ct oral contrast swallow study performed in prone position with diluted non - ionic contrast ( 1:20 dilution of iohexol in normal saline ) revealed a small fistula tract between the esophagus and the proximal left mainstem bronchus just distal to the carina [ figure 7 ] . post - processing of this contrast swallow study with volume rendering depicted the fistulous communication in 3d [ figure 8 ] . axial contrast enhanced ct ( a ) at a level just beyond the carina demonstrates defect in the posterior wall of the proximal left mainstem bronchus ( thick white arrow ) . a virtual bronchoscopy image ( b ) shows the site of the fistula opening ( thin white arrow ) oral contrast ct swallow- axial ct performed with patient in prone position shows the fistula tract ( white arrow ) opacifi ed by the diluted oral contrast volume rendering technique ( vrt ) images- frontal ( a ) and lateral ( b ) projections derived from post - processing of the contrast swallow examination show the fistula tract between the esophagus and the proximal left mainstem bronchus esophagobronchial fistulae are uncommon and difficult to diagnose . in the elderly , they are most frequently seen with an intrathoracic malignancy and are most commonly associated with malignancy of the esophagus . in a large case series , subsequent ulceration and necrosis of the malignant tissue leads to tissue breakdown and fistula formation . non - malignant causes are infrequent and include trauma ( blunt , penetrating , or iatrogenic ) , chronic inflammation ( chronic infections like tuberculosis and histoplasmosis , crohn 's disease ) , late presentation of a congenital fistula , and rarely poisoning . over the years , iatrogenic trauma has become a commoner cause for such fistulae compared to infections . tuberculosis as a cause for these fistulae has been infrequently reported in the western literature . however , in the indian subcontinent where the incidence of tuberculosis is high , chest tuberculosis should be high in the list of differentials when a non - malignant fistula is encountered . in nonmalignant conditions , traumatic bronchial or tracheal wall necrosis or necrotizing inflammation is responsible for the fistulization . fluoroscopic oral contrast swallow examination with barium is the initial investigation of choice for evaluation of dysphagia and suspected fistulae , even though endoscopy is needed for definite evaluation . if esophageal perforation is suspected , an iodinated contrast medium should be used as barium extravasation can lead to mediastinitis . if there is no frank leak seen , then barium can be given as it produces better radiographic quality being of higher density than iodine . if respiratory fistula is suspected , barium still may be used as small quantity of barium in the tracheobronchial tree is harmless . however , ionic iodinated contrast medium should not be used as they can cause chemical pneumonitis and only non - ionic iodinated contrast medium should be used in such cases . the best initial agent to use in both these scenarios of perforation or fistula is a non - ionic iodinated contrast agent . fluoroscopy allows for dynamic evaluation of esophageal motility as well as evaluation of its lumen . even though barium swallow fluoroscopy examination has the advantage of being a real - time study , fistula tracts may not always be detected . if detected , depiction of the three - dimensional course of the fistula may be difficult in spite of use of multiple projections . also , being a luminal study , it would fail to show changes in the wall of the esophagus and in the mediastinum that are shown accurately by ct . no large case series has been published about its efficacy in adult fistulae , but few case reports have highlighted its role . the patient should be given a mouthful bolus of the preparation and asked to swallow it promptly on instruction to do so . since the oral and pharyngeal phases of deglutition take no more than 2 seconds , the acquisition can be triggered immediately after the instruction to the patient to swallow has been given . the patient position may be changed to better opacify the fistula tract as we used prone and right decubitus positions in our cases to better opacify the fistula tracts . a recent study demonstrated that ct contrast swallow was better tolerated and more sensitive than fluoroscopy at detecting post - esophagectomy anastomotic leaks . post - processing these studies with maximum - intensity projection and with volume rendering allows a three - dimensional evaluation of the fistula tract . supplementing oral contrast swallow to the chest ct protocol in cases where fistulae are suspected can improve the diagnostic ability of ct and also better demonstrate these fistulae .

we report two cases of esophagobronchial fistulae diagnosed by multi - detector computed tomography ( mdct ) oral contrast swallow examination . it is helpful to supplement the ct study with an oral contrast swallow as it aids in confirmation of a suspected fistula and also demonstrates the fistula tract better . we present the clinical details and the imaging findings on mdct of two cases of esophagobronchial fistulae one secondary to chronic chest tuberculosis and the other secondary to a squamous cell carcinoma of the upper esophagus followed by discussion of the etiology , pathogenesis , and imaging of these fistulae .

Introduction Case Reports Case 1 Case 2 Discussion Conclusion

an individual , as a consumer researcher , wants to buy a refrigerator . with appliances of most sorts , people regard best practice decision making as consulting trustworthy comparison websites and magazines , ones that go beyond expressing opinions , or recording likes , to numerically rate the alternative products on a set of attributes or criteria . they want these decision support tools to give them ratings that can be trusted because they are produced free of any conflict of interest or other biases the consumer does not know , and does not want to know , why this refrigerator is given a 4*/80% rating on reliability , and a 3*/60% rating on environmental impact , and another one the opposite ratings . feeling justified in assuming a common sense , lay understanding of the terms reliability and environmental impact , they do not have neither the time nor motivation to find out more about what these concepts mean , in terms of the mechanical functioning of the refrigerator , the quality of its components , the emissions it produces or whatever else contributes to these ratings made by the expert assessors some may wish to establish whether consumers have made an informed decision by seeing how well they score on a test of refrigerator knowledge . giving considerable and fixed weight to knowledge in their measures of decision quality , consumers decisions might be regarded as poor quality , because their knowledge sub - score is low . in contrast , consumers may regard themselves as having made good decisions , indeed the best possible decisions they could make , given the time and cognitive effort they are willing to devote to research into the decision - making process including accessing and accumulating knowledge deemed important , even essential , by others . but surely health care decisions are different from buying refrigerators ? choosing between surgery and medical options for newly - diagnosed cancer , or pain management for chronic osteoarthritis , is not like buying a household appliance , is it ? in fact , nothing really changes for the individual , whom we now conceptualize as a researcher conducting a continuing , informal n - of-1 study into his / her health . the affective and emotional differences between the two situations may well produce differences in the decision - making process , but the patient accepts that this will not necessarily enhance the quality of the decision as he / she defines it . patients may become interested in finding out more about their medical condition than they would about refrigerators , and actually do so . but unless it leads to a change in a performance rating for an available option on one of their criteria especially the bean for a criterion they weigh heavily the additional information they now possess is decision - neutral . people - as - researchers may feel better informed in some sense , but they realize they will not necessarily be in a position to make a better decision and therefore have not ended up more decisionally empowered . they may even simply have become more anxious and regretful about the opportunity costs of acquiring the information , in the form of the foregone benefits from other activities in which they could have engaged . informed decision ? according to themselves and us , absolutely , since they have consulted a transparent set of option performance ratings on relevant criteria , originating from a source that they have decided is the most trustworthy . their decision quality score may well be low according to an instrument that weights highly the knowledge that they are assumed by others to need to make an the growing number of condition - specific decision quality instruments being developed , notably by karen sepucha and colleagues , all give very heavy weight to a knowledge subcomponent . there could be no clearer confirmation of the issue at stake here than the title of one of the background papers to these projects : how does feeling informed relate to being informed? trust is crucial here . in either shared or unshared decision making , trust relates to the inputs into decision making , since we have left behind the notion of an agency relationship , previously dominant in conceptualising medical practice . trust is always a matter of degree , rather than a binary all or nothing , whether it relates to the beans provided by the clinician , or by a decision support tool . even if there is only one , dubious , source , it will be the most trustworthy . unless , that is , the person rates his / her own estimates as more trustworthy than the best source , since so we envisage an individual regarding the respected consumer magazine s beans on refrigerators as the most trustworthy in relation to that purchase decision . people s task in health care decisions , given a restricted willingness to devote time and energy to processing information , is to assess the trustworthiness of the available sources of beans for the outcomes and other criteria important to them . they would expect a clinician , or a team developing the ratings for a decision aid , to be highly trustworthy and to be provided with evidence for this , especially in the case of an aid . the key information the person - as - researcher requires is labelling that ensures he / she will get what it says on the tin when they open an aid . with this meta - information , they can make an informed choice about which tins of what size to open . the other major problem with any imposed information requirement is that it condemns many on the continuum of health literacy , and especially health numeracy , to receiving little or no help . we fully support attempts to reduce health illiteracy and innumeracy , especially their decision - focused forms . however , it is too much to expect of a decision support tool or a clinician to overcome the limitations of previous education and socialization in these respects . moreover , it is important to accept that even if aid users are able to register and report the relative numbers of sad and smiley faces in frequency diagrams , or repeat back 1 in x statements about which there is considerable doubt this does not in any way ensure that they can meaningfully incorporate the numerical probabilities they have correctly registered ( say 10% and .05% , or 1 in 10 and 1 in 2000 ) , into their decisions . this is not to say that a decision aid should not contain help in this respect , including guidance on how the person can best avail themselves of what it offers , and information on the bases of that offering . it is to suggest that much of this should be provided on an opt - in basis . nothing in what we have said is intended to imply that the community is not entitled to apply community - level criteria and weights to what it provides , or allows to be provided , to whom , under what conditions , and at what cost , in the pursuit of goals such as efficiency , equity and justice . formal laws and regulations ( including those on informed consent and clinician liability ) and resource allocation policies ( including reimbursement decisions ) will be the context in which the individual decision is made , and they will frequently be in conflict with what an individual sees as best for him / herself , given personal criteria and weights . external consequences for others may trump individuals preferences , as in the case of infectious diseases . trickier are the issues of social responsibility or morality which are not dealt with formally . apart from issues of environmental and social impact ( such as those arising from hormonal treatments and opioids ) , there are all those that arise in resource - constrained and interdependent systems simply as a result of those constraints and interdependencies . in these cases , we say two things . first , it is not the function of individual decision support tools to mandate the inclusion or exclusion of social criteria in an individual s set , such as concern for others health , or insist that these be given specific weights . second , that in order to be regarded as having made a high - quality decision , the individual should not be required to be informed about the social criteria they do select , other than having the processed beans available to them from a trustworthy source . normative checklists for decision support tools , such as those constructed in accordance with the guidelines of the ipdasi collaboration , are clearly intended to promote person - as - patient empowerment . but most decision aids that comply with these guidelines are designed for use only within the context of shared decision making , in which the person is assigned the status of patient . in many cases , the support can be accessed only within the clinical encounter , or with provider permission . they all perpetuate the idea that only a decision informed in a particular way and to a particular extent can be a good decision . we do not need the concept of an informed decision , only that of a good , better or best possible decision . for none of these will there be a definition that is not multi - dimensional and therefore preference - sensitive . there can only be one answer : the patient s or the person s if they are not a patient .

most guidelines for clinical practice , and especially those for the construction of decision support tools , assume that the individual person ( the patient ) needs to be in possession of information of particular sorts and amount in order to qualify as having made an informed decision. this often implicitly segues into the patient having made a good decision. in person - centred health care , whether , in what form , and with what weight , information is included as a criterion of decision quality is a matter for the person involved , to decide in the light of their own values , preferences , and time and resource constraints .

Person-centred decision making Trust The wider contexts of person-centred health care

pancreatic intraepithelial neoplasia ( panin ) is a nomenclature for microscopic proliferative epithelial lesions of the pancreas which was proposed in 2001 by hruban et al . . panin is assumed to be a precursor lesion of invasive ductal carcinoma ( idc ) . precursor lesions of idc also include intraductal papillary mucinous neoplasm ( ipmn ) and mucinous cystic neoplasm . panin is divided into three grades , panin-1 , -2 , and -3 , according to the cytological and architectural atypia . panin-1 is further subdivided into flat ( panin-1a ) and papillary types ( panin-1b ) . this hypothesis has arisen from the observation of the resected specimens , in which synchronous or metachronous occurrence of panin and idc were seen . panin had been reported to be more common in the pancreas with idc than without [ 2 , 3 ] . it has also been reported that idc occurred in the remnant pancreas several years after partial pancreatectomy for high - grade panin had been performed [ 4 , 5 ] . several studies have demonstrated higher frequencies of genetic alterations such as k - ras mutation or dpc4 inactivation in higher - grade panin lesions [ 6 7 8 9 ] . thus , it is hypothesized that pancreatic ductal lesions may progress from a histologically normal duct through flat lesion ( panin-1a ) , papillary lesion ( panin-1b ) , atypical lesion ( panin-2 ) to severely atypical lesion ( panin-3 ) . panin-3 is likely to develop eventually into idc because panin-3 and idc share critical genetic abnormalities . however , the histological evidence that panin-3 invades beyond the basement membrane of pancreatic ductal epithelium , that is , the moment panin-3 becomes idc , has not been captured yet . as defined by the consensus guideline , panin is a microscopic papillary or flat , non - invasive epithelial neoplasia that is usually < 5 mm in diameter . , panin-3 obstructed several branch pancreatic ducts and subsequently caused pancreatitis which developed clinical symptom and was detectable as a pancreatic mass on imaging studies . since pancreatic cancer was suspected , further examinations were performed . histological examination of the resected specimens showed panin-3 slightly invading beyond the basement membrane of the ductal epithelia accompanied by fibrotic changes caused by occlusion of branch ducts . a 65-year - old woman had been admitted to a former hospital for acute pancreatitis , which recurred 2 months thereafter . she was referred to our center for further examination of her pancreas after the second pancreatitis had been relieved . she was slightly thin , showing a body mass index of 20.2 , and had neither smoking nor drinking habits . laboratory data showed no abnormalities including serum levels of amylase and tumor markers related to pancreatic diseases . abdominal ultrasound revealed a low echoic mass of 13 mm in diameter in the pancreatic body without upstream dilatation of the main pancreatic duct ( mpd ) ( fig . endoscopic ultrasound showed a low echoic mass of 20 mm in diameter in the pancreatic body ( fig . since pancreatic cancer was suspected in the pancreatic body , she underwent endoscopic retrograde pancreatography , which showed a strictured segment of 2 mm in length in the mpd at the pancreatic body ( fig . cytological examination of the pancreatic juice obtained during endoscopic retrograde pancreatography revealed adenocarcinoma ( fig . 1 g ) . the patient was diagnosed with pancreatic body cancer without obvious vascular involvement ( t3 , n0 , m0 , stage iia , according to the uicc classification ) and underwent distal pancreatectomy . the resected specimens were fixed in formalin and cut at a slice thickness of 5 mm as shown in fig . histological examination revealed papillary growth of intraductal epithelia with significant nuclear atypia which was classified as panin-3 according to the definition of the consensus guideline mainly in the branch ducts ( fig . panin-3s were located separately in branch ducts with normal epithelia in the mpd between them ( fig . 2b , h , i ) , indicating that they originated from different sites of the branch ducts . histologically , this case was diagnosed as idc of the pancreatic body ( t1 , n0 , m0 , stage i ) . high - grade panin ( panin-3 ) is recognized as a best - defined precursor lesion of idc based on genetic as well as histological observations . panin-3 has been reported to share some genetic alterations with idc [ 6 7 8 9 ] and to be found usually in the pancreas with idc [ 2 3 4 5 ] . however , the direct evidence of the moment when panin-3 invades beyond the basement membrane to become idc has not been captured yet . this is the first case report in which microinvasion of panin-3 was histologically confirmed in the resected pancreatic specimens . considerable ambiguities had existed in the distinction between panin and ipmn , therefore the definitions of these two lesions were revised at the consensus meeting held at johns hopkins hospital in 2003 . according to the revised definitions , panin is a microscopic , papillary or flat epithelial neoplasm arising in the pancreatic duct and differs from ipmn , which is defined as a grossly visible , mucin - producing , predominantly papillary epithelial neoplasm . in this case , pancreatic epithelial neoplasm itself was not identified in any radiological examinations , and mucin production was not evident in pancreatic ductal epithelia by microscopic observation . a few reports had suggested that minimally invasive carcinoma should be distinguished from invasive ipmn because this subgroup of ipmn showed apparently better prognosis [ 11 , 12 13 ] . however , the precise definition of minimally invasive carcinoma has not been established [ 14 , 15 ] and has been recently proposed by some studies [ 16 , 17 ] . in this case , the patient has been alive without any signs of recurrence for more than 3 years . since there has been no concept of minimally invasive panin to date , this case is classified as idc in spite of excellent outcome . a new notion that minimally invasive panin should be distinguished from idc might be widely accepted in the future . in this case , panin-3 lesions were located separately in several branch ducts without involving the epithelia of the main duct between them . this fact indicates that panin does not arise from one small portion of pancreatic ductal epithelium and spread creeping along the epithelia , but arises multicentric . it is well known that ipmn , another precursor lesion of idc , occurs multicentric . as the whole pancreas may be involved in the genetic alteration which is related to carcinogenesis , it is not difficult to understand that panin occurs independently in different sites of the pancreas . several cases dealing with multiple occurrence of idc have been reported as rare clinical manifestation [ 18 , 19 ] . this observation could explain the rapid growth of pancreatic ductal carcinoma as follows : microinvasion initially occurs in several pancreatic ducts ; each invasion independently progresses and eventually makes a fusion mass . this observation could also explain the multiple occurrence of idc when microinvasions occur independently distinct from each other . unlike ipmn , panin-3 is hardly detected in clinical practice because panin-3 itself is not visible on imaging examination . in this case , panin-3 was successfully detected due to pancreatitis caused by the intraductal growth of panin-3 and subsequent occlusion of the pancreatic ducts . pancreatitis might be a candidate for the diagnostic clues to the early detection of pancreatic ductal carcinoma within the preinvasive stage . there are other known diagnostic clues to early detection , such as pancreatic cysts or dilatation of the mpd . in this case , however , neither pancreatic cyst nor dilatation of the mpd was observed . the detection of pancreatic ductal carcinoma in the preinvasive stage is the most promising way of improving patient survival . we hope that our report will contribute to the further understanding of early development of pancreatic ductal carcinoma .

high - grade pancreatic intraepithelial neoplasia ( panin-3 ) is recognized as a precursor lesion of invasive ductal carcinoma ( idc ) . however , histological evidence that panin-3 invades beyond the basement membrane of pancreatic ductal epithelium , that is , the moment panin-3 becomes idc , has not been captured yet . this may be because panins which are microscopic papillary or flat lesion rarely develop clinical symptoms and are not detectable on imaging examination . on the other hand , most idcs were found in the advanced stage with massive invasion . in this report , panin-3 obstructed several branch pancreatic ducts and subsequently caused pancreatitis which developed clinical symptom and was detectable as a pancreatic mass in imaging studies . a 65-year - old woman was referred to our institution for further examination of her repeated pancreatitis . abdominal ultrasound revealed a low echoic mass of 13 mm in diameter in the pancreatic body without upstream dilatation of the main pancreatic duct ( mpd ) . endoscopic retrograde pancreatography showed a strictured segment of 2 mm in length in the mpd at the pancreatic body . cytological examination of pancreatic juice revealed adenocarcinoma and distal pancreatectomy was performed . a resected specimen revealed a whitish mass of 15 mm in diameter in the pancreatic body , which was identified as pancreatitis by histological examination . papillary growth of panin-3 was seen mainly in the branch ducts . each panin-3 was located separately in the branch ducts with normal epithelia in the mpd between them . in three adjacent branch ducts , panin-3 was observed to be invading microscopically beyond the basement membrane .

