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brugada syndrome (brs) is characterized by the presence of characteristic electrocardiographic (ecg) changes (coved st - segment elevation 2 mm with a right bundle - branch pattern) in the right - sided precordial leads (leads v1 through v3), either at baseline, or after administration of a class i (sodium channel blocking) antiarrhythmic drug associated with cardiac arrest or sudden death in the absence of demonstrable structural heart disease. clinical characteristics include spontaneous or induced ventricular tachycardia (vt) or ventricular fibrillation (vf), atrial fibrillation, a family history of sudden death at age < 45 years, similar ecg abnormalities in family members, syncope, or nocturnal agonal respiration. the only proven therapeutic option for prevention of sudden death in brs patients is the implantable cardioverter - defibrillator (icd), although recent data have also demonstrated the efficacy of anti - arrhythmic agents (mostly quinidine) that block the transient outward potassium current (ito) in patients who can tolerate these medications long - term. electrical storm in brs patients (brugada storm) is a rare but devastating clinical entity. acute management of this condition usually includes medical therapy directed at augmenting the slow inward calcium current (ical) (intravenous isoproterenol), and possibly counter - acting the effects of ito (quinidine and disopyramide) [2, 4 ]. current practice guidelines give ablation a class iib recommendation for treatment of patients with brugada storm. prior studies proposed the use of endocardial ablation of focal premature ventricular complexes (pvcs) within the right ventricular outflow tract (rvot) or the right - sided his - purkinje system as a means of preventing vt / vf episodes in brs patients. subsequent animal studies suggested that radiofrequency (rf) ablation of the epicardial surface of the rvot may be more effective in eliminating vt in brs. rf ablation of epicardial ventricular foci may prevent future vt / vf episodes in brs patients. newer insights into the pathophysiology of ventricular arrhythmias in brs has led to use of other ablation targets such as late potentials, as well as low - voltage, fractionated electrograms on voltage mapping. in this paper we describe a novel combined (endocardial and epicardial) ablation in the rvot performed in a young man with brugada storm. informed consent was obtained from the patient for being included in this report. a 34-year - old male with a history of brs with a dual - chamber icd and six prior shocks for documented vf his initial diagnosis of brs was based on the presence of brugada pattern on surface ecg and recurrent episodes of nocturnal (non - exertional) syncope. an electrophysiology (ep) study revealed a prolonged h - v interval of 75 ms, and a dual - chamber icd was implanted. at initial presentation, cardiac magnetic resonance imaging did not reveal any structural heart disease or late gadolinium enhancement, and diagnostic coronary angiography revealed normal coronary anatomy and absence of luminal stenosis. he continued to have occasional episodes of palpitations but did not have any shocks for the first five years post icd implant. after receiving four appropriate shocks (all for vf episodes while asleep) over a period of two years, he was admitted and started on extended - release quinidine gluconate 324 mg every 8 hours, but this medication was stopped due to qtc prolongation (from 430 to 535 ms after the 3rd dose), dizziness, and gastrointestinal distress. he suffered two more vf shocks from his icd about ten months later, and was admitted and started on cilostazol 100 mg twice daily. however, after two months of therapy, the patient stopped taking the cilostazol citing persistence of palpitations. after being admitted to the coronary intensive care unit (cicu), he received another vf shock, and iv isoproterenol (titrated to a sinus rate of 120 beats per minute) was started overnight. interrogation of his device showed that all of his vf episodes were triggered by premature ventricular complexes with short coupling intervals (fig. 1). after ruling out other causal factors including fever, electrolyte abnormalities, and myocardial ischemia, the patient was offered the option of either restarting medical therapy or ablation. given the acute presentation of brugada storm, an ep study was performed, but no pvcs were present at baseline, and no pvcs or vt could be induced with burst pacing or programmed stimulation. a three - dimensional (3-d) map of the rvot, pulmonic valve, and pulmonary artery was constructed using an ensite navx (endocardial solutions, st jude medical, minneapolis, mn, usa) system. after a coronary angiogram revealed normal coronary arteries, percutaneous epicardial access was obtained, and a 3-d epicardial map of the rvot and lvot was also created using the ensite navx system. low voltage, complex, fractionated electrograms and late potentials in the rvot were identified up to 185 ms after the qrs complex on the endocardial surface (fig. 2), and up to 246 ms after the qrs complex on the epicardial surface (fig. these areas were targeted for rf ablation using a 3.5 mm irrigated tip ablation catheter (thermocool sf, biosense webster inc., south diamond bar, ca, usa) (figs. 4, 5a a total of 107 ablation lesions were delivered, of which twenty - eight ablation lesions were delivered on the endocardial surface, and seventy - nine ablation lesions were delivered on the epicardial surface of the rvot. towards the end of the procedure, we noted widening of the qrs duration and increased st elevation. emergent blood tests revealed hyperkalemia with a serum k level of 8.2 meq / l, serum bicarbonate of 18 meq / l, serum lactic acid of 3.3 mmol / l, a serum ph of 7.32, elevated serum creatinine of 1.35 mg / dl, and creatine kinase of 1220 u / ml. review of the anesthesiologist s records revealed that the patient was receiving sevoflurane during the case, with intermittent use of intravenous ephedrine. aggressive treatment with intravenous 50% dextrose followed by iv insulin, iv calcium chloride, iv sodium bicarbonate, and oral sodium polystyrene sulfonate were given, and the ecg abnormalities resolved. the patient was monitored in the cicu after the procedure, and did not have any recurrence of hyperkalemia or ventricular tachyarrhythmias. he was discharged on day five post - procedure after being restarted on oral cilostazol 100 mg twice daily as per referring physician preference. he was briefly admitted two weeks after the procedure with pericarditis which resolved after a one - week course of ibuprofen.fig. 1initiation of ventricular fibrillation in our patient. top panel a surface electrocardiogram (ecg) showing premature ventricular complexes likely arising from the right ventricular outflow tract. bottom panel b intracardiac electrogram from implantable cardioverter - defibrillator interrogation of the same event fig. 23-d voltage map showing late fractionated potentials in the right ventricular outflow tract (rvot) endocardium. note the unipolar electrogram at the selected site (green dot) showing a delayed fractionated signal which extends 185 ms (ms) beyond the r wave (yellow arrowheads) fig. 33-d voltage map of the right ventricular utflow tract (rvot) and anterior rv free wall showing late fractionated potentials in the rv epicardium. note the unipolar electrogram showing a delayed fractionated signal which extends 246 ms (ms) beyond the q wave (yellow arrowheads). note that the low - voltage signals are localized over the anterior wall of the rv (labeled epirv) and right ventricular outflow tract, while the mapped portion of the left ventricular epicardium (labeled epilv) shows a normal voltage map (purple) fig. 4bipolar electrograms showing late fractionated potentials in the right ventricular outflow tract (rvot) endocardium (panel a), late fractionated potentials in the rvot epicardium (panel b), and isolated diastolic potentials (yellow arrows) in the rvot epicardium (panel c) which were targeted for ablation. in each panel, the electrograms on the left are before ablation, while the electrograms on the right are after ablation fig. 5 a 3-d map of the endocardial surface of the right ventricular outflow tract (rvot) and proximal pulmonary artery (deep blue) and epicardial surfaces of the rvot and rvot (gray). radiofrequency ablation using an irrigated tip catheter was performed endocardially (red dots) and epicardially (white dots). b 3-d ablation map (white dots) superimposed on the electroanatomic voltage map of the right ventricular outflow tract (rvot) and anterior rv wall. areas with low - voltage, late, fractionated electrograms were targeted for ablation. the endocardial ablation map (red dots) in the rvot is also shown for reference purposes initiation of ventricular fibrillation in our patient. top panel a surface electrocardiogram (ecg) showing premature ventricular complexes likely arising from the right ventricular outflow tract. bottom panel b intracardiac electrogram from implantable cardioverter - defibrillator interrogation of the same event 3-d voltage map showing late fractionated potentials in the right ventricular outflow tract (rvot) endocardium. note the unipolar electrogram at the selected site (green dot) showing a delayed fractionated signal which extends 185 ms (ms) beyond the r wave (yellow arrowheads) 3-d voltage map of the right ventricular utflow tract (rvot) and anterior rv free wall showing late fractionated potentials in the rv epicardium. note the unipolar electrogram showing a delayed fractionated signal which extends 246 ms (ms) beyond the q wave (yellow arrowheads). note that the low - voltage signals are localized over the anterior wall of the rv (labeled epirv) and right ventricular outflow tract, while the mapped portion of the left ventricular epicardium (labeled epilv) shows a normal voltage map (purple) bipolar electrograms showing late fractionated potentials in the right ventricular outflow tract (rvot) endocardium (panel a), late fractionated potentials in the rvot epicardium (panel b), and isolated diastolic potentials (yellow arrows) in the rvot epicardium (panel c) which were targeted for ablation. in each panel, the electrograms on the left are before ablation, while the electrograms on the right are after ablation a 3-d map of the endocardial surface of the right ventricular outflow tract (rvot) and proximal pulmonary artery (deep blue) and epicardial surfaces of the rvot and rvot (gray). radiofrequency ablation using an irrigated tip catheter was performed endocardially (red dots) and epicardially (white dots). b 3-d ablation map (white dots) superimposed on the electroanatomic voltage map of the right ventricular outflow tract (rvot) and anterior rv wall. areas with low - voltage, late, fractionated electrograms were targeted for ablation. the endocardial ablation map (red dots) in the rvot is also shown for reference purposes in view of his clinical presentation of brs and the occurrence of unexpected hyperkalemia and metabolic acidosis with the use of sevoflurane, genetic testing for brs - related mutations as well as latent neuromuscular disorders was offered to the patient, but he declined. close monitoring of his clinical status and interrogation of his icd over a period of forty - one months post - ablation have revealed no further episodes of vt / vf. brs is an autosomal dominant genetic disorder with incomplete penetrance, but the precise genetic basis of this condition is known in only ~30% of the affected individuals. to date, mutations in 19 different genetic loci have been associated with brs, which either cause loss of function of the sodium channels or the l - type calcium channels, or cause a gain in function of the potassium channels. mutations in the scn5a gene account for ~15% of brs patients, leading to either loss of expression or altered function of the -subunit of the cardiac sodium channel. the majority of symptomatic brs patients are male, and male gender is associated with a sevenfold higher risk for cardiac arrest in patients with brs. although icds are the mainstay for prevention of sudden arrhythmic death in patients with brs, they do not reduce the frequency of malignant ventricular arrhythmias. multiple icd shocks, albeit appropriate, can be psychologically devastating to the patient [11, 12 ], may cause acute myocardial injury, myocardial dysfunction due to changes in transmembrane potential, and have pro - arrhythmic effects. medications such as quinidine may be effective in reducing ventricular arrhythmias in brs patients, but adverse effects and drug drug interactions may preclude their long - term use. this is especially true for younger patients, who may need these medications for decades. reduction in the inward sodium current (ina) in brs patients accentuates the action potential (ap) phase 1 notch caused by the transient outward potassium current (ito), resulting in loss of the dome pattern of phase 2 (inhibition of inward calcium current) of the cardiac ap and shortening of the ap duration (fig. regions with higher ito density (such as the rvot epicardium) are most susceptible to the loss - of - function ina channel mutations present in the majority of brs patients. studies from canine cardiac preparations suggest that ito currents are more prominent in male hearts compared to females, which may explain the higher prevalence of the brs phenotype in males. in addition, differential effects of the reduction in ina in the rvot epicardium and endocardium result in a voltage gradient between the rvot endocardium and epicardium. these perturbations lead to marked transmural and epicardial dispersion of repolarization, which, combined with the conduction delay caused by reduced ina, leads to phase 2 reentry. these phase 2 reentry circuits lead to development of pvcs with short coupling intervals, which can be triggers for vt / vf in brs patients [9, 18 ] (figs. 7, 8).fig. 6loss of action potential dome results in a transmural voltage gradient and sets the stage for phase 2 reentry(reprinted from the journal of the american college of cardiology volume 39, issue 12, antzelevitch with permission from elsevier) fig. 7coved - type st - segment elevation and subsequent non - sustained polymorphic ventricular tachycardia caused by premature beats induced by phase 2 reentry in a canine model of brugada syndromereprinted by permission from macmillan publishers ltd : nature reviews cardiology, shimizu. 8surface telemetry strips showing coupled premature ventricular complexes (blue arrows) as the initiating event prior to the onset of ventricular fibrillation (top panel) and conversion to normal sinus rhythm after a shock (red arrow) from the implantable cardioverter - defibrillator loss of action potential dome results in a transmural voltage gradient and sets the stage for phase 2 reentry (reprinted from the journal of the american college of cardiology volume 39, issue 12, antzelevitch with permission from elsevier) coved - type st - segment elevation and subsequent non - sustained polymorphic ventricular tachycardia caused by premature beats induced by phase 2 reentry in a canine model of brugada syndrome reprinted by permission from macmillan publishers ltd : nature reviews cardiology, shimizu. surface telemetry strips showing coupled premature ventricular complexes (blue arrows) as the initiating event prior to the onset of ventricular fibrillation (top panel) and conversion to normal sinus rhythm after a shock (red arrow) from the implantable cardioverter - defibrillator reduced ina in brs patients may also manifest a prolonged pr interval, as well as a prolonged hv interval and increased qrs duration (due to slowing of his purkinje and ventricular conduction). reduction in intraventricular conduction velocity causes development of low - voltage, high - frequency signals that occur after the qrs complex on intracardiac electrograms, which are more pronounced in the rvot epicardium in brs patients. these high - frequency late potentials may be due to localized, concealed phase 2 reentry, and the low - voltage fractionated signals are thought to correspond to the second ap upstroke after the accentuated notch, which occurs heterogeneously within the rvot epicardium. such delayed and fractionated electrogram signals have also been demonstrated on endocardial mapping of the rv [19, 20 ]. late potentials as seen on signal - averaged ecg were shown to be an independent predictor of arrhythmic events in a small study of brs patients, but larger studies exploring this association are lacking. ablative techniques for management of brs patients initially targeted the short coupled pvcs in the rvot, which may be triggers for the development of vt / vf episodes. haissaguerre and colleagues reported a series of seven patients (three of whom had brs) with documented polymorphic vt or vf, who underwent an ep study and mapping to detect focal ectopic ventricular mapping revealed that the earliest electrogram relative to the onset of the ectopic qrs complex was located within the rvot (two patients) or the right - sided purkinje system (one patient). endocardial ablation of these sites resulted in non - inducibility of vt / vf post - ablation, and was associated with reduced recurrence of vt / vf episodes during short - term (mean 7 6 months) follow - up. unfortunately, a significant number of brs patients undergoing ep study may not demonstrate focal pvcs at baseline, even with aggressive stimulation protocols, thereby limiting the usefulness of this approach [7, 23 ]. recent studies have suggested that epicardial substrate modification may be effective in preventing ventricular arrhythmias in brs patients. in a series of nine symptomatic patients with type 1 brugada pattern and multiple vf episodes, nademanee and colleagues showed that epicardial mapping revealed a unique pattern of abnormal, low - voltage, fractionated potentials that occurred late (50100 ms after the end of the surface qrs complex) which were localized to the anterior aspect of the rvot. rf ablation of these potentials led to non - inducibility of vt / vf episodes, normalized the brugada pattern on surface ecg, and prevented vt / vf recurrence in the majority of patients. these authors hypothesized that these areas of slow conduction represent areas of delayed depolarization due to micro - anatomic or functional derangements in conduction between cardiomyocytes, which lead to the characteristic st - segment elevation pattern seen in brs. this study led to case reports of epicardial ablation for prevention of ventricular arrhythmias in brs patients refusing icd implantation and recurrent vt / vf. recently, brugada, pappone and colleagues also reported that ablation of low - voltage areas on the epicardial surface of the anterior rvot and anterior rv free wall was associated with non - inducibility of vt / vf in