Introduction Case Report Discussion

ductal adenocarcinoma of the prostate was first reported by melicow and pachter in 1967 as an endometrial carcinoma prostatic utricle . since then , ductal adenocarcinoma of the prostate has been found to account for 0.27.5% of all prostate carcinomas . a 73-year - old man was referred to our hospital due to an elevated prostate - specific antigen ( psa ) level of 23.4 ng / ml . he had no remarkable medical history . the hematological and biochemical data showed no abnormal findings aside from the elevated psa levels . in february 2016 , a prostate needle biopsy detected gleason score 4 + 4 adenocarcinoma in his left prostate . computed tomography ( ct ) and magnetic resonance imaging ( mri ) showed a higher density on his left peripheral zone ( fig 1a , b ) . in may 2016 , radical prostatectomy with lymph node resection histologically , there were many large , clear - edged cells and cancer cells with low differentiation forming a circular shape . based on these findings , ductal adenocarcinoma and gleason score 4 + 4 = 8 acinar adenocarcinoma with positive surgical margin were diagnosed . the patient has not experienced recurrence or biochemical recurrence in the 10 months since radical prostatectomy . histologically , there were many large , clear - edged cells and cancer cells with low differentiation forming a circular shape . based on these findings , ductal adenocarcinoma and gleason score 4 + 4 = 8 acinar adenocarcinoma with positive surgical margin were diagnosed . no adverse perioperative events were observed . the patient has not experienced recurrence or biochemical recurrence in the 10 months since radical prostatectomy . ductal adenocarcinoma of the prostate was first reported as endometrial carcinoma of the prostatic utricle in 1967 . recent studies have suggested that ductal adenocarcinoma of the prostate developed from the ductal epithelium , based on findings from immunohistochemical and electron microscope analyses . histologically , ductal adenocarcinoma of the prostate is characterized by high cylindrical epithelium collate papillary or etat cribriform . the histological differences between ductal adenocarcinoma and acinar adenocarcinoma are thought to be clear . in this case , although the prostate needle biopsy showed acinar adenocarcinoma , the surgical specimens showed ductal adenocarcinoma . the first is a mixed type with acinar adenocarcinoma and accounts for < 75% of ductal prostate specimens . mixed - type ductal prostate adenocarcinomas account for 5.06.6% of all prostate cancer cases , and pure - type ductal prostate adenocarcinomas account for 0.40.8% of all prostate cancer cases . because ductal carcinomas account for 90% of all prostatic carcinoma cases , our case was assumed to be pure type . because of its extension toward the urethra , the tumor was not palpable on a digital rectal examination and showed a low psa level . ductal adenocarcinoma of the prostate usually extends toward the urethra and shows macrohematuria and urinary symptoms at an early stage . reported that ductal adenocarcinoma of the prostate showed a significantly poorer prognosis than acinar prostate adenocarcinoma in nonmetastatic cases . however , in metastatic cases , there were no prognostic differences between these 2 groups . other reports have found no marked differences in the 5-year survival rate between ductal adenocarcinoma and gleason score 810 acinar adenocarcinoma . reported therapies of ductal adenocarcinoma of the prostate are also the same as for acinar adenocarcinoma , including radical prostatectomy , androgen deprivation therapy , and radiation therapy or a combination of these therapies . reported that pure ductal adenocarcinoma tended to extend into the submucosal urethra ; as such , pure ductal adenocarcinoma carries a higher risk of a positive surgical margin in the urethra . although we are not performing adjuvant therapy in this patient at present , careful observation including ct , mri , or positron emission tomography - ct should be performed , as psa does not always accurately represent cancer progression .

ductal adenocarcinoma is an unusual variant of adenocarcinoma of the prostate . a 73-year - old male was referred to our hospital for the further examination of an elevated prostate - specific antigen level of 23.4 ng / ml . radical prostatectomy ( rp ) was performed based on the diagnosis obtained by a prostate needle biopsy . the rp specimen revealed ductal adenocarcinoma of the prostate with positive capsular penetration . we herein report a rare case of ductal adenocarcinoma of the prostate .

Introduction Case Presentation Operative Procedure and Pathological Findings Postoperative Course Discussion Statement of Ethics Disclosure Statement

patients treated with salvage chemotherapy had a response rate of only 9% and a median time to tumor progression ( ttp ) of 9 weeks . although bevacizumab can offer a significantly higher response rate of 55% and a period of clinical stabilization with a median ttp of 26 weeks , the tumor remains in the brain and continues to proliferate despite clinical and radiological appearances of improvement . as a result , bevacizumab has a questionable impact on the overall survival of patients [ 3 , 4 ] . therefore , new and novel treatments are needed for patients with recurrent glioblastoma who failed initial treatment with radiotherapy and temozolomide . the novottf-100a device is a new treatment approved by the united states food and drug administration ( fda ) for recurrent glioblastoma . the device emits alternating tumor treating electric fields ( ttfields ) via 2 pairs of transducer arrays placed orthogonally on the scalp . the ttfields work by interrupting tumor cells during mitosis , resulting in violent blebbing during cytokinesis , asymmetric chromosome segregation , and aneuploidy [ 5 , 6 ] . these cell biology effects ultimately result in apoptosis or slippage into a g0 state of the tumor cell , while simultaneously making it susceptible to immunogenic cell death . in the pivotal phase iii clinical trial , the novottf-100a device had a similar efficacy when compared to salvage chemotherapy , but without the toxicities associated with systemic chemotherapies [ 7 , 8 ] . here , we report a patient who had failed bevacizumab therapy for a recurrent cystic glioblastoma and , at the time of bevacizumab continuation , received add - on ttfields therapy by using the novottf-100a device . this treatment combination eventually resulted in the disappearance of cystic enhancement together with a marked reduction of the cyst size and the cerebral edema in the surrounding brain . the patient is a 76-year - old right - handed woman who came to the brain tumor center for an evaluation of her recurrent glioblastoma after bevacizumab failure . her initial neurological problems occurred 9 months prior to presentation and consisted of mental confusion and comprehension problems manifesting as a fluent aphasia . a gadolinium - enhanced head mri at an outside hospital showed a cystic enhancing mass in the left temporal lobe of the brain . she then received 6 weeks of external beam fractionated radiotherapy to 6,000 cgy ( 200 cgy in 30 fractions ) with concomitant daily temozolomide at 75 mg / m , followed by adjuvant temozolomide 200 mg / m for 5 days on a monthly basis . after 5 cycles of adjuvant temozolomide , new cystic enhancement was discovered while performing a head mri ( fig . 1a , b ) , and she was placed on 4 mg of dexamethasone 4 times / day . bevacizumab was subsequently started at a dose of 10 mg / kg every 2 weeks . after 2 cycles of bevacizumab , there was only a partial decrease in the gadolinium enhancement and the size of the cystic tumor ( fig . additional pathology testing revealed a negative idh1 immunohistochemical labeling but a positive olig2 , egfr amplification and methylated mgmt promotor status . because ttfields interrupt tumor cells during mitosis and have no appreciable overlapping toxicity with bevacizumab , we proceeded to add to her bevacizumab treatment ttfields therapy using the novottf-100a device . the treatment compliance was recorded by sensors , embedded within the transducer arrays and can be downloaded by computer for review in clinic visits . after a total of 6 cycles of bevacizumab plus ttfields therapy , with a respective mean and median compliance of 17.6 hours ( 73% of the day ) and 18.4 hours ( 77% of the day ) ( range 3.6 to 22.8 h ) , there was a near complete resolution of gadolinium enhancement and a 65% reduction in the size of the cystic tumor ( fig . 1e , f ) . however , there were also recurrent tumors detected in the left internal capsule and the medial left frontal brain ( fig . 2a , b ) , which were located outside of the prior radiation treatment fields . therefore , the recurrent tumors were treated with fractionated cyberknife radiosurgery to 2,100 cgy ( 700 cgy in 3 fractions ) . despite this radiosurgery intervention , the patient continued to deteriorate neurologically and her deterioration was most likely caused by the microscopic invasive glioblastoma . she eventually died 15 months after her first recurrence and 22 months after initial diagnosis . the addition of novottf-100a to bevacizumab therapy in our patient appears to be safe and may provide added efficacy after initial incomplete response to bevacizumab alone . the rationales to combine ttfields therapy with bevacizumab are threefold . first , there is no overlapping side effect and , therefore , this combination does not appear to cause synergistic or additive toxicities . in patients with glioblastoma , we retrospectively analyzed 20 patients treated with this combination and found no instance of intracranial hemorrhage . other treatment side effects are minor in severity and they include the expected scalp rash , electric shock sensation from poorly applied transducer arrays and vivid dreams , all of which resolved upon the application of corticosteroid cream and adjustment of the arrays . second , ttfields therapy mimics the effects of chemotherapy by interference with tumor cell mitosis while not having the conventional side effects of chemotherapy [ 5 , 7 ] . when first approved by the fda , bevacizumab was combined with various cytotoxic agents , including carboplatin and irinotecan , carboplatin and etoposide as well as lomustine or carmustine , but the side effects do not warrant the routine clinical use of bevacizumab combined with cytotoxic chemotherapy . third , both bevacizumab and novottf-100a are listed in the national comprehensive cancer network practice guideline for recurrent glioblastoma . therefore , there appear to be strong rationales to combine novottf-100a with bevacizumab for recurrent glioblastoma . the resolution of our patient 's cystic tumor is a notable response to novottf-100a and bevacizumab after initial bevacizumab failure . although the response assessment in neuro - oncology criteria deem it as nonmeasurable , cystic tumors can not be measured in a traditional bidimensional fashion for response assessment , unless there is an associated solid nodule measuring 10 mm or greater in diameter . however , the disappearance of enhancement in the cystic tumor is still remarkable . in their retrospective series of 51 recurrent high - grade gliomas treated with bevacizumab and irinotecan , zuniga et al . reported only 8% of patients with a complete response while the majority had either a partial response ( 63% ) or no response ( 29% ) . similarly , when bevacizumab was used as a single agent , kreisl et al . and friedman et al . these data indicate that bevacizumab alone rarely results in a complete radiographic response , and the partial response seen in a majority of patients suggests that there are probably multiple proangiogenic pathways activated in the glioblastoma . a prior post hoc analysis of the response characteristics from the pivotal phase iii trial indicates that secondary glioblastomas as well as low or no dexamethasone usage are potentially important predictors of response in patients treated with the novottf-100a device alone [ 7 , 17 ] . first , secondary glioblastomas may have a different genetic composition that makes the tumor cells more susceptible to ttfields . indeed , verhaak et al . have shown that most of the secondary glioblastomas fall in the proneural genotype with amplification of pdgfra and olig2 as well as mutations in idh1 and tp53 . although our patient 's tumor appears to be a primary glioblastoma by the lack of idh1 immunohistochemical labeling , there could still be unidentified genetic mutations that make her tumor susceptible to ttfields therapy . indeed , 9 out of 14 responders in the phase iii trial did not have prior low - grade histologies , and they could have genetic mutations or epigenetic alterations that make them susceptible to novottf-100a monotherapy . second , the slower growth rate of our patient 's tumor may have helped to allow sufficient time for the ttfields to effect a treatment response . this is because the median time to response for ttfields therapy is longer than that for chemotherapies , 8.4 versus 5.8 months , respectively , as noted in the prior post hoc response analysis . last , our patient 's dexamethasone was completely discontinued 2 months after initiation of combination ttfields and bevacizumab therapy , but a near complete resolution of gadolinium enhancement and a significant reduction of the cystic tumor were not detectable until 6 months into treatment . consistent with this observation , the inverse relationship between response and dexamethasone dosage is probably a consequence of removing the immunosuppressive effect of dexamethasone , which would allow for better control of the glioblastoma by the patient 's immune system . therefore , allowing sufficient treatment time and removing dexamethasone are key parameters to maximize the probability of a response from ttfields . the pattern of relapse from combination ttfields and bevacizumab therapy is unknown . at the time of tumor recurrence , this type of distant recurrence of glioblastoma could be the consequence of progressive development of the invasive phenotype , intracranial inhomogeneity of the ttfields , or incomplete coverage of certain parts of the brain by ttfields . these hypothesis - generating observations would be important for future studies to correlate the location of the relapsed disease and the distribution of the electric fields within the brain .

the novottf-100a device emits alternating tumor treating electric fields ( ttfields ) that interfere with cytokinesis and chromosome segregation during mitosis . because it has a similar efficacy to cytotoxic chemotherapy , the device has been approved by the united states food and drug administration for the treatment of recurrent glioblastoma . although bevacizumab has been in use for recurrent glioblastoma , patients who experience incomplete or no response to bevacizumab may be predisposed to early bevacizumab treatment failure . however , the addition of ttfields therapy may augment the efficacy from bevacizumab . we report a patient with recurrent cystic glioblastoma who received add - on ttfields therapy due to an incomplete response to single - agent bevacizumab . after 6 cycles of therapy , a resolution of cystic enhancement was noted , together with reduction of the tumor cyst and resolution of most of the cerebral edema in the surrounding brain . however , the patient also suffered from relapsed disease at locations distant from the original glioblastoma and the corresponding radiation fields received at initial diagnosis . we conclude that combination ttfields and bevacizumab therapy is safe and may be efficacious for patients with recurrent glioblastoma . a further study would be needed to determine the relapse pattern and the distribution of the electric fields in the brain .

Introduction Case Report Discussion

plaque - induced gingivitis is one of the most frequent periodontal diseases , affecting more than 90% of the population , regardless of age , sex or race20 . brazilian epidemiologic studies show a high prevalence of gingival inflammation , ranging from 74% to 100% , although the mean individual percentage of gingival bleeding varies from 28% to 35%5 . however , the inability of the normal adult population to perform adequate toothbrushing has led to the search for chemotherapeutic agents in order to improve plaque control12 . these chemicals , mainly triclosan and chlorhexidine , have been used as mouthrinses or added to dentifrices to avoid plaque formation and development of gingivitis10,12,14,22 . as some of these substances may have undesirable side effects , such as tooth staining and taste alteration , phytotherapic agents with antimicrobial and antiinflammatory properties have been investigated7,16,17 . the use of natural products in the prevention and treatment of oral conditions has increased recently and could be of benefit to low - socioeconomic level urban and rural communities4 . among the various currently available herbal agents , aloe vera , popularly known as " babosa " , is a plant commonly found the northeast of brazil . its foliage , extract and resin present antimicrobial , antiinflammatory and healing properties and are indicated to hepatic and stomach diseases9 . the antimicrobial effect of a dentifrice containing aloe vera has been demonstrated in an in vitro study , in which this phytotherapic agent inhibited the growth of diverse oral microorganisms , such as s. mutans , s. sanguis , a. viscosus and c. albicans 7 . the only study available evaluating the clinical effects of aloe vera showed a significant reduction of gingivitis and plaque accumulation after use of a mouthrinse containing this natural product20 . to the present date , there is no reported controlled trial evaluating the efficacy of a dentifrice containing aloe vera in the control of plaque and gingivitis . therefore , the purpose of the present study was to assess the antiplaque and antigingivitis effects of this phytotherapic agent compared to a fluoridated dentifrice . thirty adult subjects from the university of fortaleza , brazil ( 15 male and 15 female , aged 35 to 43 years ) were enrolled in this double - blind , parallel , controlled clinical trial . all randomly screened participants were informed about the nature of the study and signed an informed consent form in compliance with the guidelines of the brazilian national health council . the protocol was approved by the institutional ethics committee ( report cotica / unifor : 407/ 2006 ) . the subjects were entered in the study if they had gingival bleeding index ( gbi)1 > 40% , presence of at least 20 natural teeth and absence of supragingival calculus and other plaque retentive factors . subjects with medical disorders or probing depth > 3 mm , individuals under antimicrobial therapy at least 1 month prior to the study and using mouthrinses or dentifrices containing substances with antiinflammatory properties , as well as smokers and pregnant women were excluded from the trial . the participants were assigned to either the test group ( n=15 ) or the control group ( n=15 ) by random permutation of three . the test group used a herbal dentifrice containing aloe vera ( forever bright , forever living products , tempe , az , usa ) and the control group used a fluoridated dentifrice ( sorriso dentes brancos , kolynos do brasil , so paulo , sp , brazil ) , with no antiinflammatory properties , but with color and taste similar to those of the test dentifrice . a single , previously calibrated examiner3 scored the gingival bleeding index ( gbi)1 and the plaque index ( pi)19 , which were recorded on the buccal , mesial , distal and lingual surfaces of all teeth . the values of 4 sites of each tooth were averaged to determine the gbi and pi for each subject . in addition to this examination , the hard and soft oral tissues were visually inspected for the presence of any adverse reaction by the same examiner . after the initial examination , all teeth of each subject were polished with pumice and flossed to eliminate plaque remnants . a personal " kit " containing a new toothbrush ( leader ; facilit odontolgica e perfumaria ltda , rio de janeiro , rj , brazil ) and the test or control dentifrice was given to all participants . they were instructed to brush their teeth for 1 min , three times a day , using the bass technique , and to refrain from any other oral hygiene procedures throughout the period of the clinical trial . verbal and written instructions about the correct use of dentifrice were given to all subjects as well . the tubes containing the dentifrices were previously coded to warrant that neither the examiner nor the volunteers knew their content , which was revealed only after completion of the study . the subjects were asked to return their dentifrice tubes , that were weighted by a digital balance ( filizola , modelo bp6 , indstrias filizola s.a . , so paulo , sp , brazil ) , previously and after the trial , so that compliance could be indirectly evaluated . student 's t - test was used to evaluate statistical differences between the weights of dentifrice tubes on days 0 and 30 ( =0.01 ) . mann - whitney test was performed to evaluate statistical differences between control and test groups on days 0 and 30 ( =0.01 ) . in each group , the mean scores of gbi and pi were compared between baseline and the end of the trial by the wilcoxon test ( =0.01 ) . the test dentifrice had a good acceptance and did not show adverse effects , such as formation of abscess and ulcerations or allergic reactions . only one subject in the test group reported unpleasant taste , but he did not drop out the clinical trial . there was a significant reduction of dentifrice tube weights between days 0 and 30 in both groups ( p<0.001 ) , which indirectly indicated that the volunteers actually used the dentifrices ( table 1 ) . on day 0 , there was no statistically significant difference between the control and test groups with respect to gbi ( p=0.8774 ) and pi ( p=0.0477 ) means . these results indicated that both groups were well balanced at baseline ( tables 2 and 3 ) . at the 30th day , plaque ( p=0.2801 ) and gingival bleeding ( p=0.9346 ) were present in both groups , but the difference between them was not significant statistically ( tables 2 and 3 ) . comparing the means between baseline and day 30 in each group , there was statistically significant difference in both the gbi ( p=0.002 , control group and p=0.001 , test group ) and pi indexes ( p=0.002 , control group and p=0.001 , test group ) ( tables 2 and 3 ) . aloe vera is a natural product contained in herbal dentifrices with commercial appeal on the control of plaque and gingivitis . despite its free commercial use , this phytotherapic agent does not have sufficient data to support its antigingivitis and antiplaque claims21 . to the best of our knowledge , the present study is the first report about effect of a dentifrice containing aloe vera on gingivitis . the results showed that both toothpastes were efficient on plaque reduction ( 23% in test group and 19% in control group ) . this percent difference was not significant at the end of the trial . in spite of a reported in vitro inhibitory effect of aloe vera against microorganisms from supragingival biofilm7 , its in vivo antiplaque effect in the present study was not satisfactory . it is interesting to note that this previous study7 evaluated a dentifrice that also contained other antiplaque agents , which probably masked the effect of aloe vera . the test and control dentifrices reduced gingivitis significantly , although the fluoridated dentifrice presented a higher percent reduction on bleeding areas ( 56% versus 53% ) . the gbi is a generally used dichotomous index to evaluate gingivitis , but it does not access the severity of gingival inflammation . studies evaluating the reduction of gingivitis by a grading index could be interesting to complement these results . gingival index uses a scale in which color changes in the gingival tissues precede bleeding on probing ; however this parameter is not necessarily an accurate indicator of gingivitis18 . the findings of the present study are in agreement with those of villalobos , et al.20 ( 2001 ) , who observed a significant reduction on plaque and gingivitis after a 30-day use of mouthrinses containing aloe vera associated to toothbrushing . this study20 also showed an additional antiplaque and antigingivitis effect of this phytotherapic agent , which was not observed in the present clinical trial . villalobos , et al.20 ( 2001 ) used a higher concentration of aloe vera ( 50% ) , which could explain the better effect of this phytotherapic agent when compared to our findings . although the manufacturer does not inform the concentration of aloe vera in the product used in the present study , the percentage of therapeutic agent in a dentifrice usually range from 0.4% to 1.0% of the total formulation6 , which was probably the concentration used in our study and could explain those results . furthermore , villalobos , et al.20 ( 2001 ) used a mouthrinse containing only aloe vera as the active agent , favoring its action without interference of other components . the test dentifrice used on the present trial contains other agents that can promote a moderate antiplaque effect , such as menthol and sodium lauryl sulfate12,21 . since the last two components are also present in the control dentifrice and considering that no difference was found between groups the antagonism of diverse substances with similar effects , in the same product , should be considered as well . this fact has been highlighted by wua and savitt21 ( 2002 ) and confirmed by other clinical trials comparing fluoridated and herbal dentifrices11,15 . home - use dentifrice studies are often influenced by a number of factors which can mask the superiority of a test agent over the controls . participants in clinical trials may experience some improvement associated not specifically to the therapeutic properties of the test agent but rather related to a behavior change - hawthorne effect15 . subjects enrolled in oral hygiene studies usually improve their toothbrushing , irrespective of the product they receive11,13,15 . although the volunteers of the present study were not aware of which dentifrice they were using , another important factor is the novelty effect , which is the motivation of oral hygiene practice by the use of a new substance . on the other hand , lack of compliance in the correct use of dentifrice can occur as well15 . in order to minimize its occurrence , the participants were asked to bring the dentifrice tubes at the end of the trial to be weighed , so we could evaluate indirectly subject compliance . the significant difference between weights in both groups , before and after the study , indicates that the participants used the products , but does not confirm if they were used correctly . however , it may not be sufficient to show the superiority of the test dentifrice over the control toothpaste15 . further long - term studies must be performed to evaluate the antigingivitis effect of this herbal dentifrice . if its real benefit is confirmed , the use of aloe vera should be advantageous in cases where patients have little motor skills and toothbrushing is compromised . within the limits of this clinical study , it may be concluded that the dentifrice containing aloe vera did not show any additional effect on plaque and gingivitis control compared to the fluoridated dentifrice .