the electrophysiology lab, and normalization of the surface ecg in 14 brs patients who had received an icd. the use of ablation (whether epicardial or endocardial) for the management of brugada storm has rarely been reported. nakagawa and colleagues reported a case of a forty - one - year - old man with brugada storm who was found to have a pvc originating from the posterolateral wall of the rvot that consistently initiated his vf episodes, and endocardial rf ablation of this pvc eliminated his vf episodes, in spite of the co - existence of other pvc morphologies in the rvot and rv inflow tract. in a series of four patients with a history of vf storm, sunsaneewitayakul and colleagues showed that endocardial rf ablation of late activated zones in the rvot seen on non - contact mapping was associated with normalization of the brugada pattern on surface ecg, however, recurrent episodes of vf (including vf storm) were recorded in three of the four patients post - ablation. cortez - dias and colleagues performed epicardial ablation in a 60-year - old woman with brs and frequent episodes of polymorphic vt / vf refractory to quinidine. they targeted bipolar electrograms with delayed fractionated potentials (up to 370 ms after the surface qrs complex) on the anterior rvot wall. the surface ecg normalized six weeks after ablation, and no recurrent vt / vf episodes were observed over a 6-month follow - up. a combined (epicardial endocardial) ablation strategy for brugada storm, targeting low - voltage, fractionated, late potentials in the rvot in the absence of spontaneous or induced pvc triggers on an ep study, has not been previously reported in the literature. in addition, we report the longest follow - up duration (forty - one months) for a patient undergoing an ablation - based strategy for management of brs in the published literature. we were unable to pursue a focal ablation strategy due to the absence of spontaneous or induced pvcs in the ep lab, which (as noted) is not an uncommon scenario in brs patients. in the context of electrical storm in a young patient with brs who had already demonstrated intolerance to quinidine, we employed a combined ablation strategy consisting of ablation of both epicardial and endocardial late potentials within the rvot, to eradicate areas of delayed depolarization and thereby reduce or eliminate the substrate conducive to sustained episodes of vt / vf. we propose that the absence of pvc triggers on ep study should prompt the operator to look for late, low - voltage, fractionated potentials in the rvot, and target these sites identified with electroanatomic mapping for ablation. although the brugada pattern on surface ecg persists in this patient even after forty - one months of follow - up (fig. 9), he has had no recurrences of vt / vf since the ablation procedure. he remains on cilostazol after the ablation as per the referring clinician s preference.fig. panel c follow - up ecg after 41 months of follow - up surface electrocardiograms (ecg) of our patient. panel c follow - up ecg after 41 months of follow - up there is conflicting data in the literature regarding the utility of cilostazol for the prevention of ventricular arrhythmias in brs patients [27, 28 ]. current guidelines for management of brs do not include cilostazol as a potential therapy for patients with brs. other experts currently give a class iib recommendation for the use of cilostazol for arrhythmia suppression in brs patients. our patient continued to have persistent palpitations during the short duration (two months) of therapy with cilostazol prior to the ablation. therefore, we believe it is unlikely that this medication alone has provided such sustained control of his ventricular arrhythmias. the development of hyperkalemia, metabolic acidosis, acute kidney injury, and rhabdomyolysis in this patient was attributed to the use of sevoflurane and ephedrine during the ablation procedure. isolated cases of severe hyperkalemia and rhabdomyolysis in the peri - operative period have been reported with the use of inhaled anesthetics such as sevoflurane. it is plausible that the concomitant use of sevoflurane and ephedrine may have potentiated this risk. aggressive correction of hyperkalemia and associated cardiac arrhythmias is recommended in these situations, followed by subsequent testing for latent neuromuscular disease. our patient was offered genetic testing as well as muscle biopsy testing after this event, but he declined. bai and colleagues reported a case series of forty - nine patients with arrhythmogenic right ventricular dysplasia / cardiomyopathy (arvd / c) and recurrent vts or multiple icd therapies in spite of anti - arrhythmic medications, who underwent either an endocardial - only vt ablation (n = 23) or a combined endo - epicardial vt ablation (n = 26) in a non - randomized fashion. after follow - up for at least three years, patients in the combined endo - epicardial group had significantly more freedom from icd therapies or ventricular arrhythmias compared to the endocardial - only group (84.6% versus 52.2%, p = 0.029). recent research suggests that there is considerable overlap in clinical phenotype as well as the molecular mechanisms responsible for ventricular tachyarrhythmias among patients with brs and arvd / c. combined endo - epicardial ablation has also been shown, in small studies, to be feasible and safe for treating monomorphic vt refractory to anti - arrhythmic medications in selected patients with hypertrophic cardiomyopathy. we describe the first reported case of combined (epicardial and endocardial) ablation of abnormal, low - voltage, fractionated late potentials within the rvot (in the absence of spontaneous or induced ventricular ectopic beats on ep study) for the treatment of brugada storm. post - ablation, our patient has not had any recurrences of vt / vf over a follow - up period of forty- one months, the longest follow - up duration for any brs patient treated with ablation reported in the literature. although he remains on pharmacotherapy with oral cilostazol following the procedure, it is unlikely that cilostazol alone produced such sustained control of his ventricular arrhythmia. we propose that a combined ablation of late potentials in the rvot may be a useful approach for management of selected patients with brs.	a 34-year - old man with brugada syndrome (brs) presented with electrical storm, manifested as multiple appropriate shocks from his implantable cardioverter - defibrillator over a period of 7 hours. he had not tolerated prior treatment with quinidine, and had self - discontinued cilostazol citing persistent palpitations. after stabilization with intravenous isoproterenol, an electrophysiology study was performed but no spontaneous or induced ventricular ectopic beats were identified. a three - dimensional (3d) endocardial electro - anatomic map of the right ventricular outflow tract (rvot), pulmonic valve, and pulmonary artery, as well as a 3d epicardial map of the rvot, were created. low voltage, complex, fractionated electrograms and late potentials were targeted for irrigated radiofrequency ablation both endocardially and epicardially. post - procedure, he was maintained on cilostazol (referring clinician preference), and has had no further ventricular tachyarrhythmia episodes over the past forty - one months. we propose that this novel ablation strategy may be useful for acute management of selected patients with brs.
wild type cd1 embryos were harvested, staged and stained by wholemount in situ hybridization using established methods,. measurement of palates before dissection and after staining confirmed that no significant shrinkage occurred (data not shown). palatal explants were cultured (37c, 5% co2) using the trowell technique in dmem (sigma), 20 u / ml pen - strep (gibcobrl), 10% fbs (gibcobrl), 50 mm transferrin (sigma) and 150 g / ml ascorbic acid (sigma). for cutting and inhibitor experiments, serum - free advanced d - mem / f12 (gibcobrl) supplemented as above, was used. su5402 (calbiochem) was diluted in medium from 10 mm in dmso stock ; cyclopamine (sigma) from 20 mg / ml - ethanol stock. explants placed in a minimum volume of pbs in wells cut into 1% agarose were digitally imaged under a stereo dissecting microscope and measurements made using the ruler in imagej (from the nih imagej website) calibrated with a micrometer slide. dimensions of fixed material were within 8% of those of fresh, unfixed material (data not shown). simulations of reaction - diffusion patterning were performed using the javascript in ref with the parameters du=0.03, d u=0.02, a u=0.1, b u=0.06, c u=0, fmax=0.2, dv= 0.06, d v=0.5, a v=0.1, b v=+0, c v=0.2, gmax=0.5. wild type cd1 embryos were harvested, staged and stained by wholemount in situ hybridization using established methods,. measurement of palates before dissection and after staining confirmed that no significant shrinkage occurred (data not shown). palatal explants were cultured (37c, 5% co2) using the trowell technique in dmem (sigma), 20 u / ml pen - strep (gibcobrl), 10% fbs (gibcobrl), 50 mm transferrin (sigma) and 150 g / ml ascorbic acid (sigma). for cutting and inhibitor experiments, serum - free advanced d - mem / f12 (gibcobrl) supplemented as above, su5402 (calbiochem) was diluted in medium from 10 mm in dmso stock ; cyclopamine (sigma) from 20 mg / ml - ethanol stock. explants placed in a minimum volume of pbs in wells cut into 1% agarose were digitally imaged under a stereo dissecting microscope and measurements made using the ruler in imagej (from the nih imagej website) calibrated with a micrometer slide. dimensions of fixed material were within 8% of those of fresh, unfixed material (data not shown). simulations of reaction - diffusion patterning were performed using the javascript in ref with the parameters du=0.03, d u=0.02, a u=0.1, b u=0.06, c u=0, fmax=0.2, dv= 0.06, d v=0.5, a v=0.1, b v=+0, c v=0.2, gmax=0.5.	we present direct evidence of an activator - inhibitor system in the generation of the regularly spaced transverse ridges of the palate. we show that new ridges, or rugae, marked by stripes of sonic hedgehog (shh) expression, appear at two growth zones where the space between previously laid - down rugae increases. however, inter - rugal growth is not absolutely required : new stripes still appear when growth is inhibited. furthermore, when a ruga is excised new shh expression appears, not at the cut edge but as bifurcating stripes branching from the neighbouring shh stripe, diagnostic of a turing - type reaction - diffusion mechanism. genetic and inhibitor experiments identify fibroblast growth factor (fgf) and shh as an activator - inhibitor pair in this system. these findings demonstrate a reaction - diffusion mechanism likely to be widely relevant in vertebrate development.