the effect of aloe vera on the reduction of plaque and gingivitis was evaluated in a randomized , parallel and double - blind clinical trial . subjects were randomly allocated to the test group ( n=15 ) dentifrice containing aloe vera - or the control group ( n=15 ) fluoridated dentifrice . plaque index ( pi ) and gingival bleeding index ( gbi ) were assessed at days 0 and 30 . subjects were asked to brush their teeth with the control or test dentifrice , three times a day , during a 30-day period . there was a significant reduction on plaque and gingivitis in both groups , but no statistically significant difference was observed among them ( p>0.01 ) . the dentifrice containing aloe vera did not show any additional effect on plaque and gingivitis control compared to the fluoridated dentifrice .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSION

there have been sporadic cases reported since it was first described by lennox et al . , in 1952 . we report an additional case of pmc of the skin in a 70-year - old male presenting with a swelling in the lateral canthal region of his left eye , and review the pertinent literature . a 70-year - old male noticed a swelling near the the left canthus since one - and - a - half years . a raised , freely mobile , firm skin mass of approximately 3.0 2.5 cm was present on the lateral canthal region on the left eye . gross examination revealed a well - circumscribed , spherical soft tissue mass , partially skin covered , measuring 2 cm in diameter . the cut surface of the tumor had a gray - brown gelatinous appearance [ figure 1 ] . microscopically , the sections revealed a tumor in the dermis composed of tumor cells arranged in nests , glands , and cribriform patterns [ figure 2 ] . the cells were seen floating in large pools of mucin separated by thin fibrovascular septa [ figure 3 ] . [ figure 4 ] further investigations , including a colonoscopy , ultrasonographic examination of the abdomen , and computerized tomography ( ct ) scans of the chest , abdomen , and pelvis were found to be normal . thus , the lesion in the skin was reported to be pmc of the skin . gross specimen revealing a spherical , well - circumscribed tumor with a gelatinous cut surface . tumor in the dermis composed of cells arranged in nests , glands , and cribriform patterns , floating in mucin lakes ( h and e , 20 ) small nests of adenocarcinoma lying in pools of extracellular mucin that were separated by fibrocollagenous septae ( h and e , 40 ) photomicrograph showing pools of pas positive , diastase - resistant mucin between the tumor nests ( pas stain , 20 ) primary mucinous carcinoma of the skin ( pmcs ) is a rare adnexal neoplasm with sweat gland differentiation . this tumor was first described by lennox et al . , in 1952 , and later designated by mendoza and helwig , in 1971 . . recently studied approximately 200 cases of pmcs documented in the literature and found that the mean annual age standardized incidence of pmcs during the period 1978 to 2003 was 0.07 per million person - years . although some debate exists as to the apocrine or eccrine origin of this tumor , most authors favor the eccrine differentiation based on the evidence obtained from the immunohistochemical studies and electron microscopic ultra structural analysis . it has been suggested that pmcs may develop as a progression from abnormal apocrine or eccrine ducts , analogous to the progression seen in the mucinous carcinoma of the breast . a ductal in - situ component can occasionally be seen ranging from ductal hyperplasia to frank carcinoma in - situ . rosai originally suggested that the ductal proliferation continues until the overproduction of mucin results in islands of tumor cells breaking off and floating in the mucinous pools . primary mucinous carcinoma of the skin is slightly more common in men than women and typically affects people in the age range of 50 to 70 years . the large majority of specimens of pmcs arise on the face , especially on the eyelids and scalp . the primary lesion of mesenchymal chondrosarcoma ( mcs ) is often solitary , and the history is often of a small cutaneous lesion that grows slowly for many months or years and suddenly enlarges . by the time it is first brought to medical attention , the neoplasm is a nodule that varies in diameter from about 0.7 8.0 cm . mucinous carcinoma rarely originates in the skin ; the majority of examples in the skin are actually metastatic to it . common sites of origin of mucinous carcinomas are the breast , gastrointestinal tract , salivary glands , lacrimal glands , nose , paranasal sinuses , bronchi , renal pelvis , and ovary . metastatic lesions from the breast or colon are most likely to mimic mucinous carcinoma of the skin . differentiating primary mucinous carcinoma from metastatic tumors , particularly from these sites , the absence of expression of ck20 by immunohistochemical staining may exclude the diagnosis of metastatic colorectal mucinous carcinoma . it is important to recognize , however , that the likelihood of mucinous carcinoma from the breast , metastasizing to the skin ( much less on the face or eyelid ) is exceedingly low . cases of pmcs have been found to be estrogen receptor , progesterone receptor , and gcdfp-15 positive . qureshi et al . , suggest that finding an in - situ component of a tumor that stains for myoepithelial cells ( positive stains for p63 and ck5/6 among others ) can help to exclude metastatic mucinous breast carcinoma . the distinction of mcs from a mucinous carcinoma metastatic to the skin is important because the prognosis of mucinous carcinoma metastatic to the skin is poorer than that of mcs . primary lesions can be differentiated from metastatic lesions by their more organized epithelial cells , less hyperchromasia , and fewer mitoses in individual cells . in addition , in the case of metastatic carcinoma , tumor cells invade between the collagen bundles at the margin of the nodule . due to the difficulty in distinguishing these histologically , a thorough workup for metastatic lesions no primary tumor was detected in the breast , digestive tract , salivary glands , lacrimal glands , paranasal sinuses , lungs or kidneys . primary mucinous carcinoma of the skin is a slow - growing benign tumor with a high local recurrence rate . treatment of mucinous carcinoma entails local excision . on account of the high rate of recurrence , adequate excision with generous margins other treatments , such as chemotherapy and radiation , are generally not employed in the management of these tumors . patients should be counseled about the importance of frequent follow - ups for evaluation of local tumor recurrence or development of regional lymphadenopathy .

primary mucinous carcinoma of the skin is a rare adnexal tumor of sweat gland origin . a case report is presented of a 70-year - old male , who presented with a slow growing mass near the lateral canthus of his left eye . the case was clinically diagnosed as a fibroma . an excisional biopsy of the lesion revealed mucinous carcinoma of the skin . investigations excluded the possibility of metastatic mucinous carcinoma . thus , the lesion in the lateral canthus region was diagnosed as primary mucinous carcinoma of the skin , a rare site of occurrence .

INTRODUCTION CASE REPORT DISCUSSION

incidental detection of small renal masses is increasing . this has led to an increase in biopsy of small renal masses , a proportion of which needle biopsy of small renal masses is controversial owing to the risk of seeding malignant cells along the needle tract . needle tract seeding is a rare event ; the incidence is estimated to be less than 1 in 10,000 cases of all biopsies . eight other cases of needle tract seeding in a renal mass biopsy have been described in the medical literature , two as recently as 2013 ( table 1 ) . we report our experience of a man with renal cell carcinoma ( rcc ) seeding along a biopsy tract and compare the circumstances and biopsy techniques with reported cases in the literature . a 66-year - old man was incidentally found to have a 32-mm right lower pole renal mass on a computed tomography ( ct ) scan ( fig . two samples were obtained by use of a 16-gauge temno core biopsy needle ( carefusion , san diego , ca , usa ) and a 22-gauge francine needle . histopathology revealed a well - circumscribed 30-mm clear cell rcc , predominantly fuhrman grade 2 with focal areas of grade 3 . there was an area where the capsule was interrupted that corresponded to a hemorrhagic area on the cortical surface ( fig . a tumor deposit was also noted in the perinephric fat . these features suggested that the tumor deposit in the fat was likely due to tumor seeding rather than a metastasis and that the tumor seeding could have resulted from the needle biopsy . his tnm staging was pt3a nx mx , at least stage 3 disease ( american joint committee on cancer , 7th edition , 2010 ) and his leibovich score was 5 ( intermediate risk ) . six months after the operation , there was no radiological evidence of tumour recurrence on a ct scan . aside from the potential for false - negative results , a key risk of renal mass biopsy is seeding of the biopsy tract with malignant cells . several factors in theory could affect the risk of biopsy tract seeding , such as needle size , the number of needle passes , and the use of a coaxial needle . biopsy tract seeding has been reported in renal mass biopsies using needles as fine as 23-gauge and as large as 14-gauge . theoretically , a larger - bore needle would increase the risk of seeding owing to an increased area of defect on the surface of the tumor and an increased circumference or surface area of the needle . however , because of the scarcity of cases , it is difficult at this stage to accurately determine a relationship between needle size and the risk of seeding . it is also difficult because of underreporting to associate the risk of needle tract seeding with the number of needle passes through a tumor . use of a coaxial needle allows multiple passes through the renal mass with only one pass through the surrounding normal tissue . this theoretically reduces the risk of needle tract seeding into normal tissue and potentially reduces patient discomfort as well . although it is interesting to note that a coaxial needle was not used in any of the currently reported cases of needle tract seeding after renal mass biopsy ( table 1 ) , there are just too few cases to establish a firm relationship between the risk of biopsy tract seeding and the use of a coaxial needle . visualization of larger coaxial needles on ultrasound or ct may be easier than with smaller biopsy needles , and this may improve accuracy . histological evidence of biopsy tract seeding may not always be found after definitive surgery to remove the renal mass . seeding into excised perinephric tissues can be found soon after surgery but seeding into surrounding muscle , fascia , and skin may only be apparent months , or even years , after surgery . as was seen with this case , the biopsy needle traversed skin , subcutaneous tissue , multiple muscle and fascia layers , and perinephric fat before reaching the renal lesion ( fig . thus , the tumor could theoretically seed into one or more of these tissues ; seeding as superficial as the subcutaneous tissue has been reported ( table 1 ) . this delayed presentation may increase the risk of adverse outcomes such as further metastasis and poorer prognosis . time to presentation or diagnosis of tumor seeding after renal mass biopsy has ranged from 24 days to 84 months in previously reported cases where tumor seeding was not found on the initial histopathological analysis ( table 1 ) . in conclusion , a common feature in all reported cases of needle tract seeding from a renal mass biopsy is that a coaxial needle was not used . however , because of the paucity of cases , there is currently no satisfactory association between the risk of needle tract seeding and needle size or the number of needle passes . it is important to consider that histopathological evidence of needle tract seeding may not be apparent in all cases , especially if seeding occurred beyond the excised tissues .

a 66-year - old man underwent computed tomography - guided needle biopsy of a suspicious renal mass . two months later he underwent partial nephrectomy . histology revealed a 30-mm clear cell renal cell carcinoma , up to fuhrman grade 3 . an area of the capsule was interrupted , which corresponded to a hemorrhagic area on the cortical surface . under microscopy , this area showed a tongue of tumor tissue protruding through the renal capsule . a tumor deposit was found in the perinephric fat . these features suggest that tumor seeding may have occurred during the needle biopsy .