k channels are modular proteins, composed of a central pore module (pm) surrounded by sensor domains that perceive external stimuli and convert them into changes of pore activity (gating). this feature and simple assessment of their performance by electrophysiological measurements make ion channels amenable for testing the hypothesis that the pore of voltage - gated k (kv) channels acquired the voltage - sensing domain (vsd) throughout evolution. first, it suggests that single domains, once detached from the original channel, can function independently ; for instance, the pm of the bacterial kvlm channel forms a functional k channel when separated from its vsd domain (santos., 2008). second, regulatory modules, which control k channels, are also found in proteins with completely different enzymatic functions. a vsd displaying the hallmarks of those of kv channels is connected to a soluble phosphatase in the sea squirt ciona intestinalis phosphatase (ci - vsp) protein (murata., 2005). third, modularity also implies that domains of different classes of channels can be swapped. so far, attempts to exchange entire membrane modules between k channels have been unsuccessful, leaving this third implication unfulfilled (caprini., motif of the vsd and the p loop with most of the transmembrane domains of the pore, could be exchanged between k and na channels (lu. it was concluded that membrane modules have coevolved so intimately in kv proteins that it is impossible to exchange them entirely without losing functionality of the chimeric channels. here, we show that it is possible to obtain a voltage - gated channel in one step by fusing two full - length modules : the vsd of ci - vsp and the pm of the viral k channel kcv (plugge., 2000). these two membrane modules are evolutionarily unrelated, and there is not any a priori indication that they should be able to make a voltage - sensitive channel. the ciona vsd regulates a soluble enzyme, yet it remains speculative if this sensor would gate a channel pore. the second component, the viral channel kcv, is clearly not designed to be controlled by external stimuli, such as voltage. apart from a 12amino acid - long n terminus, the channel comprises only the bare pm and has neither additional membrane nor cytosolic domains (plugge., 2000). all constructs were inserted into bamhi and xhoi restriction sites of psgem vector (a modified version of pgem - he). in vitro transcription was performed on linearized plasmids using t7 rna polymerase (promega), and crnas were injected (50 ng per oocyte) into xenopus laevis oocytes, as reported previously (plugge., 2000). mutations were inserted by site - directed mutagenesis (quikchange site - directed mutagenesis kit ; agilent technologies) and confirmed by sequencing. chimeric constructs were generated by overlapping pcr from ci - vsp (provided by d. minor, university of california, san francisco, san francisco, ca) and pbcv-1 kcv. kvsynth1 includes amino acids 1239 of ci - vsp and amino acids 194 of kcv. two - electrode voltage - clamp (tevc) experiments were performed as indicated previously (plugge., 2000) using an amplifier (geneclamp 500 ; axon instruments) and filtered at 5 khz. data acquisition and analysis were done using the pclamp8 software package (axon instruments). electrodes were filled with 3 m kcl and had a resistance of 0.20.8 m in 50 mm kcl. the oocytes were perfused at room temperature (2527c) at a rate of 2 ml / min with a bath solution containing 50 mm kcl (or nacl), 1.8 mm cacl2, 1 mm mgcl2, and 5 mm hepes, adjusted to ph 7.4 with koh (or naoh). mannitol was used to adjust the osmolarity of the solution to 215 mosmol / l. bacl2 diluted from 1-m stocks was added to the solution as indicated. the standard clamp protocol consisted of steps from the holding voltage of 20 mv to voltages in the range of 100 to + 100 mv ; tail currents instantaneous and steady - state currents were sampled after 10 ms and at the end of the voltage step, respectively. for single - channel recordings, the vitelline membrane of the oocytes was removed mechanically before the experiment. bath and pipette solution for measurements with kcvpbcv1 contained 100 mm kcl, 1.8 mm cacl2, 1 mm mgcl2, and 10 mh hepes, adjusted to ph 7.4 with koh. the slightly different k concentrations (150 mm for kvsynth and 100 mm for wt) do not impede comparison. according to pagliuca. (2007), the difference in conductance for the wt would be 20% at most, which is well within the error bars. currents were recorded with an amplifier (dagan3900 ; dagan corporation) and a digitizer unit (lih8 + 8 ; heka). all constructs were inserted into bamhi and xhoi restriction sites of psgem vector (a modified version of pgem - he). in vitro transcription was performed on linearized plasmids using t7 rna polymerase (promega), and crnas were injected (50 ng per oocyte) into xenopus laevis oocytes, as reported previously (plugge., 2000). mutations were inserted by site - directed mutagenesis (quikchange site - directed mutagenesis kit ; agilent technologies) and confirmed by sequencing. chimeric constructs were generated by overlapping pcr from ci - vsp (provided by d. minor, university of california, san francisco, san francisco, ca) and pbcv-1 kcv. kvsynth1 includes amino acids 1239 of ci - vsp and amino acids 194 of kcv. two - electrode voltage - clamp (tevc) experiments were performed as indicated previously (plugge., 2000) using an amplifier (geneclamp 500 ; axon instruments) and filtered at 5 khz. data acquisition and analysis were done using the pclamp8 software package (axon instruments). electrodes were filled with 3 m kcl and had a resistance of 0.20.8 m in 50 mm kcl. the oocytes were perfused at room temperature (2527c) at a rate of 2 ml / min with a bath solution containing 50 mm kcl (or nacl), 1.8 mm cacl2, 1 mm mgcl2, and 5 mm hepes, adjusted to ph 7.4 with koh (or naoh). mannitol was used to adjust the osmolarity of the solution to 215 mosmol / l. bacl2 diluted from 1-m stocks was added to the solution as indicated. the standard clamp protocol consisted of steps from the holding voltage of 20 mv to voltages in the range of 100 to + 100 mv ; instantaneous and steady - state currents were sampled after 10 ms and at the end of the voltage step, respectively. for single - channel recordings, the vitelline membrane of the oocytes was removed mechanically before the experiment. bath and pipette solution for measurements with kcvpbcv1 contained 100 mm kcl, 1.8 mm cacl2, 1 mm mgcl2, and 10 mh hepes, adjusted to ph 7.4 with koh. the slightly different k concentrations (150 mm for kvsynth and 100 mm for wt) do not impede comparison. according to pagliuca. (2007), the difference in conductance for the wt would be 20% at most, which is well within the error bars. currents were recorded with an amplifier (dagan3900 ; dagan corporation) and a digitizer unit (lih8 + 8 ; heka). to test whether the voltage - dependent movement of the ciona vsd is able to gate a channel, we created the fusion protein kvsynth1 by adding amino acids 1239 of ci - vsp to the full - length kcv sequence (amino acids 194). the fusion protein and its individual components have been expressed in xenopus oocytes and tested by tevc. 1 a) generate small currents, not different from typical water - injected oocytes. oocytes expressing kcv alone (fig. 1 b) show the typical k currents, which have been characterized previously (moroni., 2002 ; gazzarrini., 2004, 2006 ; kang., 2004 ; abenavoli., the i - v relationship of kcv is linear at moderate voltages. at extreme voltages, the ohmic behavior is lost because of a fast gating mechanism that occurs at the selectivity filter and results in a negative slope conductance (abenavoli., 2009). kvsynth1 generates currents that are very different from those of its single components and shows the typical properties of a delayed - rectifier k channel, slowly activating at positive potentials (hille, 1992) (fig. depolarizing voltages generate large time - dependent currents on top of a small instantaneous component. the corresponding steady - state i - v relationship shows a strong outward rectification. to quantify the degree of rectification, we use the ratio of steady - state currents at + 60/100 mv (i+60mv / i100mv). for kvsynth1, it is 13.3 2 (average of six oocytes) ; this ratio is 1 0.1 in kcv. (a) vsd is the voltage - sensing domain (amino acids 1239) of ci - vsp ; s1s4 indicate the four transmembrane domains of the vsd. (b) kcv is the full - length (amino acids 194) viral k channel ; tm1, tm2, and p indicate, respectively, the two transmembrane domains and the pore loop of kcv. voltage protocol : vh, 20 mv ; test voltages, + 100 to 100 mv (200 mv for kcv) ; tail, 80 mv. test pulse length : a and c, 8.5 s ; b, 0.6 s. (bottom) corresponding steady - state i - v relationships. replacement of external k with na shifts the reversal voltage of the fully activated kvsynth1 current by 80 mv 6 (sd, n = 3 oocytes) (fig. 2 a) and reduces the outward current, another typical feature of kcv (plugge., 2000). the estimated relative permeability pna / pk of 0.05 0.01 closely resembles the value of 0.03 0.01 reported previously for kcv (chatelain., 2009). another feature of kcv that is preserved in kvsynth1 is the aforementioned negative slope conductance of the open channel, in this case already visible at approximately 100 mv (fig. 2 b showing clear flickering behavior with a consequent reduction of the unitary channel conductance at voltages negative of approximately 100 mv. current reversal potential recorded from the same oocyte was 20 mv in 50 mm [k]out () and 98 mv in 50 mm [na]out (). voltage protocol : prepulse voltage step at + 60 mv, followed by testing voltages from + 60 to 180 mv. (left) representative opening burst at different membrane voltages, with arrows denoting the baseline. the low - pass filter was set to 20 khz, the sampling rate was 100 khz, and traces were digitally filtered at 2 khz for clarity. (right) comparison of the i - v curves of kvsynth1 in 150 mm k () and kcv in 100 mm k (). (c) nernst plot : current reversal potential (erev) of macroscopic kvsynth1 current plotted as a function of external k concentration (lg[k]out). black line is the linear regression to erev mean values (n = 3). steady - state currents recorded in () control solution (50 mm k) and () plus 1 mm bacl2. currents recorded from a holding potential (vh) of 20 mv to the indicated test voltages. test pulse length was 8.5 s. the addition of barium blocks the inward current and moderately affects the outward current, as reported previously for kcv channel (plugge., 2000). (e) western blot analysis performed on total protein extracts with a custom - made antibody (8d6) recognizing the tetramer, but not the monomer, of kcv. the expected molecular mass of kcv and kvsynth1 tetramers are 42.5 and 149 kd, respectively. the single - channel i - v relationship (i - v curve) of kvsynth1 reported in fig. 2 b together with that of kcv for comparison shows that at membrane voltages more negative than 60 mv, the current of kvsynth1 decreases, resulting in a negative slope conductance. the kcv channel shows this behavior only at more negative voltages, beyond 100 mv (abenavoli., 2009). this is consistent with the observation that the instantaneous currents of kvsynth1 in tevc experiments (fig. the conductance of the synthetic channel (112 25 ps between 50 and + 50 mv) is only slightly smaller than that of kcv (133 8 ps, also reported previously by abenavoli. this confirms that the general filter structure and function are not dramatically altered by the addition of the voltage sensor. 2 summarizes other kcv properties preserved in kvsynth1, including k selectivity shown by the slope of 58.5 mv in the nernstian plot of fig. 2 c, voltage - dependent barium block of the instantaneous inward current shown in fig. 2 e shows a western blot decorated with an antibody raised against the kcv tetramer that does not recognize the monomer. the same antibody recognized kvsynth1 wt at about the predicted molecular mass of the tetramer (149 kd). interestingly, mutation f305a in the k channel signature sequence (gfg) of kvsynth1 prevented antibody recognition, indicating that either the protein is not synthesized or, more likely, does not form a stable tetramer., kvsynth1 displays the pore properties of kcv, such as selectivity, fast gating, and overall pore architecture ; these are not modified by the addition of the vsd. the new property introduced by the vsd is a strong voltage dependency of the currents. the voltage sensor closes the channel at negative potentials and opens it with a slow kinetics at voltages more positive than 0 mv (t1/2 = 450 74 ms at + 60 mv ; n = 4 oocytes) (fig. 3 a was determined from the tail currents after subtraction of the instantaneous component (inset of fig. even though full saturation of the open probability could not be reached for the presence of endogenous conductances in oocytes, the activation curve could be fitted with a boltzmann equation. kvsynth1 has an apparent half - activation voltage (v1/2) of + 56 mv, and channel opening is caused by the movement of the equivalent of about one electronic charge across the membrane (z = 0.92). these parameters strongly resemble those reported for the sensing currents of ci - vsp, either with or without the phosphatase (murata., 2005 ; villalba - galea., 2008). to confirm that the vsd is responsible for the voltage - dependent properties of kvsynth1, we replaced two of the four arginines in the s4 segment, r229 and r232, with glutamines. in ci - vsp, this double mutation eliminates the sensing currents and generates a voltage - insensitive phosphatase (murata., 2005 ; murata and okamura, 2007). in kvsynth1, the double mutation r229q / r232q results in a very small and quasi - ohmic current (3b) ; the mutated vsd has apparently lost its tight control on channel opening and voltage dependency. another interesting mutation that shifts the q - v curve of ci - vsp to the left by 46 mv is the single neutralization of the gating charge r217 to q (villalba - galea., 2008). the same mutation in kvsynth1 resulted in a channel that is already more open at the holding voltage because the activation curve is shifted by 38 mv to the left (fig. 3, c and d). judging from the behavior of these mutants, we conclude that the properties of the vsd are transmitted to the gating of the pore. to examine further details of the causal interplay between vsd and channel gating, we tested whether kvsynth1 also reveals the mode shift, which has been described for the sensor domain (villalba - galea., 2008). during a long - lasting depolarization conformation in which the voltage dependency is markedly shifted toward negative potentials. to test this, we measured the tail currents of kvsynth1 r217q after holding the same oocyte at two different preconditioning voltages, 100 and + 40 mv for 5 s. tail currents were collected at 80 mv after prepulse at different voltages that were kept short (1.2 s) to limit the effect on the relaxation. 3 e shows that the resulting itail / v curve is shifted to more negative potentials when the oocyte is preconditioned at + 40 mv. the observed negative shift of 30 mv (n = 3 oocytes) matches quite well the direction and the amplitude of the shift reported for the vsd (villalba - galea., 2008). these data further confirm that the properties of the sensor are transmitted to the gating of the pore. (a) activation curve of kvsynth1 constructed from tail currents (inset) after subtracting the instantaneous current offset. 1 c. datasets (n = 6) were jointly fitted to a two - state boltzmann equation (dotted line) of the form y = (1 + e 1/2), where z is the effective charge ; v1/2 is the half - activation voltage ; and f, r, and t have their usual thermodynamic meaning. (b) steady - state i - v relationships of the single mutant f305a (), the double mutant r229q / r232q (), and the wt kvsynth1 (). currents were recorded from a holding potential (vh) of 20 mv to the indicated test voltages (pulse length, 600 ms). (c) exemplary currents recorded from wt kvsynth1 (control) and its r217q mutant. to compare currents from different constructs, the traces have been normalized to the current value recorded at + 60 mv and expressed in arbitrary units (a.u.). voltage protocol as in fig. 1 c. (d) activation curve of the r217q mutant () constructed as in a from four datasets : v1/2 = 18 mv and z = 1.1. the curve of the wt (dotted line) is replotted from a for comparison. the same r217q - expressing oocyte was subjected to the following protocol : preconditioning (5 s) at either 100 or + 40 mv and test pulses from 120 to + 30 mv. tail currents (at 80 mv) are plotted for preconditioning at 100 mv () or at + 40 mv (). the test pulses were kept short (1.2 s) to maintain the effect of the preconditioning voltages ; hence, data in e do not reflect full activation of the channel. considerable evidence supports the role of the s4s5 linker in kv channels as the transducing element between sensor movement and pore gating (long., 2005). in kvsynth1, the vsd is directly connected to kcv after removal of the 16amino acid linker that in ci - vsp couples the vsd to the phosphatase (fig. the 12amino acid - long n terminus of kcv acts here as the s4s5 linker of kv channels and transmits the conformational changes of the sensor to the pore. to study if and how the linker influences gating, we varied its length and composition by combining the n terminus of kcv and the linker of the vsd, as shown in fig. 4 b. all constructs produced functional channels, apart from that with the 28amino acid linker that barely expressed and was not further analyzed. 