INTRODUCTION CASE REPORT DISCUSSION

despite its ubiquity in cell membranes , there is little quantitative information on the effects of small mole fractions of cholesterol on the phase diagrams , molecular organization , and surface viscosity of mixtures of phospholipids and cholesterol . understanding cholesterol s effects in altering the local molecular organization and rheology of lipid monolayers may give clues to the mechanisms that stabilize nanometer - scale rafts in complex lipid - cholesterol mixtures ; the raft hypothesis has emerged in recent years as a general organizing principle for the structure of eukaryotic cell membranes . rafts are hypothesized to result from nanometer - scale phase separation of ordered and viscous domains in which cholesterol , saturated lipids , and membrane proteins preferentially accumulate , surrounded by a sea of less viscous , unsaturated lipids with greater protein diffusivity . however , the fundamental physics underlying raft formation is still being developed , especially how interactions between saturated phospholipids and cholesterol determine membrane phase behavior , viscosity and diffusivity . the interactions of cholesterol and saturated phospholipids also play an important , but poorly understood , role in the properties of human lung surfactant ( ls ) , a lipid - protein monolayer necessary to reduce the surface tension in the lung alveoli . at present , even the existence of cholesterol in native ls is questioned , as the lung lavage required to harvest ls inevitably causes blood and cell debris to be coextracted , potentially contaminating ls with cholesterol . this lack of consensus is reflected in the composition of replacement lung surfactants , which are used to treat neonatal respiratory distress syndrome ( nrds ) , which occurs in 20 00030 000 premature infants each year in the u.s . survanta and curosurf , two clinically approved animal extract replacement surfactants for treatment of nrds , have all cholesterol removed after harvesting . infasurf , the third clinically approved surfactant , retains 4 - 5 wt % cholesterol . resolving this controversy is difficult , as there is little information on the effects of small cholesterol fractions on the organization and dynamics of phospholipid monolayers . our previous work has shown that the surface viscosity of dipalmitoylphosphatidylcholine ( dppc ) monolayers decreases by an order of magnitude per wt % cholesterol , up to about 3 wt % , suggesting that the cholesterol - containing infasurf would have significantly different monolayer dynamics than cholesterol - free survanta and curosurf . interfacial viscosity is believed to have a significant effect on monolayer collapse , surfactant spreading during breathing , and transport from the type ii epithelial cells , where surfactant is produced , to the alveolar air water interface . interfacial viscosity may also be important during the instillation of replacement surfactant and the reopening of bronchial airways . however , the origin of this dramatic effect of cholesterol on dppc viscosity is not yet known . cholesterol may also play a role in lung surfactant inactivation in acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) . ards occurs as a rapid onset of respiratory failure and affects about 150 000 people per year in the u.s . with a mortality rate of 3040% . the pathogenesis of ards is not fully understood , but in both ali and ards , surfactant is inactivated by some primary pathogenesis such as lung inflammation , trauma , pulmonary infection , near - drowning , etc . the cholesterol content of ards patients shows an increase in cholesterol content and in vitro , cholesterol levels greater than 10 mol % increase the minimum surface tension at monolayer collapse , which may exacerbate lung damage . grazing incidence x - ray diffraction ( gixd ) shows that up to 7 mol % added cholesterol does not change the basic alkane packing of dppc , but does decrease the extent of ordering , or coherence area , suggesting an increased number of lattice defects . we postulate that the free area available for diffusive transport in a two - dimensional analog of the classic cohen and turnbull free volume model of viscosity is inversely proportional to the number of molecules in a coherence area . using this relationship , the surface viscosity data for all surface pressures and cholesterol fractions collapses to a simple logarithmic relation with no adjustable parameters . this suggests that the decreased molecular ordering caused by the incompatibility of cholesterol with the alkane chain lattice is the origin of the orders of magnitude decrease in surface viscosity . 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( dppc , r - enantiomer ) and dihydrocholesterol ( avanti , alabaster , al ) with 0.1 wt % texas - red dhpe ( n-(texas red sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , invitrogen , grand island , ny ) were mixed in the appropriate ratios and diluted to 0.2 mg / ml in hplc - grade chloroform ( fisher scientific , st . dihydrocholesterol ( chol ) was used instead of cholesterol to minimize oxidation but has little impact on phase behavior . surface pressure area isotherms were recorded at 25 c using a teflon trough ( nima , coventry , england ) with custom designed stainless steel ribbons which reduce film leakage at high surface pressures . a filter paper wilhelmy plate ( riegler and kirstein , gmbh ; potsdam , germany ) was used to measure surface pressure . the open area of the trough used was 125 cm and each complete compression / expansion cycle took about 8 min ( 0.42 cm / s ) . for fluorescence imaging , the langmuir trough was mounted on a nikon optiphot optical microscope with a custom designed stage equipped with long working distance objectives designed for fluorescent light . a dichroic mirror / barrier filter assembly directed the excitation light onto the monolayer films at a normal angle of incidence and filtered the emitted light and the images were detected by a silicon intensified ccd camera . videos of the monolayer film were recorded during the compression - expansion cycle directly onto the computer using the pinnacle studio capture software . the continuous , fluid lipid phase appears bright due to the preferential segregation of the texas red dye , while the better ordered domains exclude the dye molecules and appear dark . two - dimensional gixd experiments were carried out at the chemmatcars station at beamline 15-id at the advanced photon source , argonne national laboratory . dppc / chol at the appropriate ratios were dissolved in chloroform solution at 1 mg / ml and spread dropwise onto the air / deionized water interface in a custom langmuir trough , which was temperature - controlled at 22 c. after waiting 30 min for solvent evaporation , the monolayers were compressed to the desired surface pressure ( 20 , 30 , or 40 mn / m ) and annealed for an additional 30 min . the trough was enclosed in a helium - filled chamber and the oxygen level was constantly monitored during exposure to the x - ray beam . the analysis of gixd data for two - dimensional films at the air - water interface follows that of kaganer et al . it is well established that the bragg peaks correspond to ordering within the alkane chains of the lipid tailgroups ; the organization of the headgroups is inferred from changes in the tailgroup lattice in response to changes in surface pressure and cholesterol fraction . circular ferromagnetic probes ( microbuttons ) of diameter 20 m , thickness 1 m , with button holes of diameter 3.5 m were fabricated by photolithography . . a uniform magnetic field of magnitude , b , and orientation , , was generated by the output of two independent pairs of electromagnets controlled by a custom labview code to exert a controlled torque , l , on a microbutton of moment m and orientation . to measure the frequency - dependent linear viscoelastic response , a sinusoidal magnetic field was applied to generate a time varying applied torque . the microbutton orientation was determined from bright field images of the holes in the microbuttons as a function of applied torque , to determine the rotational resistance . from the rotational resistance , the linear viscoelastic surface moduli were obtained from the solution of the hydrodynamic problem of a rotating cylinder within a viscoelastic monolayer atop a viscous subphase . in terms of measured experimental properties , the surface loss modulus , g is the out of phase component of the rotational resistance , the surface storage modulus , g , is in the in - phase component as in conventional 3-d rheology ( figure 6b ) . the surface viscosity for the newtonian response we observed over the frequency range of 0.110 hz is s = g/ , or for the 1 hz frequency used in figure 6 , s = g/2. the same exponential dependence of surface viscosity on surface pressure was obtained using a 100 m probe showing that continuum values of elasticity and viscosity were being measured . the 100 m probe is more than an order of magnitude larger than the domain sizes we have seen ( figures 5 and 6 ) . recent results using macroscopic wire rings ( 10 cm diameter ring , 0.7 mm diameter wires ) showed identical trends with surface pressure and good agreement for similar monolayers . the maximum surface viscosity that we could measure with our rheometer was 100 pams ; the surface viscosity of pure dppc at 40 mn / m was greater than this and was not measured . freshly - cleaved mica substrates ( s&j trading inc . ; glen oaks , ny ) connected to a computer - controlled dipping mechanism in a commercial circular nima l - b trough ( biolin scientific , inc . , linthicum heights , md ) were pulled through the monolayer at 5 mm / min at a constant surface pressure of 20 mn / m . transfer ratios were determined by recording the interfacial area change of the trough during transfer and comparing this to the surface area of the mica substrate . a transfer ratio of 1 means that these areas are equal ; only films with transfer ratios of 1 were examined . the mica substrates were glued to stainless steel discs and affixed to the magnetic holder of an mmafm-2 afm ( digital instruments ; santa barbara , ca ) with a cantilever tip ( asylum research , ac160ts ; santa barbara , ca ) designed for tapping mode operation . two - dimensional gixd was carried out at chemmatcars , sector 15-id at the advanced photon source , argonne national laboratory on dppc monolayers with various mole fractions of dihydrocholesterol ( chol ) . figure 1 shows the gixd intensity maps for ( a ) pure dppc at 20 mn / m and ( b ) 6.4 mol % chol / dppc monolayers at 40 mn / m . figure 2 shows the qz - integrated intensity profiles ( arbitrary units ) for dppc / chol monolayers at 20 , 30 , and 40 mn / m for 0 to 7 mol % chol ( each spectra offset by 1000 ) . two - dimensional x - ray maps of ( a ) pure dppc at = 20 mn / m and ( b ) 6.4 mol % chol in dppc at = 40 mn / m . two bragg reflections are visible , the degenerate reflection at positive qz and lower qxy and the at higher qxy and qz = 0 , indicating nearest neighbor tilt . the peak remains at the same location for all cholesterol or surface pressures ( dotted lines ) . the basic motif of the dppc lattice is unchanged by cholesterol , although the tilt is reduced with increasing cholesterol . cholesterol broadens both peaks consistent with a decrease in the size of the correlated areas in the monolayer . we assign the bragg peak in figure 1 at lower qxy and positive qz as due to the degenerate ( 11 ) and ( 11 ) reflections of distorted hexagonal packing ; the second peak at higher qxy and qz = 0 is due to the nondegenerate ( 02 ) reflection . as the ( 11 ) reflection is located at qz > 0 , and the ( 02 ) reflection is centered at qz = 0 , the alkane chains are tilted in the nearest neighbor ( nn ) direction . regardless of composition or surface pressure , all ordered areas in the monolayers had the same distorted hexagonal packing with nn tilt ( figure 3b , inset ) . ( a ) d11 and ( b ) d02 as a function of cholesterol and surface pressure . d11 decreases with increasing surface pressure and cholesterol fraction . this is consistent with a decrease in tilt , which decreases d11 , while the alkane chain packing normal to the chain axis , hence d02 remains the same . one gray and one white circle are the two alkane chains in a rectangular unit cell of dimensions a and b. translation of the pair of gray and white circles by a and/or b generates the lattice . the spacing between the dotted lines corresponds to the d - spacings in table 2 and a , b. the qz - integrated intensity profiles ( figure 2 ) show that the ( 11 ) bragg peak broadens and moves to higher qxy with increasing cholesterol content ( dotted lines ) for all surface pressures , while the ( 02 ) peak also broadens with increasing cholesterol content but remains at constant qxy . from the qij values in table 1 , the real space lattice dimensions are dij = 2/qij ( table 2 and figure 3 ) . the tilt angle , , for nearest neighbor tilt is given by tan = qz[q112 ( q02/2 ) ] . the coherence length is determined from the full width at half - maximum ( fwhmij ) of each peak after correction for the instrumental resolution , lij = ( 0.92)/(fwhmij ) ( table 2 ) . q02 and q11 are the lattice parameters in fourier space for the two strong reflections from the qz averaged data . all d - spacings are referenced to a two molecule rectangular unit cell with a = d10 = [ d112 ( 2d02 ) ] and b = 2d02= d01 ( see inset to figure 3b ) . the error in a is larger than b as the ( 11 ) reflection is much broader than the ( 02 ) reflection ( see figure 2 ) . is the tilt angle of the alkane chains in degrees with respect to the water surface , tan = qz[q112 (q02/2 ) ] from table 1 ( see inset to figure 3b ) . the chain area is the cross sectional area of the chains in the direction perpendicular to the chains , or ( ab cos )/2 . l02 and l11 are the coherence lengths in the ( 02 ) and ( 11 ) directions , lij ( 0.92)/(fwhmij ) . figure 3a shows that , at a fixed surface pressure , adding chol decreases d11 while figure 3b shows that d02 remains constant . for pure dppc , d11 decreases from 4.79 to 4.58 as the surface pressure increases from 20 to 40 mn / m ; d02 is constant at 4.30 ( table 1 ) . cholesterol and surface pressure influence the lattice in a similar way ; the same linear relationship between d11 and the tilt angle holds over the range of surface pressures and cholesterol fractions examined ( figure 4 ) . decreases with increasing surface pressure , and with some scatter , increasing cholesterol fraction from 35 for pure dppc at 20 mn / m to 18 for 7 mol % chol at 40 mn / m . this change in tilt causes a decrease in the area per dppc molecule at the air water interface from 49.9 1 to 43.9 1 . molecular tilt angle measured from the monolayer normal , , decreases in the same manner with increasing cholesterol fraction as with increasing surface pressure ( over this range of , sin tilt tilt ; to convert to degrees of tilt , = 180(tilt/ ) ) . black symbols 20 mn / m surface pressure , open symbols 30 mn / m and gray symbols 40 mn / m . with some scatter , the tilt decreases with increasing cholesterol fraction . this linear relationship between d11 and is the same for changes in cholesterol fraction and surface pressure , which suggests that the local alkane packing of dppc does not change with added cholesterol , but that both surface pressure and cholesterol act to decrease the mismatch between the lipid headgroup and tailgroup area in the same way . from the values in table 2 , we determine a rectangular two - molecule unit cell of dimensions , a = d10 = [ d112 ( 2d02 ) ] and b = 2d02 = d01 ( figure 3b inset ) , with a decreasing from 5.8 0.1 at 20 mn / m , to 5.4 0.1 at 40 mn / m ; b remains constant at 8.6 0.05 . these unit cell dimensions are consistent with a hexagonal packing of the alkane chains , which are tilted to accommodate the mismatch in projected area between the dppc headgroup and the close - packed chains . as is the case for other lipids , accommodation of the larger dppc headgroup area occurs by dilation of the alkane chain area via an increase in the tilt angle . tilt costs little favorable alkane chain contact energy , as tilt occurs without changing the distances between the alkane chains . tilt in the nn direction causes a , which is measured in the plane of the monolayer , to increase . however , b remains constant as this spacing does not change with tilt if the alkane chains retain their close - packed configuration . increasing the surface pressure provides a uniform compression of the dppc headgroup , which results in a decrease in the headgroup - chain incompatibility , and hence the tilt , without altering the alkane chain packing . the area per alkane chain perpendicular to the alkane chains , 20.5 0.3 = ( ab cos )/2 , is constant within the experimental error for all surface pressures and also for all cholesterol fractions . pure chol has an untilted hexagonal lattice with a d spacing of 5.7 , and an area per molecule of 35 , much larger than the 20.5 area per alkane chain we measure . the invariance of d02 ( or b ) , and the linear relationship between d11 and is consistent with the bulk of the chol not intercalating uniformly into the dppc lattice as it does at higher mole fractions , but separating primarily into a second phase . figure 5 shows afm images of dppc / chol monolayers transferred to mica substrates at 20 mn / m showing two distinct morphologies . at 0.8 mol % chol , dark gray , 10100 nm circular areas are dispersed in extended light gray domains . the dark gray nanodomains localize preferentially at the boundaries of the light gray domains ( arrows ) . increasing the chol fraction causes the circular nanodomains to percolate into linear structures ( 3.7 mol % ) , although the width of the linear structures remains 10100 nm . the linear structures eventually break up the light gray domains ( 5 mol % ) . afm force spectroscopy showed that the nanodomains were more compliant and easier to deform than the dppc domains , suggesting that the nanodomains are disordered and do not contribute to the gixd signature of the monolayer . the alkane chains of dppc prefer to be untilted to maximize the van der waals contact between the chains , but are frustrated by the conflicting cross - sectional area requirements of the phosphocholine headgroup . this mismatch requires the tailgroup lattice to dilate , which responds by the lower energy tilt deformation in order to fill space efficiently . however , chol has a complementary shape to dppc , with a relatively small alcohol headgroup and a relatively large sterol ring tailgroup . hence , this shape complementarity suggests that chol can relieve the packing frustration of dppc by making up some of the mismatch between the headgroups and alkane chains . palmitic acid ( pa ) and hexadecanol ( hd ) , which also have complementary shapes with dppc , also lead to a decreased tilt at a given surface pressure , but do not show nanodomains in afm images . hd is the same as the c16 alkane chain of dppc , which allows pa and hd to be incorporated into the dppc lattice . pa and hd increase the correlation length of the mixed crystal and the surface viscosity . tapping - mode afm images of dppc / cholesterol monolayers transferred by langmuir - blodgett deposition to mica substrates at 20 mn / m . for 0.8 mol % cholesterol , dispersed , circular 10100 nanodomains appear ( darker gray indicates more compliant relative to the lighter gray background phase ) , which preferentially locate at the boundaries of the light gray domains ( arrows ) . for 3.7 mol % chol , the circular nanodomains have condensed into linear features begin to break up the light gray domains , but the light gray domains remain continuous . for 5.0 mol % chol the nanodomains make up a cocontinuous network separating the light gray domains , while decreasing the size of the light gray domains to 100200 nm . fluorescence images of dppc monolayers with 0.0 , 0.2 , and 0.4 mol % cholesterol at coexistence between the disordered le ( light ) and ordered lc ( dark ) phases at 10 mn / m . contrast is due to doping the monolayer with 0.1 wt % of the fluorescent ( n-(texas red sulfonyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , ( invitrogen ) which segregates to the more fluid le phase . the domain width decreases with increasing cholesterol , indicative of a decrease in line tension . although the shape of cholesterol can relieve the frustration between the area of the headgroup and alkane chains of dppc , the sterol rings can not efficiently pack into the alkane chain lattice . this leads to a second type of frustration ; the decrease in tilt due to the accommodation of the area mismatch results in disrupting the alkane chain lattice , which is likely higher in energy than decreasing the tilt . the combination of the sterol rings of chol and more disordered dppc chains gives the nanodomain phase excess tailgroup area , which may relieve part of the packing frustration of the headgroups in the adjacent ordered dppc domains , albeit at longer range than if the cholesterol intercalated within the dppc lattice . the size of domains in typical phase - separated monolayers is governed by a competition between line tension , , which leads to larger domains , and entropy and electrostatic interactions , which favor smaller domains . fluorescence images ( figure 6 ) at the coexistence surface pressure ( 910 mn / m ) show that increasing chol leads to a dramatic decrease in the width of the domains , which means a large decrease in . this decrease may be sufficient to stabilize the nanodomains against coalescence . an additional factor stabilizing the nanodomains may be the energy of reducing the tilt in the adjacent dppc domains . the time for diffusive mixing of the nanodomains with the bulk is of order seconds over the 100 nm length scales of the nanodomains , so even with electrostatic interactions slowing bulk coalescence , molecular diffusion should eliminate the nanodomains in minutes if this structure were not stable . this stability may also be an indication that we are seeing an example of the recently proposed 2-d analogs of 3-d microemulsions , in which compositional variations within a single phase are due to coupling between monolayer curvature ( i.e. incompatible area requirements of headgroups and tails ) and composition . the evolution of the structure between discrete circular nanodomains ( analogous to spherical micelles in 3-d ) to extended linear structures ( rod - like micelles ) to an interconnected network ( bicontinuous microemulsion ) suggests this possibility . figure 7a shows that the coherence length , l02 , in the untilted direction for pure dppc is about 70 lattice repeats , or > 300 , more than five times that in the tilted direction , l11 60 or about 12 lattice repeats ( table 2 ) . for both 20 and 30 mn / m , l02 decreases monotonically with increasing cholesterol fraction to 20 lattice repeats , but l11 only decreases to 10 lattice repeats . at 40 mn / m , l02 does not monotonically decrease with cholesterol fraction , the scatter in l02 is much greater than at lower surface pressures , and l02 is always less than expected from the results for the lower surface pressures . this is likely due to a decrease in film stability caused by a combination of leakage under the trough barriers and slow monolayer collapse during the 3 - 5 h required for gixd . the afm images in figure 5 show that the average dppc ( light gray ) domain size decreases from microns to 100200 nm with cholesterol . even with the decreasing domain size , the positional ordering given by the coherence lengths are orders of magnitude smaller than the domain size for a given cholesterol fraction . however , the orientational order extends for tens of microns as shown by the spiral domain textures in figure 6 . dppc / chol monolayers have nanometer - range positional order and micron - range orientational order , similar to tilted smectic c liquid crystals and other langmuir films that are classified as hexatics . ( a ) coherence lengths , l11 , in the ( 11 ) or tilt direction , and l02 , in the ( 02 ) or untilted direction , normalized by their respective lattice constants , d11 and d02 . l02 decreases significantly with cholesterol fraction , but is less affected by surface pressure . l02 for 40 mn / m has more scatter and is nonmonotonic with cholesterol fraction , likely the result of film instabilities due to trough leakage and monolayer collapse at higher surface pressure . ( b ) the surface viscosity of dppc / chol monolayers measured with a microbutton magnetic rheometer decreases exponentially with cholesterol fraction for a given surface pressure for small mole fractions of cholesterol , then plateaus in the same fashion as l02 . ( surface viscosity for pure dppc at 40 mn / m was too high for the viscometer to measure . ) ( c ) free area model for dppc / chol monolayers ( eqs 6 - 8 ) provides an excellent correlation between the surface viscosity and the number of correlated molecules , ( l02l11)/ab , over the entire range of cholesterol fraction . the lines are linear regression fits of eq 7 to the data ( p < 0.01 ) . ( d ) normalizing to a reference state ( taken to be that of pure dppc for 20 and 30 mn / m and 0.4% chol for 40 mn / m surface pressures ) , collapses the data onto a single universal curve relating surface viscosity to the molecular organization . the line is a linear regression fit of eq 8 to the data with p < .001 showing that the data is well described by the free area model . figure 7b shows that the surface viscosity , s , of dppc / chol monolayers decreases exponentially with increasing cholesterol fraction at a given surface pressure . in previous work , we found that the exponential dependence of surface viscosity on surface pressure was well - correlated by a free area the free - volume model was developed to explain the divergence in the viscosity of a liquid at the glass transition . the premise underlying the model is that in order to diffuse , a molecule in a liquid or other condensed phase has to have sufficient free volume , that is , volume not occupied by other molecules , in order to escape the cage formed by its neighboring molecules . each molecule has a minimum van der waals volume , v0 , and moves randomly with thermal velocity u , within confining cages of diameter d0 defined by its nearest neighbors . cohen and turnbull calculated the probability for fluctuations in free volume relative to the average free volume , v. if the local fluctuation in free volume exceeds v0 , a hole is created in the confining cage sufficiently large to allow a diffusional jump of the solute molecule into the hole . diffusion occurs if another molecule fills the hole left by the solute molecule before the original molecule returns to its starting position . the probability p(v0 ) that the free volume , vf , rearranges to give a void volume , v0 , large enough for a molecule to diffuse ( at constant energy ) is given by1thus giving a diffusivity2 in which g is a geometric factor . the parameter b in eqs 1 and 2 is to take into account overlaps of free volume , and cohen and turnbull suggest a range from 1/2 b 1 . in three dimensions , the diffusivity can be related to the bulk viscosity , , via a generalized stokes - einstein relationship , d = kt / f . for spherical particles , the friction factor , f , is given by the stokes drag on a sphere of diameter a : f = 3a . this leads to the free volume model for the viscosity:3 in applications of the model , the free volume is the difference between the measured volume per molecule , v , and v0 : vf = v v0 . in applications of the model , v0 is a fitting parameter ; theoretically , v0 is related to the volume per molecule at the glass transition where the viscosity diverges . for a 2-dimensional film of constant thickness , l:4 in analogy to the free volume , af( ) a0 , in which a( ) is the area per molecule determined at a surface pressure , , from a surface pressure - area isotherm and a0 is taken to be a fitting parameter . in 2-dimensions , the diffusivity and free area can be related to the surface viscosity , s , via the saffmann - delbrck model for a cylinder diffusing within a viscous monolayer , surrounded by a viscous subphase , to give an equation analogous to eq 3 in terms of the free area:5from fitting dppc viscosity data , we found that a0 40 , which is roughly equal to ab cos , the molecular area of a dppc molecule in a close - packed , untilted lattice ( table 2 ) . however , cohen and turnbull did not speculate on the molecular origins of af(19 ) as they were modeling an unstructured liquid . however , for semicrystalline monolayers , we postulate that the free area is proportional to the number of defects in the lattice , which is inversely proportional to the number of correlated molecules at that composition and surface pressure:6 lattice defects , such as dislocations , vacancies , and grain boundaries decorrelate the lattice , which creates free area and pathways for diffusion . we define as the free area ( or number of defects times the area per defect ) per number of correlated molecules ; we take to be constant , independent of concentration . combining eqs 5 and 6:7 in which = ba0/. linear regression to eq 7 ( figure 7c ) shows that for 20 , 30 , and 40 mn / m , the slopes , = 0.0134 0.0007 , 0.0131 0.002 and 0.0196 0.007 are the same within the experimental error . s0( = 20 mn / m ) = 0.09 0.02 , s0( = 30 mn / m ) = 0.37 0.2 , and s0( = 40 mn / m ) = 0.6 0.5 pms , although the physical significance of the surface pressure variation of s0 is not given by our model . the pearson correlation coefficient , r , for the three lines are 0.99 , 0.95 and 0.81 , respectively , giving a statistically significant fit of eq 7 to the data with p < 0.001 , 0.001 , and 0.05 , respectively . to better compare the data at different surface pressures , the surface viscosity and correlated area are normalized relative to a reference composition , xref , at the same :8ref(xref, ) is the surface viscosity and ( ( l02l11)/ab)ref is the number of molecules in the coherence area at xref ( taken to be pure dppc for = 20 and 30 mn / m and 0.4% chol for = 40 mn / m ) . linear regression to eq 8 gives = 0.0133 0.007 , which is the same as the fits to eq 7 . the pearson correlation coefficient is 0.91 , giving p < 0.001 , showing that eq 8 gives a statistically significant representation of the surface viscosity over the almost three orders of magnitude change in surface viscosity ( figure 7b ) . for a0 40 , 1500 ( for b = 1/2 ) 3000 ( for b = 1 ) . for pure dppc , ( l02l11)/ab 450 , giving af 36 from eq 6 , which is consistent with the variation in area per molecule , a(x, ) = a0 + af(x, ) , measured from dppc isotherms . adding cholesterol decreases ( l02l11)/ab to 100 , thereby increasing the free area per molecule to 1530 , resulting in the dramatic decrease in surface viscosity . these values of af are in agreement with the assumptions behind the cohen and turnbull free volume theory , which postulates that vf v0 , hence af a0 . in summary , gixd shows that increasing the chol fraction at constant surface pressure , or increasing the surface pressure at constant chol fraction , decreases the tilt of the dppc lattice , while leaving the alkane chains close - packed with an invariant area per molecule normal to the alkane chain direction . this confirms afm images that show cholesterol does not intercalate homogeneously into the dppc lattice but is expelled to disordered nanodomains that break up the dppc domains . the nanodomains evolve from isolated , circular 10100 nm diameter domains to 1050 nm wide linear nanodomain aggregates to an interconnected network structure with increasing cholesterol . however , the monolayer is homogeneous at micron - scale optical images ; macroscopic phase separation does not occur as at lower surface pressure ( figure 6 ) . hence , the dppc / cholesterol monolayer is better described as a nanostructured , single - phase monolayer , rather than a mixture of two distinct phases , similar to recently proposed two - dimensional microemulsions . as such , this system is analogous to 3-d nanostructured surfactant - oil - water bicontinuous microemulsions , or copolymer systems that separate into nanometer - scale regions more enriched in one or the other monomer , which are considered single phase . adding cholesterol does not change the local hexagonal dppc chain packing , but does reduce the molecular tilt , suggesting that cholesterol relieves some of the packing frustration between the dppc headgroup and tailgroups . most important to the monolayer dynamics , the extent of order , or the number of correlated molecules , decreases with increasing cholesterol fraction at all surface pressures , suggesting an increased number of lattice defects that create free area for visco - diffusive transport . at all cholesterol fractions , the positional order is much shorter ranged than the orientational order , consistent with an overall hexatic organization . we show that a simple model that proposes that the free area available for visco - diffusive transport is inversely proportional to the number of correlated molecules , which collapses the surface viscosity data for all surface pressures and cholesterol fractions onto a single universal curve . molecular defects and the associated decrease in domain size caused by the incompatibility of cholesterol packing into the alkane chain lattice enhance visco - diffusive transport in monolayers . our model and data show that the extent of molecular correlations is an excellent predictor of the effects of the cholesterol on the surface viscosity of model lung surfactant monolayers .

adding small fractions of cholesterol decreases the interfacial viscosity of dipalmitoylphosphatidylcholine ( dppc ) monolayers by an order of magnitude per wt % . grazing incidence x - ray diffraction shows that cholesterol at these small fractions does not mix ideally with dppc but rather induces nanophase separated structures of an ordered , primarily dppc phase bordered by a line - active , disordered , mixed dppc - cholesterol phase . we propose that the free area in the classic cohen and turnbull model of viscosity is inversely proportional to the number of molecules in the coherence area , or product of the two coherence lengths . cholesterol significantly reduces the coherence area of the crystals as well as the interfacial viscosity . using this free area collapses the surface viscosity data for all surface pressures and cholesterol fractions to a universal logarithmic relation . the extent of molecular coherence appears to be a fundamental factor in determining surface viscosity in ordered monolayers .

Introduction Materials and Methods Results and Discussion Conclusions

pollution is predicated to all tiny particles in the air which are produced due to human or natural activities ( 1 ) . since the centralization of human activities is associated with metabolic human interactions in urban areas , different pollutants enter into air easily and cause the urban environment more vulnerable ( 2 ) . it is such that , world health organization ( 1992 ) , has addressed the air pollution as a serious problem ( 3 ) . improper use of fuel and inappropriate topography of tehran caused air pollution due to the entrance of approximately 1.5 million tons of pollutants annually . hence , the combination of natural and artificial factorscauses tehran to be one of the most polluted cities inthe world , standing by mexico city , beijing , cairo , sao paulo , shanghai , jakarta , and bangkok ( 4 ) . air pollution has also caused painful events all around the world : the pollution event in meuse ( 1930 ) , in which 63 people experienced respiratory problems ; or the death of more than 400 people in london due to the contaminated thick smog in 1952 are two examples of such irrecoverable events ( 1 ) . accor - ding to an environmental program of united nations 48 % of premature deaths are due to exposure to particulate matter in both outdoor and indoor environments , with potentially 500000 excess deaths annually due to particulate matter in outdoor situations ( 5 ) . likewise , the air quality control agency s report indicates that more than 4500 people are dying every year in tehran due to air pollution ( 6 ) . the most important sources of air pollution can be divided into four key groups including overcrowding ( 7 ) , economic growth ( 8) , natural factors ( exp : geographical situation and topography , temperature inversion etc.)(9 ) , and mobile and stationary sources ( exp . non - standards and poor fuel consumption of motor vehicles , industries in and around the city , home heating and cooling systems ) ( 1 , 10 ) . depending on the type and sources of pollutants , metropolises around the world took different measures to control the air pollution . among developing countries , south africa has benefited from a collection of best legal solutions in order to reduce the air pollution ; among them are objective and standard setting , status quo assessment and priority area delineation , control strategy preparation and implementation ( 11 ) . china , as a developed country has also taken effective measures to control air pollution including integrated monitoring program on acidification of chinese terrestrial systems ( impacts ) , permanent control of vehicles emissions , increasing the quality of fuels , taking advantage of new technologies , developing transportation systems , providing five - year plans to understand causes and sources of pollution , current status , effects , and control of acid rain ( 1216 ) . in italy , special measures have been taken for energy management in industry , transportation systems and domestic systems ( 17 ) . economic and industry growth plus overcrowding in india , caused city planners to take serious measures including banning polluting vehicles , developing roads , escalating standards and regulations enforcement and etc . just some of them have been successful to decline air pollution , while the others failed to effectively and efficiently control air pollution . in south africa , despite numerous plans and policies for controlling air pollution , they failed due to some major challenges such as lack of relationship between district and local municipalities , lack of relationship between provincial and local authorities , plan s integration , public s roles , technical capabilities in programs , extending a partial focus on some polluter sources and lack of systemic approach in air pollution planning , plans and climate change management , shortage of funding and etc.(11 ) . improper prioritization of environmental intervention , lack of funding , unwillingness of the countries to plan with a systemic approach , lack of enforcement and poor communication between all public and private sectors are the most principal factors causing the failure to successfully control air pollution ( 1821 ) . from 1334 , lots of case and cross - sectional plans , programs and projects had been developed individually or as a part of a national document to control the air pollution of tehran . they are including the first five - year development plan ( 19881993 ) , clean air plan ( 1995 ) , transportation emission reduction project ( 1997 ) , comprehensive plan of tehran s combating air pollution ( 1997 ) , second five - year development plan ( 19962000 ) , third five - year development plan ( 20012005 ) , 20 year visionary plan ( 2005 ) , forth five - year development plan ( 20062010 ) , tehran s master plan ( 2007 ) , fifth five - year development plan ( 20112015 ) and master plan of metropolises combating air pollution ( 2011 ) ( 22 ) . despite the fact that all these plans had been precisely codified , the present evidence implies that expected results based on reducing the air pollution have not been met due to lack of an integrated stewardship responsible for regulating , coordinating and monitoring the process and the gained results of air pollution control plans ( 23 ) . hence it is necessary to analyze the air pollution controlling plans with a pathologic approach to identify a set of practical solutions for tehran . this paper bears a twofold purpose : first , pathologically analyze air pollution control plans of tehran from the viewpoint of experts ; and secondly offer appropriate and effective solutions for controlling the air pollution . a descriptive case study method was used in 2012 for pathologic analysis of air pollution control plans in order to offer solutions for tehran metropolis as the capital city of iran . the research team provided a semi - comprehensive review of the literature which contained more than 70 articles of different metropolises all around the world for developing an initial conceptual framework as a springboard for developing the interview questions . the scope of the review is limited to developed and developing metropolises ( china , italy , india and south africa ) which faced the same air pollution difficulties as in tehran . by developing the framework , a qualitative content analysis was drawn to identify the manifest and latent contents relating to different air pollutants and plans for their control . the key contents around which the interview questions were organized consists of 1 ) causes and sources of air pollution , 2 ) challenges & obstacles towards effective performance of air pollution control plans and 3 ) the most effective controlling solutions for air pollution in metropolitan areas . a semi - structured face - to - face interview was done to survey tehran s air pollution control plans , from the aspects mentioned above . the participants were chosen by snow - balling sampling and according to their context of work ( practicing professionals and tehran , tarbiyat modarres and shahid beheshti university professors ) . the interviews were continued until the saturation in data and no more information was gathered by interviewing and about 80% of data were repeated by experts , so about 14 experts , professors and managers interviews were practical and applied for data analysis . the context of the interview consists of the extent to which air pollution control plans of iran are appropriate and the ways to cope with the air pollutants in different sectors ( industry , transportation systems and domestic system ) with regard to other metropolises experience in the very same field . in addition , they were asked to provide more information about the relevant issues , if possible . as the interview sessions precede , the quality , relevancy and comprehensiveness of questions were developed simultaneously . all the opinions ( meaning units ) were reviewed , condensed and labeled as a code through a back and forth movement between the whole and part of the interview texts . then the codes with the same meaning grouped together under higher order heading to create categories ( 27 sub - categories and 8 categories ) in a way that each group of codes dealt with a specific issue or content area . next , the primitive title and content of all categories and sub - categories were discussed by the article research team . finally , the underlying meanings which were the latent contents of the categories were formulated into four main themes . the analysis results of all 14 viewpoints from practicing professionals were categorized into four main themes . the first theme is demonstrated in table 1 as causes and sources of air pollution of tehran metropolis . as it is depicted in the table , the most vital sources of air pollution corresponds to the process of policy making , lack of attention to environmental changes , topogherafic status of tehran , pollutant due to mobile and stationary sources and the matter of improper energy management . the second theme is demonstrated in table 2 as challenges & obstacles towards effective performance of air pollution control plans of tehran metropolis . causes and sources of air pollution of tehran metropolis challenges & obstacles towards effective performance of air pollution control plans of tehran metropolis as it is shown , the most important challenges of tehran s air pollution management are focused on nine areas which we abstracted them into two wider categories including firstly , air pollution stewardship challenges and secondly , environmental ( political , economic , social and technical ) challenges . some factors such as lack of information for evidence - base decision making , poor vertical and horizontal coordination among urban developmental goals and plans , improper funding , follo - wing unfit pattern and mores are categorized as stewardship challenges , on the other hand political intervention in plans provision and performance , sanctions against iran , unaffordability of some families to exchange their non - standard cars with new ones and some more are identified as environmental challenges , which are presented in the table 2 in detail . in order to demonstrate the challenges importance , the amount of their repetitions were accounted and shown in table 3 in percentage : the third theme is demonstrated in table 4 as the most effective controlling solutions for air pollution in the metropolitan areas . with regard to the fact that air pollution is one of national planning priorities in metropolitan areas , the most effective controlling solutions for air pollution in the world the forth theme is demonstrated in table 5 as the most effective controlling alternatives for air pollution in tehran metropolis which is broken into two main categories , first of all technical & infrastructural factors , then managerial & administrative factors . planning based on reliable evidences , involving all responsible key stakeholders in planning process , developing the parks & green sites , monitoring the amount & type of different pollutant in the air by the help of new technology , fair distribution of facilities and development , non - individual based planning , considering the implementation & monitoring capacities in contemporary with planning , improving the mutual understanding and communication among authorities and citizens , considering revision loops for plan , developing environmental standards and mores are categorized as technical and infrastructural factors . furthermore , improving the cooperative & mutual communications among the education deputy of tehran municipality and the other authorities forculturalization & informing , developing public participation capacities , are categorized as managerial & administrative factors . tables 5 , there are those alternatives which suites to tehran s status and were mentioned by the interviewees . in order to estimate the alternatives importance , the amount of their repetitions were accounted and shown in table 6 in percentage . the most effective controlling alternatives for air pollution in tehran metropolis the priorities of most effective controlling alternatives for air pollution in tehran metropolis our findings confirm that tehran s air pollution has been due to five major reasons including improper policy making , no attention to the pollutants changes , geographical situation , mobile and stationary sources and nonstandard energy production . paying inadequate and incoherent attention to the sources of pollution caused the authorities face with some important challenges including those which could be due to not having a unique and organized stewardship and of course those challenges could be due to the environmental changes which are not controllable by the air pollution management authorities . as all metropolises confront such challenges , their authorities implement different plans in order to manage air pollution . some of the best and effective controlling solutions are focused on culturalization , infrastructural development such as public transportation ( 16 ) and applying economical control levers in china ( 13 ) , utilizing participatory capacities and strategic planning in south africa ( 11 ) , applying solar battery to produce clean energy in italy ( 24 ) , identifying emission limits for automobiles and industry in india ( 3 ) . in iran , same as other metropolises , several plans are prepared and some are implemented , but , unfortunately , thanks to some challenges mentioned above , they failed to control tehran s air pollution as it was expected to be achieved in the plans . to solve the challenges and improve the quality and effectiveness of controlling air pollution plans , should name paying more attention to the matter of stewardship in the field of air pollution management , cultural and infrastructural development , focusing on effective management system doing systematic studies before planning in other words systematizing the plan preparation process , standardization of fuel and energy production and consumption , enhancing public participation capacity , developing appropriate coordination and controlling mechanism , realization of social justice through capacity evaluation in regional scope , standardizing and utilizing new technologies and professional experts , stopping manufacture of the low quality automobiles and reducing import tariff of the high quality ones , applying economical control levers , making transparency in deaths statistic due to air pollution , providing environment organization s box to centralize polluting industries penalties , and legitimizing air pollution control plans to enhance their implementation s guarantee ( 22 ) . our findings about the air pollution control plans challenges replicate the finding of a survey on controlling air pollution plans in south africa conducted by niaiker et al . ( 11 ) . in the viewpoint of niaiker poor standards and regulations to support plans implementation , poor cooperation among stakeholders ( key authorities , planners , policymakers , citizens etc . ) , poor resource generation and poor attention to the type and source of pollution are the most important challenges caused the failure of control plans . also , asadollah ( 23 ) mentions that poor and disorganized stewardship is a key factor which leads to the failure of tehran s air pollution control plan . furthermore , our finding about controlling alternatives for tehran s air pollution is similar to the findings of a survey on the same subject in this metropolis ( 19 ) . he believed that developing an appropriate evaluation and monitoring mechanism to enforce different responsible authorities through evaluate the performance of them , realization of social justice and improving the participation capacities are success factors of plan implementation . these finding can help tehran s authorities to look at plan preparation process with a new approach and would be practical for every developing large cities which are confronting such problems . in other words , authorities can positively benefit the viewpoints of the practicing professionals who consider the plan s deficiencies from unlike aspects in diverse levels of initials in advanced studies requirements , plan s preparation to plans implementation . we considered only the most important challenges of tehran s air pollution control plans in order to offer effective controlling solutions . further , more researches should be done to determine priority of the offered solutions in accordance with tehran s facilities and requirements . controlling air pollution of tehran needs a serious attention from policymakers to make an effectual enforcement through applying a systemic cycle of preparation and revising effective and comprehensive plans , implementing enforcement and evaluating the environmental impact of the plans through involving all stakeholders 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 .

abstractbackgroundthe centralization of human activities is associated with different pollutants which enter into environment easily and cause the urban environment more vulnerable . regarding the importance of air pollution issue for tehran metropolis , many plans and regulations have been developed . however , most of them failed to decline the pollution . the purpose of this study was to pathologically analyze air - pollution control plans to offer effective solutions for tehran metropolis.methodsa qualitative content analysis in addition to a semi - structured interview with 14 practicing professional were used to identify 1 ) key sources of tehran s air pollution , 2 ) recognize challenges towards effective performance of pertinent plans and 3 ) , offer effective solutions.resultsrelated challenges to air - pollution control plans can be divided into two major categories including lack of integrated and organized stewardship and pest challenges.conclusionfor controlling the air pollution of tehran effectively , various controlling alternatives were identified as systematization of plan preparation process , standardization and utilization of new technologies & experts , infrastructural development , realization of social justice , developing coordination mechanisms , improving citizens participatory capacity and focusing on effective management of fuel and energy.controlling air pollution in tehran needs a serious attention of policymakers to make enforcements through applying a systemic cycle of preparation comprehensive plans . further , implement the enforcements and evaluate the environmental impact of the plans through involving all stakeholders .