4 c shows exemplary currents from constructs with 4, 6, and 20amino acid linkers and their corresponding i - v relationships. the first and most cogent observation is that there is a minimum linker length required for efficient control of the voltage sensor on the pore, and that the transition is quite sharp. the construct with 4 amino acids is quite similar to the original channel kcv, whereas that with 6 amino acids shows the typical features of kvsynth1, rectification and slow kinetics. extending the linker up to 12 amino acids does nt change significantly the properties of kvsynth1 (not depicted), whereas trespassing this length results in a gradual disappearance of the properties, as shown by the construct with a 20amino acid linker, with a predominant instantaneous component and an increase in inward current (see also the corresponding i - v curves in fig. 4 c). 4 d, where we have plotted the degree of rectification (i+60mv / i100mv) as a function of linker length, for all measurable constructs. interestingly, rectification was not appreciably influenced by the amino acid composition because two constructs with a linker of the same length, 20 amino acids, but of different sequence (see fig. the length of the linker connecting the vsd to the pore affects the degree of rectification in kvsynth1 constructs. (a) expanded view of the sequences of the two sequences that where variably combined to form the kvsynth1 linker. (b) list of linkers that have been tested in kvsynth1, their amino acid (aa) length, and amino acid sequences (color coded as in a). (c) exemplary current traces recorded from kvsynth1 constructs with the 4, 6, and 20amino acid linkers and their corresponding i - v relationships (color coded). to compare currents from different constructs, the traces have been normalized to the current value recorded at + 60 mv and expressed in arbitrary units (a.u.). voltage protocol as in fig. 1 c. (d) the degree of current rectification (i+60mv / i100mv) is plotted as a function of linker length for all functional constructs. experimental data (with the exclusion of the value corresponding to the construct with a 4amino acid linker that loses the rectification) have been interpolated with a logistic function (black line) in which the lower asymptote was set to 1, corresponding to the value measured in kcv that lacks rectification (dotted line). the present data show that the coupling of ci - vsd to kcv is necessary and sufficient to transform a voltage - independent k channel into a kv - type outward rectifier. first, it shows unambiguously that the shallow voltage dependency of the s4 of ciona vsd is sufficient to gate a channel pore. the activation curves of kvsynth1 closely reflect the charge movement obtained with the isolated vsd in its wt and mutant form. hence, the voltage - dependent features of the sensor are fully transmitted to the gating of the channel pore. kvsynth1 exhibits the features of an outward rectifier, which conducts best at depolarizing voltages. this gain of function over the ohmic kcv conductance can be explained with a model according to which the coupling with vs closes the pore of kcv ; the movement of the vs then causes a slow and voltage - dependent opening at positive voltages. from the shallow voltage dependency of kvsynth1, it can be speculated that the movement of a single vsd is probably sufficient for opening of the channel. because the typical pore gating features of kcv are still maintained in kvsynth1, the movement of the vsd is most likely operating a cytosolic gate of the channel ; the latter is operated by salt bridge interactions at the cytosolic entrance of kcv (tayefeh., 2009). previous data implied that the pore and the voltage - sensor domains of kv channels presumably required a tight coevolution at their interacting surfaces to achieve voltage - dependent gating (long. in contrast, the present data show that a naive electromechanical coupling between an unrelated vsd and a pore is already sufficient for a voltage - dependent gating of the channel pore. the distinct coevolution between the sensor and the pore in modern kv channels is therefore more likely a means to a fine - tuning of channel gating and not a basic structural requirement. another remarkable finding is that the effective voltage control of the vsd over the pore is a function of the linker length. it is evident that the linker must be longer than 4 amino acids and performs best with a length between 6 and 12 amino acids. this is in the range of linker length in kv channels (long., 2005). efficient coupling can be achieved in kvsynth1 with completely unrelated linker sequences, such as the n terminus of kcv and the c terminus of ciona vsd (see fig. 3). even though we can not exclude that both linkers generate a similar fold, the data suggest that the length of the linker is more important for coupling of the two domains than its particular sequence. in this respect, the data are in good agreement with the idea that a rigid connection, which is presumably better provided by a short linker, favors the mechanical coupling between voltage sensor and pore (long., 2005 ; lee., third and lastly, the data show that a primitive channel with poor control on gating can acquire sophisticated voltage regulation via a naive fusion with a regulatory element from another source. this finding makes it likely that the appearance of kv channels indeed occurred as a result of a singular evolutionary step, as suggested previously (chanda and bezanilla, 2008).	the modular architecture of voltage - gated k+ (kv) channels suggests that they resulted from the fusion of a voltage - sensing domain (vsd) to a pore module. here, we show that the vsd of ciona intestinalis phosphatase (ci - vsp) fused to the viral channel kcv creates kvsynth1, a functional voltage - gated, outwardly rectifying k+ channel. kvsynth1 displays the summed features of its individual components : pore properties of kcv (selectivity and filter gating) and voltage dependence of ci - vsp (v1/2 = + 56 mv ; z of 1), including the depolarization - induced mode shift. the degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. this highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.
acute neurogenic pulmonary edema (npe) is an underdiagnosed yet a common clinical entity. presence of preoperative npe presents a dilemma to the neuroanesthetist due to the divergent goals of management of raised intracranial pressure and pulmonary edema and also the possible adverse interaction of the two conditions when they co - exist. we report a patient with acute obstructive hydrocephalus due to cerebellar metastatic lesion who presented with npe that resolved on placement of the ventriculoperitonial (vp) shunt. a woman, about 50year old, presented with progressive headache, holocranial and continuous type, associated with multiple episodes of vomiting and swaying gait of two months duration. her magnetic resonance imaging (mri) brain revealed fourth ventricular obstruction with obstructive hydrocephalus. as the patient was drowsy though responding to verbal commands and oriented, an emergency vp shunt was planned. she had an unremarkable past history with no previous history of tuberculosis or respiratory illness. she had a pulse rate of 65 per minute and her blood pressure was normal. chest x - ray showed slight haziness in the right lung suggestive of pulmonary edema- [figure 1 ]. the peripheral oxygen saturation (spo2) was around 84% and the arterial blood gases showed a pao2 53 mmhg. the spo2 increased to 92% on administration of 100% oxygen through the face mask of the anesthetic circuit. as there was no other cause for the respiratory dysfunction such as infection, aspiration, or previous respiratory illness, a diagnosis of npe was considered. anesthesia was induced with propofol 2 mg / kg and oral endotracheal intubation was facilitated with vecuronium 0.1 mg / kg body weight. anesthesia was maintained with air and oxygen mixture, adjusting the fio2 to maintain an arterial saturation of > 90%, and 1% isoflurane along with atracurium and fentanyl infusions. patient remained hemodynamically stable and mean arterial blood pressure (map) was maintained at 80 mmhg. after the cerebrospinal fluid (csf) drainage spo2 gradually increased to 100% and the fio2 could be reduced to 0.5.the neuromuscular blockade was reversed at the conclusion of surgery. the patient was awake, responding to verbal commands with normal motor power and tone. postoperatively, the pao2 was 253 mmhg on oxygen supplementation with a face mask delivering a fio2 of 0.4. preoperative chest radiograph of the patient showing pulmonary infiltrates suggestive of pulmonary edema immediate postoperative chest radiograph of the patient showing resolution of pulmonary infiltrates npe may be a consequence of a number of diverse central nervous system insults, including head trauma, brain stem lesions, rupture of intracranial aneurysm, excessive irrigation during endoscopic ventriculostomy, during angioplasty for vasospasm, and postictal period. diagnosis requires a high index of suspicion, especially in the case of respiratory decompensation in neurosurgical patients. the pathogenesis of npe probably involves overactivation of the sympathetic autonomic system with pulmonary hypertension, endothelial dysfunction, and increased vessel permeability. there are two theories on how it occurs : the blast theory and the permeability