Introduction Materials & Methods Results Discussion Conclusion Ethical consideration

the syndrome is named after french neurosurgeon octave crouzon , who described this rare genetic disorder first time in 1912 . although it is encountered rarely , crouzon syndrome constitutes almost 5% of all craniosynostoses with an approximate birth prevalance of 1/25,000 - 1/50,000 . the syndrome is characterized by abnormal head shape , midfacial hypoplasia , maxillary hypoplasia , mandibular prognathism , ocular hypertelorism , proptosis , and airway obstruction due to premature fusion of multiple calvarial and skull base sutures within the first year of life . however , the clinical picture may vary greatly from mild to severe midfacial and orbital anomalies . the relationship between craniosynostosis and chiari malformation type i ( cm - i ) has been well - documented . cm - i has a tendency to accompany syndromic craniosynostosis more commonly than sporadic synostosis . the incidence of cm - i in crouzon syndrome is about 70% . herein , we present a 16-year - old boy who admitted with symptoms related to cm - i and underwent suboccipital decompression . however , on physical examination his cruzonoid features drawed attention . a 16-year - old , formerly healthy boy admitted to the outpatient clinic with occasional headache and neck pain . on physical examination it was remarked that he had characteristic features of syndromic craniosynostoses : hypertelorism , proptosis , midfacial hypoplasia , and abnormal head shape [ figures 1a and b ] . however , the patient and his family did not admit to a hospital for this reason before , despite prominent cruzonoid features . the patients neurological examination revealed no abnormalities including pinprick , touch , pain , and temperature sensations in both upper extremities . anteroposterior ( ap ) and lateral plain radiographs of head and a cranial computed tomography ( ct ) demonstrated midfacial and orbital hypoplasia , the fusion of bilateral coronal and lambdoid sutures and sagittal suture , alongside with the increase of ap diameter of the head [ figures 2a and b ] . cranial and spinal magnetic resonance imaging ( mri ) studies were obtained and it showed a 18 mm cerebellar tonsil herniation into the foramen magnum with accompanying syringomyelia between th 4 and th 7 [ figure 3 ] . surgery was planned in order to decompress the posterior cranial fossa . using a median incision , the dura was opened in y - form and after arachnoid dissection duraplasty was performed . there were no significant events in the postoperative course and the patient was discharged from the hospital with no neurological deficits . the patient was then referred to the genetics department for the further evaluation of the craniosynostosis . phenotypical features like the typical dismorphic facies , ocular proptozis and hypertelorism , parrot - like nose , and frontal bossing alongside with the patients mothers history of four recurrent intrauterine fetal losses supported the diagnosis of crouzon syndrome in our patient . regular follow - up examinations were scheduled for the observation of orbital deformities and vision . control mri showed complete resolution of the tonsillar herniation and significant reducement of the syringomyelia both in length and thickness [ figures 4a and b ] . four years after the operation the patient is still doing well with no neurological or ocular deficits and with the relief of his symptoms at the admission . ( a and b ) photographs show characteristic features of syndromic craniosynostoses in our patient : hypertelorism , proptosis , midfacial hypoplasia , and abnormal head shape . ( published with permission and informed consent of the patient ) ( a ) anteroposterior ( ap ) and lateral plain radiographs of head shows increase of ap diameter of the head and midfacial - orbital hypoplasia . fusion of multiple calvarial sutures is also remerkable , ( b ) axial cranial computed tomography scan demonstrated the abnormal head shape and the fusion of bilateral coronal and lambdoid sutures alongside with sagittal suture sagittal t2-weighted magnetic resonance imaging shows a 18 mm cerebellar tonsil herniation into the foramen magnum with accompanying syringomyelia between th 4 and th 7 with the largest thickness of 11 mm ( a ) sagittal t2-weighted mri of the craniocervical junction showed complete resolution of 18 mm cerebellar tonsil herniation , ( b ) sagittal t2-weighted thoracal mri showed relative resolution of the accompanying syringomyelia between th 4 and th 7 with the largest thickness of 7 mm the premature fusion of cerebral sutures has been postulated as the mechanism leading to the development of cm - i in patients with syndromic craniosynostosis . especially premature fusion of the lambdoid suture has been accepted as a crucial developmental anomaly , which results in a relatively small posterior fossa . cinalli et al . , have reported that cm - i is present in 70% of patients with crouzon syndrome . on contrary , they have proposed , that this relationship is due to earlier closure of sagittal and lambdoid sutures in crouzon syndrome compared to apert syndrome . hydrocephalus , jugular venous stenosis leads to venous hypertension and associated brain malformations have been postulated as the other mechanisms leading to cm - i development . mutations in three of four fibroblast growth factor receptor ( fgfr ) genes have been demonstrated to be responsible for various types of syndromic craniosynostoses including crouzon syndrome . fujisawa et al . , have demonstrated that a missense mutation in fgfr2 gene ( tyr281cys ) is responsible for the development of cm - i in patients with crouzon syndrome . , the patient or his family was never attempted to seek for professional medical help . the syndrome was only diagnosed when the patient was admitted to our department with symptoms of cm - i . strahle et al . , presented a series of patients with cm - i associated with craniosynostosis . in their series of 29 patients , however , the mean age of the whole patient group was 1.8 years ( range 2 months-9 years ) . this data shows that both craniosynostosis or craniosynostosis related cm - i are expected to be diagnosed in early childhood . cm - i could be diagnosed before the craniosynostosis in some instances , but the age of our patient at diagnosis was 16 . to our knowledge , this is the only case report indicating a late adolescent diagnosis of crouzon syndrome through clinical symptoms of an associated cm - i . surgical approach to craniosynostosis related cm - i may include cranial vault remodeling with an adequate posterior fossa decompression . however , most neurosurgeons agree with the conservative follow - up of patients with cm - i unless it is not symptomatic or associated with spinal syringomyelia . strahle et al . , reported some of his patients cm - i was resolved or regressed with cranial vault remodeling only , without posterior fossa decompression . , we do not consider cranial vault remodeling as an option , instead we applied a classical posterior fossa decompression for cm - i . strahle et al . , underlined the risk of venous bleeding due to abnormal venous sinuses and increased venous hypertension , therefore suggested a posterior fossa decompression without dural opening and c1 arcusectomy . we did not encounter any venous bleeding during the surgery , where we opened dura and performed a duraplasty . pediatric patients with cm - i should be carefully examined for the clinical signs and features of crouzon syndrome or other syndromic craniosynostosis . in mild clinical forms or in case of a late diagnosis , posterior fossa decompression without cranial remodeling should be kept in mind as a treatment option .

chiari malformation type i ( cm - i ) related to syndromic craniosynostosis in pediatric patients has been well - studied . the surgical management consists of cranial vault remodeling with or without posterior fossa decompression . there were also cases , in whom cm - i was diagnosed prior to the craniosynostosis in early childhood . we present a 16-year - old boy who admitted with symptoms related to cm - i . with careful examination and further genetic investigations , a diagnosis of crouzon syndrome was made , of which the patient and his family was unaware before . the patient underwent surgery for posterior fossa decompression and followed - up for crouzon 's syndrome . to our knowledge , this is the only case report indicating a late adolescent diagnosis of crouzon syndrome through clinical symptoms of an associated cm - i .

Introduction Case Report Discussion Conclusion

permanent postoperative hypoparathyroidism results from the unintentional removal or injury of parathyroid glands during thyroid and parathyroid surgery . permanent hypoparathyroidism is defined as persistent hypocalcemia requiring calcium and vitamin d supplementation 6 months after surgery . the risk is nominal during a minimally invasive parathyroidectomy for a single adenoma but is greatest after a subtotal or total parathyroidectomy , thyroid resection and nodal dissection for large and extensive thyroid cancers , and reoperative neck operations . even though the risk of transient hypocalcemia can be high during an extensive neck dissection , the permanent hypoparathyroidism rate is typically around 1% in the hands of experienced endocrine surgeons at high - volume centers . the accidental onset of permanent hypoparathyroidism can be agonizing for the patient and the clinician alike . for the patient , its negative impact includes a reduced quality of life , expensive lifelong medication supplementation , frequent laboratory testing , and the potential for frequent hospital admissions . in addition , the persistent absence of parathyroid hormone ( pth ) has long - term systemic effects on the body , such as the development of osteoporosis ( due to the decreased function of osteocytes ) , premature cataracts , cardiac dysfunction , and neurologic dysfunction [ 1 , 2 ] . in the late 1960s and 1970s , several new techniques were introduced in an attempt to prevent the detrimental health and social consequences of permanent hypoparathyroidism . for example , intraoperative autotransplantation of parathyroid tissue into the sternocleidomastoid muscle or the brachioradialis muscle was recommended . however , not all patients at risk of permanent hypoparathyroidism actually develop it , nor do all patients require an immediate autotransplant . in fact , an unnecessary autotransplant , if performed during parathyroid surgery , could result in persistent hyperparathyroidism . additionally , the autograft could become autonomously hyperfunctional , posing a diagnostic and treatment dilemma in the future ; wells et al . overcame that limitation and transformed our approach to prevent hypoparathyroidism by introducing autotransplantation of cryopreserved parathyroid tissue . cryopreservation permits parathyroid tissue to be stored for potential reimplantation , thereby avoiding the risk of needlessly implanting parathyroid tissue during initial surgery . the clinician is able to accurately determine whether any residual parathyroid tissue will recover function or whether a delayed autotransplant will be needed . the disadvantages of cryopreservation of parathyroid tissue include the potential of graft failure and the risk of graft - dependent hypercalcemia . this paper provides an up - to - date , comprehensive review of the cryopreservation of parathyroid tissue and its current role in thyroid and parathyroid surgery . the clear indication for an autotransplant of cryopreserved parathyroid tissue is permanent postoperative hypoparathyroidism . in the hands of experienced endocrine surgeons , the risk is particularly low in patients with sporadic primary hyperparathyroidism ( phpt ) , most of whom have a single parathyroid adenoma . nonetheless , certain patients have a higher risk of developing permanent hypoparathyroidism after their initial neck surgery ( table 1 ) . most commonly at risk are patients with multigland parathyroid hyperplasia , especially those with familial primary hyperparathyroidism . such patients may undergo either a subtotal ( 3.5-gland ) parathyroidectomy or a total parathyroidectomy with an immediate autotransplant . both a subtotal parathyroidectomy and a total parathyroidectomy can result in aparathyroidism , so cryopreservation of parathyroid tissue is recommended at the time of the initial surgery . in addition , it has been reported that the use of intraoperative parathyroid hormone ( iopth ) monitoring during parathyroid surgery can accurately predict patients at risk of developing postoperative hypocalcemia . a drop of > 80% of iopth at 10 minutes is a significant factor for postoperative hypocalcemia . therefore , cryopreservation of parathyroid tissue should be considered during parathyroid surgery when the iopth drop > 80% . patients with end - stage renal disease are at high risk of developing secondary ( shpt ) and tertiary ( thpt ) hyperparathyroidism . such patients have persistent stimulation of the parathyroid glands secondary to abnormalities in the metabolism of calcium , phosphorus , and vitamin d , resulting in multigland parathyroid hyperplasia . they have an inherently high risk of disease recurrence if a subtotal parathyroidectomy is performed but a high risk of permanent hypoparathyroidism if a total parathyroidectomy and an immediate autotransplant are performed . moreover , such patients may require multiple operations ; to prevent aparathyroidism as a consequence of the initial or subsequent operations , cryopreservation of parathyroid tissue is recommended . the risk of aparathyroidism is nominal with only a thyroidectomy but increases with more extensive surgical resections . routine central neck dissections for thyroid cancer have a complication rate of permanent hypoparathyroidism of up to 14% . given such risks , the role of prophylactic central neck dissections for papillary thyroid cancer continues to be debated . an immediate intraoperative autotransplant is preferred during a neck dissection for thyroid cancer , yet cryopreservation of parathyroid tissue fragments is also warranted given the multiple operations that may be required in the future . subsequent operations increase the risk of permanent aparathyroidism , an outcome that cryopreservation of parathyroid tissue may prevent . in contrast to initial neck operations , the risk of permanent hypocalcemia after a redo neck operation is not minuscule , but rather as high as 30% [ 6 , 10 ] . the risk of aparathyroidism is higher during a redo neck operation because the viability of the remaining parathyroid glands can not be adequately determined . during the initial surgery , parathyroid glands left in situ may have unknowingly been devascularized or damaged . during a redo neck operation , removal or inadvertent injury of any remaining functional gland this scenario can be particularly problematic in patients who develop hyperparathyroidism after previously undergoing a thyroidectomy ; during a redo neck operation , any parathyroid gland removed must be assumed to be the last viable parathyroid gland and should be autotransplanted immediately to optimize transplantation success . a fragment of the parathyroid tissue may be cryopreserved for a possible delayed autotransplantation if future neck reoperations result in aparathyroidism . other common reasons for parathyroid reoperations include persistent hyperparathyroidism ( hypercalcemia < 6 months after the initial surgery ) and recurrent hyperparathyroidism ( hypercalcemia > 6 months after the initial surgery ) . operative success for redo parathyroid surgery is < 90% and is fraught with an 18% rate of causing permanent hypoparathyroidism . immediate autotransplantation of parathyroid tissue to prevent hypoparathyroidism impedes determination of surgical outcome , which is unpredictable during redo parathyroid surgery . accurately assessing the number of parathyroid glands previously resected , or the viability of the remaining parathyroid glands , is difficult . cryopreservation of parathyroid tissue , rather than an immediate autotransplant , allows the surgeon to appropriately predict surgical outcome , the functionality of any remaining parathyroid tissue , and the need for a delayed parathyroid autotransplant . operative failure and disease recurrence are infrequent in patients with sporadic phpt ; reoperations are more commonly needed in those with shpt and thpt . up to 15% of patients with shpt and thpt tend to have more than four parathyroid glands , increasing the risk of operative failure . cryopreservation of their parathyroid tissue is especially important , because the long - term renal effect of prolonged shpt and thpt predisposes patients to transient hypocalcemia from bone hunger . before any parathyroid autotransplant , it is critical to differentiate transient from permanent hypoparathyroidism . similarly , patients with thyroid cancer who require repeating operations are at risk of hypoparathyroidism , because their remaining parathyroid gland function can not properly be assessed . furthermore , if parathyroid glands are immediately autotransplanted into the neck muscles during the initial thyroid surgery , the function of those glands can not be properly evaluated . during reoperations for thyroid cancer , a portion of any inadvertently removed or devascularized parathyroid glands should be sent for cryopreservation before any immediate autotransplant . patients who develop aparathyroidism during subsequent operations might later benefit from a delayed autotransplant of the cryopreserved parathyroid remnant . is dissected into 30 to 40 pieces of 1 1 1 mm . the supernatant is decanted ; about 10 tissue fragments are transferred into each sterile freezing vial to be resuspended in the freezing media . several media have been proposed in the literature since the initial report by wells et al . . the typical freezing medium contains roswell park memorial institute ( rpmi ) 1640 solution [ 2 , 4 , 6 , 10 , 1316 ] . most institutions use an 80% rpmi 1640 solution [ 2 , 4 , 10 , 13 ] , but some use either a 60% rpmi solution 1640 or no rpmi 1640 solution . in addition , some authors recommend supplementing the rpmi 1640 solution with 2 mm of glutamine [ 11 , 16 ] and 5 g / ml of penicillin streptomycin or 50 g / ml of gentamicin . dimethyl sulfoxide ( dmso ) in a 10% concentration is added as a cytoplasmic stabilizer [ 2 , 4 , 10 , 11 , 15 ] . other reported concentrations of dmso range from 7.5% to 20% [ 16 , 18 ] . the last component of the storage medium is either a 10% to 30% autologous serum [ 2 , 10 , 11 , 15 , 16 ] or a 10% to 20% fetal bovine serum [ 4 , 11 ] . when the rpmi 1640 solution is excluded , a 90% fetal bovine serum is used . the goal of the freezing process is to preserve cellular function by maintaining cellular integrity through the temperature change . to accomplish that goal , a cooling technique developed at the mayo clinic entails placing the vials in an ice chest filled with dry ice prechilled to 55c to 60c for 1 hour to allow cooling by 1c per minute . other authors recommend placing the vials in a 60c ethyl alcohol bath or in either a 70c or a 80c freezer overnight . the vials can also be placed in a programmable freezer and cooled by 1c per minute until a temperature of 80c is reached . once the vials are cooled , they are transferred into a liquid nitrogen freezer and stored at any of several recommended temperatures : 170c [ 15 , 16 ] , 180c [ 4 , 17 ] , 190c , or 196c . the vials containing the parathyroid tissue designated for delayed autotransplantation are removed from the liquid nitrogen freezer and placed in a warm water bath . the vials are shaken at 37c [ 11 , 17 ] to 42c until the parathyroid tissue fragments are thawed . the fragments are washed in rpmi 1640 solution at 37c three times [ 10 , 11 ] . other authors recommend rinsing the fragments five times in 1 ml of rpmi 1640 solution with a 20% autologous serum . the rpmi wash is performed to rinse the dmso , which is toxic to cells at room temperature . the patient 's nondominant forearm is chosen for the parathyroid tissue reimplantation under local anesthesia . dissection is continued until a flexor muscle , preferably the brachioradialis muscle , is exposed . one to three parathyroid graft fragments are transplanted into separate muscle pockets . to maximize the chances of graft function , care must be taken to not cause an intramuscular hematoma , which can compromise graft function . functionality is determined not only clinically but also biochemically ( by sampling blood from the grafted and nongrafted antecubital veins , to determine the pth level at both sites ) . the parathyroid graft is reported as fully functional , partially functional , or nonfunctional ( table 2 ) . clinically , the graft is considered fully functional when the patient remains eucalcemic and asymptomatic after all the calcium and vitamin d supplementation has been discontinued . a graft is also considered fully functional when the pth ratio between the grafted and nongrafted arms is greater than 1.5again , after all the calcium and vitamin d supplementation has been discontinued . the graft is considered partially functional when the patient continues to require calcium supplementation , with or without vitamin d supplementation , with a pth ratio of greater than 1.5 . the graft is considered nonfunctional when the patient is hypocalcemic and requires calcium supplementation , with or without vitamin d supplementation , with a pth ratio of less than 1.5 . in renal patients , the functionality of the parathyroid graft is determined by their pth levels , independent of calcium and vitamin d supplementation ( since most renal patients require supplementation ) ( table 2 ) . the potential benefits of cryopreservation are limited by the reduced functionality of cryopreserved grafts , as compared with immediately autotransplanted grafts . cryopreserved grafts retain functionality in < 70% of patients ; fresh autografts > 90% [ 2 , 15 ] . the cryopreservation process may induce cellular necrosis and impair cellular viability and , ultimately , cellular function . earlier studies found no difference in cell viability and secretory capacity between fresh and cryopreserved parathyroid tissue grafts [ 6 , 19 ] . yet other studies demonstrated that , even though the percentage of viable cells did not necessarily differ between fresh and cryopreserved tissue , cryopreservation decreased the total number of live cells by > 70% . that effect on live cell yield was the same whether the parathyroid tissue was frozen as tissue fragments or as dispersed cells . recently , other authors reported that the cryopreservation process decreased total cell viability and that decreased cell viability was associated with increased storage time . both viability and function were drastically reduced with a storage time of 22 months [ 15 , 17 ] . to counterbalance the effects of cellular necrosis , some authors routinely perform histologic examination of the cryopreserved tissue and autotransplant parathyroid tissue according to the percentage of necrotic cells . in addition , cryopreserved parathyroid tissue should be utilized as soon as it is deemed necessary , since a longer storage time limits delayed autotransplant success . the devastating effect of permanent aparathyroidism has been , for the most part , ameliorated by parathyroid gland cryopreservation and delayed autotransplants . an immediate autotransplant is preferred during thyroid surgery , yet cryopreservation of a portion of the parathyroid tissue can also greatly help patients at high risk of undergoing further surgery . cryopreservation allows the clinician to make appropriate decisions regarding the status of the remaining parathyroid glands . such enhanced decision making is important because most patients undergoing parathyroid and thyroid surgery experience only transient hypocalcemia . differentiating between transient and permanent hypocalcemia is critical , especially after a parathyroidectomy when hypocalcemia may result from bone hunger , rather than insufficiency of pth . cryopreservation facilitates appropriate surgical and clinical decisions , prevents unnecessary immediate parathyroid autotransplants , and offers a chance to cure aparathyroidism .

the risk of permanent hypoparathyroidism following thyroid and parathyroid surgery is around 1% in the hands of experienced endocrine surgeons . although this complication is rare , rendering a patient permanently aparathyroid has significant consequences on the health and quality of life of the patient . immediate autotransplantation of parathyroid glands that are injured or unintentionally removed offers the best possibility of graft viability and functionality . however , since the majority of cases of hypoparathyroidism are transient , immediate autotransplantation can complicate postoperative surveillance in certain patients , especially those with primary hyperparathyroidism . cryopreservation of parathyroid tissue is an alternate technique that was developed to treat patients with permanent hypoparathyroidism . this method allows for parathyroid tissue to be stored and then autotransplanted in a delayed fashion once permanent hypoparathyroidism is confirmed . this article provides a contemporary review on cryopreservation of parathyroid tissue and its current role in thyroid and parathyroid surgery .