defect theory, with evidence in favor of both of them. the treatment is mainly supportive using mechanical ventilation and alpha - adrenergic blocking agents for managing increased pulmonary arterial pressure. it has been hypothesized that deep levels of anesthesia might protect against the development of npe due to a more pronounced inhibition of the hypothalamic, brainstem, and spinal vasoactive sympathetic centers. an insufficient anesthesia level may not be able to inhibit the sympathetic nervous system during an injury of the central nervous system and thus predispose to development of npe. therefore, maintenance of adequate depth of anesthesia and attenuation of neuroendocrine response to intubation are important. the use of peep in neurosurgical patients is limited by conflicting reports on its effect on intracranial pressure. the presence of raised intracranial pressure and the need to provide good brain relaxation for surgery may limit the application of peep. a high fio2 was sufficient to maintain optimal blood gases without the necessity for peep in this patient. the reduced lung compliance and high intrathoracic pressure during mechanical ventilation in the presence of pulmonary edema may also pose a problem for providing brain relaxation. the cerebrogenic autonomic and neurohumoral dysregulation due to intracranial hypertension may cause intraoperative hemodynamic dysfunction. a thorough understanding of the patho - physiological mechanisms behind the development of npe helps in the management of these patients, thus preventing further complications. npe after aneurismal sub - arachnoid haemorrage was shown to resolve after endovascular coiling. and npe in this patient resolved after ventriculoperitonial shunt. in conclusion, in patients with central nervous system pathology in respiratory distress, the possibility of diagnosis of npe must be considered and the inciting pathology should be deliberated. anesthetic management must be carefully titrated considering the divergent goals of npe and intracranial hypertension.	neurogenic pulmonary edema may be a less - recognized consequence of obstructive hydrocephalus. the authors report a patient with acute obstructive hydrocephalus due to cerebellar metastatic lesion, who presented with neurogenic pulmonary edema. the edema resolved on placement of the ventriculoperitonial shunt. this report addresses the importance of recognition of neurogenic pulmonary edema as a possible perioperative complication resulting from an increase in intracranial pressure and the issues involved with anesthetic management of co - existing neurogenic pulmonary edema and intracranial hypertension.
nowadays, the life expectancy of women has increased significantly in most countries, but in some countries, such as iran, healthy life expectancy has seen no meaningful increase. according to the report of world bank in 2007, healthy life expectancy for iranian women is 59 years, but the healthy life expectancy for women in iran s neighboring countries, such as oman, qatar, russia, saudi arabia, syria, turkey, and the united arabic emirates (uae), is reported to be between 63 and 65 years, and for the countries of ireland, norway, and sweden, it has been reported to be between 73 and 75 years. with increasing life expectancy, a major challenge appears to be about how to increase the quality of life and the healthy life years, while there is increase in the noncommunicable diseases, such as obesity, cardiovascular disease, cancer, diabetes, and other threats to the health and community development. it is expected that by 2020, the noncommunicable diseases would attribute to 73% mortality and 60% of the global burden of disease. on the other hand, these diseases are preventable by limiting the risk factors and modifying the lifestyle, including following healthy diets, doing sufficient exercise, and not drinking alcohol and smoking, so that 80% of cardiovascular disease, stroke, type 2 diabetes, and cancer cases can be prevented. also, women are faced with more and different events and challenges than their coeval men. middle - aged women are vulnerable to physiologic, psychosocial, and economic factors. women often have to do things that are beyond their time and capacities. family responsibilities, work, and housework, all are the demanded preferences of women that result in lack of time for considering their health issues. in addition, factors such as their understanding of health problems, health insurance, and access to health care, social factors (education, employment, wages, and marital status), and cultural and economic factors affect women s health, and are important in planning their health program. on the other hand, first experience of chronic diseases, such as high blood pressure, arthritis, heart disease, and diabetes, occurs in the middle age in women, and as a result of physiologic reasons due to the phenomenon of menopause, they are susceptible to changes that may affect their health. although are known the basic elements of a healthy lifestyle, why middle - aged women do not follow ? timmerman believes that the internal barriers are the thoughts and feelings that surround an individual and are the reasons that make it difficult to change the individual s behavior. these internal barriers include lack of time and motivation, lack of knowledge, enjoying bad behavior, indolence, tiredness, irritability, and no belief in the fact that behavior can be successfully changed. john and ziebland carried out a large 3-year follow - up study for considering the internal and external barriers to changing diet and physical activity of participants ; they recognized that 61% of barriers were internal barriers and 9% were external barriers. also, in another study, holgado. showed that in compliance with a healthy diet, the internal barriers, such as lack of time, are considered as more difficult barriers than the external barriers, such as costs. in a qualitative study by parvizi. focusing on the theoretical explanation of health from the viewpoint of women, sweetness and difficulty in maternal role, social and cultural factors affecting women s health, and family health were the main categories of health concepts expressed. in this study, women expressed that factors such as employment, education, and health awareness have an effect on their health. in the examination of barriers to women analyzed the relationship between the personal barriers, such as lack of interest, lack of awareness, and physical - psychological problems, and the exercise motivation of the individuals, and in this study also, lack of awareness was considered as the effective factor for insufficient exercise. researches have shown that the women who were successfully involved in the health - enhancing behaviors are more likely to engage in them in the present and future. unfortunately, the individual methods, such as healthy diet, exercise, and health responsibility, are not common among middle - aged women for reducing the risks of lifestyle disorders and they risk their health by adopting undesirable life patterns and health - destructive behaviors during their life. one of the ways by which the health status of different classes in the society is improved is by designing appropriate programs for this group according to their special needs. qualitative researches help us to understand the human phenomena by emphasizing on their social context. also, this method would be applied in situations with limited understanding of the phenomenon in which the aim of the study is generating new ideas that can help to modify a topic and evidence - structure - based planning and designing. in the developing countries, such as iran, as a muslim and developing country with its special c ultural and social background, and different health status indicators compared to other countries, and its demographic transition and aging perspective in the coming years, we need to do independent research in the women s health area in order to attain the strategies for universal health planning in accordance with our special cultural and social conditions. so, the researchers carried out a comprehensive study titled health promoting behaviors of middle - aged women and exploring effective factors in order to offering strategies for their health management through a sequential mixed methods approach to provide strategies for the infrastructure of a proper and sociable program to promote the health of iranian middle - aged women by hearing their voice. in this study, the motivation and effective internal barriers on healthy lifestyle of middle - aged women had a significant role in the determinants of women health promotion, and it included the major part of women s expression. this was a qualitative study based on content analysis ; and it was part of a wider study, which used a sequential mixed - method design. qualitative researchers rely on various qualitative methods to explore the behaviors, attitudes, and experiences of people within the context of their lives. thus, we adopted a qualitative approach to gain an in - depth understanding of internal motivations and barriers affecting the healthy lifestyle of middle - aged women, and used data extracted from their statements to propose strategies for promoting their health. so, as part of the quantitative study, to examine health - promoting behaviors in 483 middle - aged women and then to determine internal motivations and barriers affecting the