1. Introduction 2. Indications for Cryopreservation 3. Cryopreservation Techniques 4. Autotransplant Techniques 5. Functionality Testing Posttransplant 6. Effects of Cryopreservation 7. Conclusion

a major feature of the movement toward hospital cost containment in the last decade has been the replacement of expensive inpatient care with less costly outpatient care . most discussions about the increasing reliance on outpatient care focus on utilization and revenue measures . a more important issue for hospital payment policy , however , is how actual costs have been affected by this shift from inpatient to outpatient services . trends in relative cost changes are not easily identified because hospital accounting systems do not easily disaggregate total cost into inpatient and outpatient components . cost - finding methodologies are complex , allowing hospitals considerable discretion in cost - allocation patterns . furthermore , an incentive was created by the prospective payment system ( pps ) to allocate costs to centers incurring outpatient charges because medicare pays a fixed amount per inpatient discharge but continues to pay for outpatients on the basis of reasonable cost . changes in relative costs have important implications in the current environment of changing methods of payment for hospital outpatient services . concern over increases in medicare outpatient expenditures led to congressional legislation in 1986 that mandated the implementation of a pps for outpatient care . an understanding of true cost increases is critical to the adoption of a plan that accurately reflects outpatient costs . in this article , we provide a methodology for disaggregating total cost into inpatient and outpatient components to examine relative changes . this is accomplished through estimation of a multiple - output total - cost function for a sample of acute care hospitals for the years 1984 - 88 . the adoption of pps by medicare in 1983 changed the payment basis for hospital inpatient care from hospital - specific costs to diagnosis - related groups ( drgs ) . under pps , hospital payments are made according to prices that are determined by averaging historic costs for specific groups of diagnoses across hospitals . the force behind this change was the federal government , the largest third - party payer for inpatient care . an intended consequence of pps was the substitution of less expensive outpatient care for inpatient care , without compromise of quality . the change in the medicare system was not the only impetus behind the increased emphasis on outpatient care . hospitals have also been influenced by other third - party payers , who have also been trying to contain costs . managed care options have been expanded , as has the use of copayments and deductibles . many plans ( including the majority of blue cross and blue shield plans ) have adopted fee schedules similar to those of pps . new techniques in cataract extraction and cardiac catheterization , for example , have made it possible for people to undergo these procedures without an overnight stay . the magnitude of the increase in outpatient utilization over the period of this study is dramatic . the annual number of outpatient visits reported by community hospitals rose from approximately 210 million to 270 million from 1984 to 1988 . the surge occurred in the number of hospitals offering services as well as the types of services being performed . in 1984 , 49 percent of community hospitals reported having an organized outpatient department . the proportion of hospitals reporting the performance of ambulatory surgery increased from 91 to 95 percent during the same period . the substantial shift from inpatient to outpatient activity is also reflected in changes in inpatient and outpatient revenue components . whereas total revenues in community hospitals increased by 44 percent from 1984 to 1988 ( from $ 156 billion to $ 224 billion ) , outpatient revenues more than doubled during the same period ( from $ 22 to $ 46 billion ) . program payments for hospital outpatient services increased by $ 2.2 billion from 1984 through 1987 , or at an average annual growth rate of 18 percent . mounting medicare payments for outpatient care led to a call for the development of a prospective payment method for such care . in 1986 , congress passed the omnibus budget reconciliation act , which directed the secretary of health and human services to develop a prospective payment plan for all types of hospital outpatient care . implementation of this law requires a system for outpatient classification , and one grouping method currently under consideration and discussed later is ambulatory patient groups ( apgs ) ( lion et al . , this scheme is similar to that of drgs , which rely on charge data for calibration of payment weights . if there is a discrepancy between the true cost of outpatient visits and the charges made for those visits , distortion could occur in the establishment of their payment rates . in this section , we describe a procedure for disaggregating total costs into inpatient and outpatient components , which includes an estimable multiple - output cost function . to identify outpatient costs , we employ the concept of incremental costs as described in baumol , panzar , and willig ( 1988 ) . outpatient costs are the incremental costs incurred as a result of outpatient activity : where oc represents outpatient costs , tc represents total costs , dis and opv represent the number of discharges and outpatient visits , respectively , and x is a vector of exogenous variables . therefore , assuming total costs are the sum of outpatient costs and inpatient costs , inpatient costs are specifically , the cost function is evaluated at the actual level of outpatient visits and at zero outpatient visits . given this breakdown of total costs , hospital - specific cost components can be obtained . the discussion then turns to a multiple - output cost function that can be used to determine the disaggregated costs described in equations ( 1 ) and ( 2 ) . one type estimates average cost per patient or per patient day as a function of various regressors that are considered to affect costs . this widely used set of behavioral cost functions is often accused of being ad hoc and of lacking foundation in the assumptions of the usual production theory . another group of models , following the work of mcfadden ( 1978 ) , employs flexible functional forms that regress total cost on output levels and input prices and , hence , are more consistent with the characteristics of the standard economic theory of production . the advantage of these models is that they are better suited for the calculation of the scale and scope economy measures that have been developed for multiple - output production . however , these models have been criticized for the large numbers of parameters that must be estimated and for excluding many factors that are known to be significant in explaining variation in costs of complex , modern hospitals . some recent work estimates hybrid cost functions that incorporate a number of desirable features from both existing types of models ( grannemann , brown , and pauly , 1986 ; vita , 1990 ) . hadley and zuckerman ( 1990 ) expand the literature with a dynamic model designed to capture the process of adjustment to pps . however , a consensus has not been reached on the appropriate form of the hospital cost function . because our major objective is to disaggregate total costs into inpatient and outpatient components , the function to be estimated is a total - cost function . as the focus is not scale or scope economies nor substitution of inputs , we chose a form that , although not derived from any particular production technology , incorporates many factors likely to be important in explaining hospital cost variation . our approach draws from the work of grannemann , brown , and pauly ( 1986 ) . having no evidence that hospitals are in longrun equilibrium , our expression is that of shortrun total costs : geographic input price variation is a major determinant of cost variation . the only input price measure available was the index of local area wage rates that has been produced by the health care financing administration ( hcfa ) for use in determining prospective payments to hospitals . to impose the assumption of linear homogeneity in input prices , the dependent variable used in the equation is the logarithm of total cost minus the logarithm of the wage index . variation in the cost to the hospital of energy and food may be partially reflected in wage rates , which must compensate workers for higher costs of living . the second- and third - order terms for the variables for the number of inpatient discharges and outpatient visits are consistent with a cost function that exhibits u - shaped average and marginal cost curves . there are two aspects of inpatient care : the number of patients and the patient length of stay . these may be entered separately , as discharge and average - length - of - stay variables , or combined into one total - days - of - care variable . we chose the former approach , although the latter would likely yield a similar result . use of our functional form allows for outpatient levels to take the value of zero , which is the case for outpatient visits for some hospitals . calculation of the incremental cost of outpatient activity also requires that the cost function be evaluated at a level of zero outpatient visits for each hospital . the vector of remaining variables , which are described in a later section , was chosen based on the results of previous studies . we make the assumption that output is exogenous as has commonly been done in other studies ( conrad and strauss , 1983 ; grannemann , brown , and pauly , 1986 ; friedman and shortell , 1988 ; hadley and swartz , 1989 ; hadley and zuckerman , 1990 ) . the majority of data used in this analysis comes from two independent sources : the american hospital association ( 1984 - 88 ) ( aha ) annual survey of hospitals and the hcfa hospital cost reporting information system ( hcris ) data files . the hcris files are cycles one through five ( 1984 - 88 ) of pps supplemented by the tax equity and fiscal responsibility act ( tefra ) data set , which was used to complete the pps data for 1984 . the sample represents all hospitals for which both aha and pps data were available , after eliminating specialty hospitals , all - inclusive - rate payers , and hospitals with fewer than 100 beds . the data bases of those 68 hospitals subject to all - payer systems of payment were not comparable with those of the larger group ; the group of small hospitals exhibits cost structures that are distinctly different from those of hospitals having 100 or more beds . this latter point was verified using the chow test for structural difference between the sample that included and the sample that excluded small hospitals ( n = 3,961 and n = 2,235 , respectively ) . this unique data set consists of 2,235 hospitals , both non - profit and proprietary . total fixed assets , drawn from the hcris data , are used as a measure of fixed capital in estimating the shortrun cost function . in doing so , it is assumed that capital stock is exogenous . to test this assumption , i.e. , to supply evidence of whether or not hospitals are in longrun equilibrium , we performed the hausman ( 1978 ) specification test for exogeneity of the capital variable for the year 1988 . if capital is exogenous , it will be uncorrelated with the error term in the cost function . the null hypothesis of no misspecification ( no correlation with the error term ) is tested by comparing two sets of parameter estimates of the cost function : one using total fixed assets and one using an instrumental variable that is correlated with fixed assets but uncorrelated with the error term . the specification test is based on the statistic where 1 and m1 are the parameter estimates and the covariance matrix from the estimation using the instrumental variable , and 2 and m2 are similar estimates from the model using total fixed assets . the m - statistic has a (k ) distribution , where k is the number of unknown parameters . because the value of m is 2.29 , and the critical value at the 1-percent level is 40.29 , we fail to reject the null hypothesis and therefore incorporate the actual value of total fixed assets into the cost function . the cost and discharge variables just described were obtained from the hcris data set ; outpatient visits were obtained from the aha data . in addition to the cost , output , and capital variables , additional explanatory variables appear in the cost function . case mix ( measured using the medicare drg case - mix index ) is included to control for output variation among inpatients that is not captured by the discharge and length - of - stay variables . this estimate of the costliness of a particular hospital 's medicare patient load was unavailable prior to the adoption of pps by medicare . although still imperfect , it improves on many earlier cost studies that relied on cruder case - mix measures . the implications of market concentration for hospital costs have been addressed in a number of recent works . many of these studies have found evidence of various forms of non - price competition ( for example , by quality or range of service offerings ) , with the general conclusion that greater market competition is associated with higher costs ( joskow , 1980 ; robinson and luft , 1985 ; white , 1987 ; hadley and swartz , 1989 ) . zwanziger and melnick ( 1988 ) demonstrate that this effect is changing in california hospitals . this was constructed using the county as the market and the number of discharges as a measure of output from which to determine market shares . ( 1987 ) found that the county is an acceptable alternative to a uniform geographic area in defining markets . scope or range of services has not been measured precisely in most cost functions ; often the interaction terms for various outputs are examined in an attempt to establish the presence of economies or diseconomies of scope . we incorporate a scope - of - services index that is calculated by cluster analysis and verified using guttman scale statistics and that follows the methodology developed by klastorin and watts ( 1982 ) ( also henderson , defiore , and stefos , 1990 ) . we have grouped the hospitals ( which are ranked on a scale of 0 to 18 , where hospitals having a higher index offer more services ) into three categories of service availability ranging from lowest to highest : basic , community , and those offering the full range of services . our service - index approach to economies of scope differs from that of grannemann , brown , and pauly ( 1986 ) , who include interaction terms between output pairs . finally , dummy variables are included for teaching status , population size , and ownership . the level of teaching activity is classified into three groups : major teaching ( affiliation with a medical school and membership in the council of teaching hospitals ) , minor teaching ( medical school affiliation only ) , and non - teaching ( neither affiliation nor council of teaching hospitals membership ) . population of a hospital 's surrounding community was coded by collapsing the metropolitan statistical area ( msa ) size into one of three groups : large urban ( more than 1 million ) , small urban ( 100,000 to 1 million ) , and rural . ownership is categorized as non - profit , profit , and government ( city , county , or state facility ) . the teaching and population variables were obtained from the aha data set ; the ownership dummy was defined from hcris data . this allows for comparison of relative costs over time . as in most of the literature on cost functions , a potential hazard for estimation of total costs on cross - sectional data is the presence of heteroscedasticity associated with output levels . a useful procedure for detecting this violation of ols assumptions in the case of a multiple - output cost function is the park - glejser test ( see vitaliano , 1987 , for another application of this test to a hospital cost function . ) glejser ( 1969 ) generalizes the test to allow for the case of heteroscedasticity , in which the error term is proportional to more than one of the explanatory variables . in addition to testing for the failure of the assumption of a constant error term variance , the test supplies an estimate of the covariance matrix of the disturbance term , u : . the inverse of the diagonal matrix contains the weights to be used in a weighted least squares ( wls ) regression . the park - glejser test indicated the presence of heteroscedasticity for the years 1984 - 86 and 1988 . consequently , wls regressions were performed for those years and ols was applied to 1987 . the belsley , kuh , and welsch ( 1980 ) diagnostics were applied ; no problems due to multicollinearity were detected . the coefficients on the discharge , outpatient visit , and los variables exhibit a highly significant pattern of positive , negative , and positive for quantity , quantity squared , and quantity cubed , respectively . the sign on the herfindahl index is negative , which is supportive of the theory of non - price competition . small rural markets tend to have lower input prices , and because the only included input price is the wage index , it is possible that the herfindahl measure could be incorporating the effect of omitted input price measures . major teaching hospitals and those located in large urban areas are more expensive , as has been shown in previous empirical work . previous authors ( grannemann , brown , and pauly , 1986 ; vita , 1990 ) failed to find evidence of complementarities among their specific measured outputs . the signs of the coefficients on our scope - of - service dummy variables also fail to indicate the presence of economies of scope . however , these results are only suggestive , and the topic is one in need of further investigation . the results of the estimated cost functions were used to disaggregate total costs into inpatient and outpatient components . equations 1 and 2 were evaluated for each hospital using the estimated cost function to determine hospital - specific values . in particular , for hospital i , the means of inpatient and outpatient costs are listed for each year by various hospital categories in table 3 . as seen in the final column , inpatient costs grew at an average annual rate of 6.6 percent and outpatient costs at a rate of 7.4 percent . this result contrasts starkly with the relative change in inpatient and outpatient revenues already discussed . revenues for outpatient services rose much more rapidly than costs in the 5 years following the introduction of pps . the results of the cost - function disaggregation procedure indicate that the growth rate of outpatient costs did not differ substantially from that of inpatient costs , despite considerable differences in the relative utilization patterns and revenue components . to further explore this finding , it is useful to consider the relationship between total cost , output levels , and unit costs . the ratio between the two components of cost may be represented as that is , the ratio of costs is equal to the product of the ratio of output and the ratio of unit costs . because the ratio of outpatient to inpatient costs remained relatively steady , and the ratio of outpatient visits to discharges rose , it follows that the ratio of the average incremental cost of an outpatient visit to the average cost of a discharge fell . table 4 lists the outpatient visit and discharge unit costs by the same hospital strata . in 1984 , the overall average incremental cost of an outpatient visit was 2.3 percent of the average inpatient cost ; by 1988 , this percentage had fallen to 1.7 . the decline in the outpatient / inpatient unit cost ratio offset the increase in the ratio of outpatient visits to inpatient discharges . because the trend in unit costs for outpatient services is a finding of this analysis that was unanticipated , an interpretation of that result is in order . ( the mean of outpatient unit costs in 1988 is $ 57 in 1984 dollars , which is 11 percent lower than the 1984 unit cost . ) an economic effect that could partially account for this is economies of scale . the output - volume change for outpatient activity from 1984 to 1988 is considerable : the average number of visits rose from 58,000 to 74,000 or 28 percent . additional econometric evidence of economies of scale appears in a study of the determinants of 1987 medicare hospital outpatient department costs by miller ( 1992 ) . one change that accounts for an increase in the use of outpatient services is the rise in the number of ambulatory surgical procedures performed in hospitals . because these operations are relatively costly , it might be expected that they would drive up the average cost of a hospital outpatient visit . despite the focus on growth in ambulatory surgery , it should be noted that such surgery comprises a relatively small portion of total outpatient volume . in 1988 , less than 4 percent of all outpatient visits reported by community hospitals were for surgical procedures ( american hospital association , 1990 - 91 ) . the percentage of hospitals reporting ambulatory surgery rose from 91 to 95 during the period of this study , and the number of freestanding ambulatory surgical centers ( ascs ) rose 670 percent from 1983 through 1990 ( prospective payment assessment commission , 1992 ) . medicare changed the incentives for utilization of these facilities beginning in 1988 by bringing hospital outpatient surgery payments more in line with the lower rates already established for ascs . the increase in the number of outpatient visits in the sample of hospitals in this study is very large . the figures in table 1 indicate that the average hospital reported 16,000 more outpatient visits in 1988 than in 1984 . the question arises whether some of this increase is the result of unbundling on the part of hospitals . some states have instituted their own non - medicare cost - containment regulations that do not permit rate increases but do allow for volume adjustments . it has been suggested that hospitals have split services into multiple billable pieces in order to increase payments . this practice could be reflected in the aha number - of - visits measure , because each appearance by an outpatient to one unit of the hospital is counted as a visit . pre - admission testing on an outpatient basis has been another response by hospitals to fixed payments per discharge . this unbundling practice is a way of increasing the payment for the same workload . to the extent that the increase in number of visits from 1984 to 1988 is partially the result of overreporting or changes in the way visits are counted , unit costs for the outpatient component for the later years would be underestimated . ( the variation in total costs resulting from outpatient activity is spread out over a larger number of visits . ) although the estimated equation is a variable - cost function that excludes capital expenditures , it is questionable whether a remaining non - capital element of measured costs that is fixed with relation to number of discharges and number of outpatient visits could be significant . similarly , the incremental - cost approach can not properly allocate costs that vary jointly with inpatients and outpatients . if a considerable amount of joint costs is assigned to inpatients by this methodology , then our measure of outpatient costs is biased downward . however , the trends that are observed during the 4-year period of study would hold , assuming that the nature of fixed and joint costs is stable over time . another approach to disaggregation of cost components is that taken by aha in its method for representing multiple hospital outputs in a single measure . discharges are adjusted ( multiplied by an adjustment factor ) such that adjusted discharges refer to the number of discharges that the hospital could serve if it were offering no outpatient or other non - inpatient services . the desired adjustment factor is the ratio of total cost to inpatient cost , which converts non - inpatient services to discharge equivalents . ( aha adjusts inpatient days by the same adjustment factor . ) assuming that total costs are the sum of inpatient costs and outpatient costs , the desired adjustment factor may be expressed as adjusted discharges are then calculated as the latter is algebraically equivalent to dividing inpatient cost by unadjusted discharges ( ic / dis ) . however , aha uses the ratio of total revenue to inpatient revenue as a proxy for the ratio of total cost to inpatient cost . hence , the aha adjustment process is a revenue - based approach to calculation of unit costs . table 5 lists the aha costs per adjusted discharge along with the implied outpatient unit costs ( oc / opv ) . this revenue - based approximation to changes in unit costs substantially overestimates outpatient costs and cost increases and underestimates those of inpatient costs as observed by comparing the results listed in table 5 with those of table 4 . finally , the adjustment factors calculated using the cost - function approach to disaggregation of inpatient and outpatient components are listed with the aha adjustment factors in table 6 . the discrepancy between cost- and revenue - based unit costs and adjustment factors is most serious among hospitals that are smaller , less urban , and non - teaching , as seen by comparing the results listed in tables 4 and 5 as well as the results in table 6 . the growth rate of hospital outpatient costs has implications from the perspective of both policy and research . regarding policy , there is a current effort by both government and private insurers to control hospital payments for outpatient services . the results of our research indicate that hospital outpatient costs are not rising nearly as rapidly as are outpatient revenues . attempts at bringing payments more in line with actual cost increases will have a serious impact on hospitals with the largest discrepancies between costs and revenues . these tend to be the smaller , rural , and non - teaching facilities in the sample . this system is similar to that of drgs , which relies on charge data for calibration of payment weights . the appropriateness of using charge rather than cost data for the annual recalibration of drg weights has been the subject of some debate . work by cotterill , bobula , and connerton ( 1986 ) using 1981 data showed little difference between use of cost and charge data . the results of the study by rogowski and byrne ( 1990 ) showed that , by 1984 , cost- and charge - data - based drg weights were less congruent . however , the authors counsel the use of charge data . in addition to the timeliness of charge data , even the best cost data are partially based on charge data , so that many of the same biases are at work in either case . price ( 1989 ) updated the issue with a study using 1986 data , which showed much larger differences between cost- and charge - based weights than previously found . results of the present work indicate that the discrepancy between outpatient costs and revenues was significant and grew during the period 1984 - 88 . if historical charge data are used for weighting of outpatient payment groups , the system could be seriously distorted in favor of those procedures for which charges have been set well above costs . if outpatient rates in general are biased upward and inpatient rates downward , the smaller hospitals would be particularly vulnerable because of their relatively smaller outpatient departments . cost - allocation patterns also have research implications , particularly for the results of studies that rely on aha revenue - ratio adjusted output measures . if relatively high outpatient revenues are not reflective of true costs , then adjusted output measures will overstate true output levels . examination of changes in unit costs demonstrates the extent to which trends in the variables are misrepresented by the revenue - adjusted measures . from table 4 , it is seen that the cost - function measure of discharge unit cost rose 43 percent from 1984 through 1988 , while the revenue - ratio measure of this variable ( table 5 ) declined by 29 percent . measures of hospital labor productivity will also be understated if based on aha adjusted output measures , although these trends are more difficult to gauge because tracking measures of labor inputs over time is confounded by a number of factors . ( it should be noted that the results of much empirical research are dependent on the reliability of aha adjusted cost and output measures . numerous studies have used the aha cost per adjusted unit of output as the dependent variable in estimations of average cost functions . researchers should be aware of changing patterns of cost allocation and of how use of revenue or charge data as a proxy for costs may affect their conclusions . improvements in hospital accounting data would be beneficial for future research as well as in construction of drg and apg weights and payment levels .

in this article , the authors estimate a multiple - output cost function for a sample of 2,235 hospitals during the period 1984 - 88 to disaggregate total costs into inpatient and outpatient components . the results suggest that outpatient cost growth is roughly proportional to that of inpatient cost , despite much higher relative growth in revenues and utilization on the outpatient side . the stability in the outpatient / inpatient cost ratio implies that the increase in the outpatient - to - inpatient utilization ratio was offset by a decline in their relative unit costs .

Introduction Background Empirical methodology Data description Results Discussion Implications Conclusion

wilms tumor ( wt ) , also known as nephroblastoma , is the most frequent renal tumor occurring in children aged 0 to 15 years , especially among those younger than 6 years . wt accounts for roughly 6% of all childhood cancers ; 5% of whom had bilateral involvement , and 1% of whom had a family history . thanks to the prospective clinical trials performed by the international society of pediatric oncology renal tumor study group ( siop - rtsg , europe ) and the children s oncology group ( cog , formerly nwtsg , north america ) , most patients ( 85% ) affected by wt can be successfully cured . however , some studies released surveys reporting that there are still a few children who may have a poor prognosis or relapse with current therapies , 50% of whom will die despite intensive re - treatment . it has been proven that genetic variants are strongly associated with the development of wt . mutations in wt1 or epigenetic defects on chromosome 11p15 contribute to the major genetic susceptibility locus . previous studies have reported several non - wt1 loci with smaller effects on the genetic predisposition for wt , including p53 gene , insulin - like growth factor - ii gene , ctnnb1 gene , and h19 . however , less than half of wt cases are attributable to known genes that are associated with wt risk ; therefore , the exact pathogenesis of most wt cases remains unknown and identification of novel genetic loci is urgently needed . the reversion - inducing cysteine - rich protein with kazal motifs ( reck ) gene , known as a transformation suppressor gene , can restrain tumor invasion and metastasis through suppression of matrix metalloproteinases ( mmps ) , including mmp-4 and mmp-9 . mmps proteolytically degrade extracellular matrix proteins , which is critical for tumor metastasis and invasion . reck is expressed in a number of normal tissues , but it appears to be down - regulated in several types of cancers , including esophageal cancer , breast cancer , lung cancer , colorectal cancer , osteosarcoma , gastric carcinoma , prostatic cancer , oral cancer , pancreatic cancer , and cholangiocarcinoma . moreover , clinical investigations have demonstrated that high expression of reck in tumor tissues usually contributes to increasing survival rates and reducing tumor invasion . moreover , hawthorm et al . analyzed wt using single - nucleotide polymorphism ( snp ) mapping array - based comparative genomic hybridization and found chromosomal deletion in 9q ( reck gene locates on 9q13-p12 ) . although it is well documented that reck has an impact on metastasis and prognosis of human cancers , reck gene snps in wt susceptibility and clinical features remains poorly characterized . to obtain adequate power for assessing the potential association , we chose snps with minor allele frequencies of > 5% . furthermore , rs10972727 and rs11788747 snps both were previously identified to be potentially associated with several types of human cancers , including oral cancer , non - small cell lung cancer , and hepatocellular carcinoma . therefore , the purpose of our study was to determine whether the 2 snps ( rs10972727 and rs11788747 ) in reck gene were associated with susceptibility to wt in chinese children . this case - control study received approval from the human research ethics board of jinan children s hospital . a total of 291study participants 97 wt children and 194 healthy controls based on an age - sex - matched pair with 1 : 2 ratio were identified at the jinan children s hospital . all the participants were han chinese . written informed consent was obtained from parents of each participant before inclusion in the study . there were 41 male and 56 female patients , and the mean age at the time of surgery was 3.3 years . the patients histopathological types and cancer grading were determined according to classification of renal tumor of childhood defined by siop - rtsg . accordingly , 43 ( 44.3% ) out of 97 cases were characterized as wt stage i , 35 ( 36.1% ) as stage ii , 12 ( 12.4% ) as stage iii , and 7 ( 7.2% ) as stage iv . according to the prognostic group , moreover , no case had bilateral wt . according to the histologic type , there were 26 cases with blastemal type wt , 8 with diffuse anaplasia , 9 with focal anaplasia , 17 with epithelial , and 37 with mixed . genomic dna was extracted by qiaamp dna blood mini kits ( qiagen , valencia , ca ) according to the instructions of the manufacturer . reck genotype polymorphisms ( rs10972727 and rs11788747 ) were determined by polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) method . pcr was carried out in a reaction mixture of 10 l containing 58 l dna template , 0.5 u of taq biocatalysts , 1.8 mmol / l mg2 + , 2.4 l dntps ( promega , madison , wi ) , and 200 nm of each primer . the 2 snps were genotyped according to the methods described by chung et al . . primers for rs10972727 were 5-gtagaagaagtgactgatcc-3 and 5-atctgactccgaagataacc-3. the primers for the rs11788747 were 5-ttctatcaggtcatggaaca-3 and 5-tgcggttaagactggagaag-3. the pcr cycling conditions were : initial denaturation at 94c for 5 min , followed by 35 cycles of 1 min at 94c , at 60c for 30 s , and at 72c for 1min , with a final extension at 72c for 10 min . to verify results from pcr - ctpp , approximately 10% of the sample analysis was a duplication and each genotype was determined by the dna sequence analysis . , hardy - weinberg equilibrium was analyzed to compare the observed and expected genotype frequencies using the standard test or fisher s exact test . the differences in distributions of genotypes and alleles between cases and controls were assessed by tests . the estimated genotype - specific risks are presented as odds ratios ( ors ) . in all cases , this case - control study received approval from the human research ethics board of jinan children s hospital . a total of 291study participants 97 wt children and 194 healthy controls based on an age - sex - matched pair with 1 : 2 ratio were identified at the jinan children s hospital . all the participants were han chinese . written informed consent was obtained from parents of each participant before inclusion in the study . there were 41 male and 56 female patients , and the mean age at the time of surgery was 3.3 years . the patients histopathological types and cancer grading were determined according to classification of renal tumor of childhood defined by siop - rtsg . accordingly , 43 ( 44.3% ) out of 97 cases were characterized as wt stage i , 35 ( 36.1% ) as stage ii , 12 ( 12.4% ) as stage iii , and 7 ( 7.2% ) as stage iv . according to the prognostic group , moreover , no case had bilateral wt . according to the histologic type , there were 26 cases with blastemal type wt , 8 with diffuse anaplasia , 9 with focal anaplasia , 17 with epithelial , and 37 with mixed . genomic dna was extracted by qiaamp dna blood mini kits ( qiagen , valencia , ca ) according to the instructions of the manufacturer . reck genotype polymorphisms ( rs10972727 and rs11788747 ) were determined by polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) method . pcr was carried out in a reaction mixture of 10 l containing 58 l dna template , 0.5 u of taq biocatalysts , 1.8 mmol / l mg2 + , 2.4 l dntps ( promega , madison , wi ) , and 200 nm of each primer . the 2 snps were genotyped according to the methods described by chung et al . . primers for rs10972727 were 5-gtagaagaagtgactgatcc-3 and 5-atctgactccgaagataacc-3. the primers for the rs11788747 were 5-ttctatcaggtcatggaaca-3 and 5-tgcggttaagactggagaag-3. the pcr cycling conditions were : initial denaturation at 94c for 5 min , followed by 35 cycles of 1 min at 94c , at 60c for 30 s , and at 72c for 1min , with a final extension at 72c for 10 min . to verify results from pcr - ctpp , approximately 10% of the sample analysis was a duplication and each genotype was determined by the dna sequence analysis . , hardy - weinberg equilibrium was analyzed to compare the observed and expected genotype frequencies using the standard test or fisher s exact test . the differences in distributions of genotypes and alleles between cases and controls were assessed by tests . the estimated genotype - specific risks are presented as odds ratios ( ors ) . in all cases , five out of 97 cases had a family history of wt , 6 cases had relapse , and 16 had metastasis . in the present study , distributions of genotypes in cases and controls were in agreement with hardy - weinberg equilibrium ( rs10972727 , p=0.349 and p=0.121 ; and rs11788747 , p=0.519 and p=0.123 , respectively ) . table 2 shows the genotype and allele frequencies of the 2 snps ( rs10972727 and rs11788747 ) in reck gene . no significant difference between cases and controls was observed for distribution of rs10972727 genotype , rs11788747 genotype , and rs11788747 allele . however , we found that the allele g of rs11788747 snp was significantly associated with an increased risk of wt ( or=0.7 , 95%ci : 0.450.99 ; p=0.042 ) . moreover , although when taking the aa genotype as a reference , we found that ag + gg genotype was not statistically significantly associated with the risk of wt , there was a pronounced trend that carriers of 1 variant combined genotype had an increasing risk of wt ( or=0.6 , 95%ci : 0.381.03 ; p=0.065 ) . further analysis was conducted to investigate the associations between distribution of the genotype and allele of rs11788747 polymorphisms and clinicopathological features of wt patients . as shown in table 3 , although there was no significant difference in distribution of the genotype , allele g carriers with advanced tumor stage ( p=0.026 ) had an increased risk of wt , as did those who had metastasis ( p=0.002 ) . table 2 shows the genotype and allele frequencies of the 2 snps ( rs10972727 and rs11788747 ) in reck gene . no significant difference between cases and controls was observed for distribution of rs10972727 genotype , rs11788747 genotype , and rs11788747 allele . however , we found that the allele g of rs11788747 snp was significantly associated with an increased risk of wt ( or=0.7 , 95%ci : 0.450.99 ; p=0.042 ) . moreover , although when taking the aa genotype as a reference , we found that ag + gg genotype was not statistically significantly associated with the risk of wt , there was a pronounced trend that carriers of 1 variant combined genotype had an increasing risk of wt ( or=0.6 , 95%ci : 0.381.03 ; p=0.065 ) . further analysis was conducted to investigate the associations between distribution of the genotype and allele of rs11788747 polymorphisms and clinicopathological features of wt patients . as shown in table 3 , although there was no significant difference in distribution of the genotype , allele g carriers with advanced tumor stage ( p=0.026 ) had an increased risk of wt , as did those who had metastasis ( p=0.002 ) . the findings of this novel study provide evidence of the effects of snps of reck on wt susceptibility and clinicopathologic status association . various lines of evidence have found that tumor stage and metastasis are the risk factors for development of wt . in the current study , we explored the association of reck gene and wt risk , and subsequently analyzed the effects of combinations of functionally related polymorphisms and clinicopathological features of wt patients . to the best of our knowledge , this is the first study examining the association of reck gene polymorphism with wt risk , although several previous studies have reported on its association with several other types of human cancers . as the most frequent renal tumor occurring in children , although wt has a high survival rate , 25% of wt children may have a poor prognosis or relapse with current therapies , 50% of whom will die despite intensive re - treatment . therefore , it is urgent for us to identify novel genetic loci that might be associated with wt risk . it has been proven that the igf pathway plays a role in development of wt . the igf pathway is a complex signaling system stimulated by insulin - like growth factors ( igfs ) , which can be produced by almost any tissue in the body and has an important effect on growth , development , and survival in many different cell types . overexpression of igf2 , activating the insulin signaling pathway , results in disorder of protein synthesis , cell cycle and cell growth , and blocks apoptosis . furthermore , yamamoto et al . indicated that reduced reck could increase igf2 expression . the interaction between reck gene and igf gene might have a potential influence on susceptibility to wt . a study by huang et al . demonstrated down - regulation of reck gene expression in patients with wt . therefore , we conducted this study to determine whether reck gene is associated with wt risk . in the current study , our findings suggest that mutant of rs10972727 in reck had an impact on increasing wt risk . in addition , allele g carriers with advanced tumor stage involving metastasis had an increasing risk of wt . advanced tumor stage and metastasis are responsible for patient mortality in most tumors [ 3739 ] . found the level of reck expression determined the prognosis of pancreatic cancer and tumors , with overexpression of reck significantly decreasing invasiveness compared with reck - negative tumors , revealing the potential value of reck as a prognostic molecular marker for pancreatic cancer . zhang et al . reported that silenced reck gene was associated with poor survival in hepatocellular carcinoma . . showed that those who carried rs10814325 with at least 1 c allele had a higher risk of hepatocellular carcinoma . , after a follow - up of salivary adenoid cystic carcinoma patients , revealed that expression of reck and mmp-2 gene were correlated with tumor progression . in our study , we found a correlation between rs10972727 in reck and tumor stage and metastasis . however , the exact physiological mechanism by which reck influences progression of wt remains unclear . firstly , the primary weakness of this study is that the small sample size may affect the power in statistical analysis . secondly , the results were not replicated in additional individuals , and this might contribute to potential false - positive errors . moreover , the method used to select potentially functional snps as targets by using a web - based tool might have led to some positive or negative errors . finally , we did not perform a functional study to further reveal the mechanism by which the genetic polymorphisms in reck affect wt risk . data from the present data indicate that rs10972727 in reck gene is not associated with wt risk in chinese children , but the association between mutant g of rs11788747 in reck and wt risk was shown . g carriers with advanced tumor stage or with metastasis might have an increased risk of wt . therefore , further research is necessary to reveal the mechanism by which the genetic polymorphisms in reck affect the wt risk .

backgroundwilms tumor ( wt ) is the most common malignant renal tumor in children . previous studies suggested the reversion - inducing , cysteine - rich protein with kazal motifs ( reck ) down - regulation might have a role in numerous human cancers . the current study was done to investigate the associations of reck single - nucleotide polymorphisms ( snps ) with the wt susceptibility in chinese children.material/methodswe analyzed 2 snps ( rs10972727and rs11788747 ) in a total of 97 wt children and 194 healthy matched controls ( 1:2 ratio ) by real - time pcr and pcr - rflp genotyping analysis.resultswe found that the g allele of rs11788747 in the reck gene was significantly associated with wt in chinese children ( or=0.7 , 95% ci : 0.450.99 ; p=0.042 ) ; as with another snp rs10972727 , however , no statistically significant difference was detected . further analysis showed there was also a statistically significant difference in genotype frequencies between terminal tumor stage ( p=0.026 ) and metastatic groups ( p=0.002).conclusionsthe present data indicate that there is a significant association between mutant g of rs11788747 in reck and wt risk . g carriers with advanced tumor stage or with metastasis might have an increased risk of wt .

Background Material and Methods Study participants and ethical statement Genotyping of RECK Statistical analysis Results Association of the 2 SNPs with the risk of WT Stratification analysis Discussion Conclusions