healthy lifestyle, we conducted in - depth, semi - structured interviews with women at the best and worst extremes of the health behavior spectrum as the community of study, according to scores given to participants in the earlier study. finally, 21 women between 40 and 60 years of age were selected by using purposeful sampling. we strived to achieve maximum variation in terms of age (40 - 60 years), education, employment, number of children, marital status, and socioeconomic background. using the questionnaires completed in the quantitative phase of the study, we selected suitable candidates and contacted them by telephone before meeting them face - to - face ; the aim of the study was explained and interviews were arranged at a convenient time and location for the participants (in a health center, at home, at workplace, or in a park). the study relied on in - depth interviews as the main tool for data collection. the interviews lasted 20 - 60 min and were digitally recorded using an mp3 voice recorder. the interviewees personal details were recorded immediately after each interview and field notes were taken of the verbal / nonverbal interactions during the interview ; these would complement other data. primary codes were assigned to each sentence / keyword identified as a unit of meaning. main categories emerged from the assimilation of primary categories ; and abstraction was applied at each stage to yield fewer categories. the following measures were taken to increase the credibility and validity of findings : (1) in - depth interviews with selected subjects were conducted at different times and locations, (2) transcripts were read several times to achieve data immersion, and (3) subjects were selected from across the community spectrum to achieve maximum diversity of age and socioeconomic / cultural status. member checking and peer examination were used to ensure the objectivity, transferability, and reliability of findings. external evaluators were asked to examine the codes / categories and report any inconsistency that might exist between the extracted categories and the interviewees remarks ; consensus about the accuracy of codes was more than 90%. this study was approved by the medical ethics committee of isfahan university of medical sciences. the study was conducted in the central iranian city of yazd, a city of nearly half a million population and well known for its highly religious and traditional culture. for adopting a healthy lifestyle, the analysis of data revealed five main themes related to the middle - aged women s internal barriers and motivation, which are as follows : the interviewees characteristics (n = 21) women s knowledge of health - promoting behaviorsimportance of health and healthy behavior for womenaffliction or fear of affliction to chronic disease and its consequencesresponsibilities of women in the family and societyskills of life management by women women s knowledge of health - promoting behaviors importance of health and healthy behavior for women affliction or fear of affliction to chronic disease and its consequences responsibilities of women in the family and society skills of life management by women one of the extracted concepts was the knowledge and awareness of women in the field of nutrition, physical activity, and health responsibility. nutritional knowledge means having information and awareness about the nutritional needs of women in this age group, familiarity with different food groups, their properties, importance of each food group for providing health, and body needs to each one of the food groups. if women had good nutritional knowledge, health - promoting behaviors related to nutrition would be observed in them and vice versa. one of the participants said : i use vegetables and dairy products or at least i try to search and find my used protein in this rows by using grains and cereals and so on and determine my diet on this basis, namely, using a complete plate of salads with different vegetables, which included different grains and vegetables and surely there is dairy in my diet. also, another participant who did not have any proper health - promoting behaviors stated : i do not know what to do and what do eat, i do not have access to anywhere, i know that exercise, proper food, and fruit are good for health but i do not know what to do. knowledge and awareness about the physical exercise and its effects on physical and psychological health is another area of healthy behavior. although most people had more knowledge about the positive effects of exercise on the mental health and human spirit, many of them had not much information about the effects of exercise on the health of cardiovascular and musculoskeletal systems and they recognized the most important effect of exercise as preventing obesity and improving physical fitness. i do exercise and go to fitness classes for almost 20 years, but it is for about 6 years that i exercise continuously because i have found very good effects of it on my mood and body. most of the women considered their daily living activities and household chores and home affairs as the physical activities, and the special programs, such as routine walking and recreational or regular physical activity, were followed by a few of them. most of the women did not have any awareness about screening of breast and cervical cancer. for example, when a participant was asked if she underwent the breast and gynecologic examinations, she replied : no, i never had a problem or pain and i did nt do that. most of the studied women had no proper understanding of preventive methods of disease, screening, and early detection of diseases, and they thought that for referring to doctor and controlling the health, one needs to have clinical symptoms and health problems. but the women themselves confessed that they need to be trained for health - promoting behaviors and stated that spreading awareness and information through the media, offering training packages to homes, and providing training programs in women s weekly religious meetings are the proper training methods. giving importance to health and considering it as a value in life is considered as another effective factor for performing the health - promoting behaviors, but most people do not care to perform health - promoting behaviors even when they know its importance. i know i must do something for my health but in fact, i did nt care about them till now. and a participant who was asked whether she had any knowledge about this matter replied : why not ; nowadays the media gives information about this matter but as i did nt have any special problem and i was healthy, so i did nt care to do it. till now, i never thought of exercise and in reality one does nt think about her aging until a part of her body starts to pain, we feel that only the others are aged. in this study, many of the participants were not aware of the importance of the role of physical factors (proper diet and physical activity and health responsibility) ; especially the young women had an unrealistic optimism that they would never face any important physical problem and this group was so indifferent to the health - promoting behaviors. chronic disease is a long - term disease that can cause changes in the body and also limits the bodily functions. chronic diseases usually need long - term treatment and have difficult recovery process. in some cases, the disease is incurable and there is not any certainty and clear treatment for it. one has to endure it for a long period of his life or until the end of his life, and cope with its complications. so, affliction or fear of affliction to chronic disease in some persons, observing the disease in the families and relations, and also observing the side effects of these diseases caused the women pay more attention to performing the health - promoting behaviors. i got dizziness in my 45 years ; i was tested and found to have high blood fat. since then, i am so careful with my diet. existence of a disease in the participants was another factor that led to health behaviors. also, considering aging, chronic diseases, and avoiding health complications in some people led to performing the desired behavior. i do blood test once a year ; i could nt say that i m afraid but if i could just do something with a little prevention, i do not want my kids to fall in trouble and i do nt want to have their care most participants got more precise in performing the healthy behaviors by observing the illness and its consequences in their surroundings. women had different ideas about cancer ; in some of them the fear of cancer made them pay more attention to screening and in some of them it prevented them from this behavior. as i am afraid of breast cancer, i have visited doctor and went twice to do mammography. while the other participant stated : i know that the breast examination and mammography is so important but i do nt follow them, i am afraid of mammography ; i am afraid that some dangerous thing is diagnosed in my breast ; now i am relaxed by knowing nothing. the rate of awareness and knowledge about the disease discovering methods were effective on the women s attitude about using them. on the other hand, disease, especially heart disease and osteoarthritis, sometimes i decide to walk at least but i leave it (because of) my back and knee pain. the prevalence of chronic diseases in middle - aged and elderly women participants was a motivating factor for considering a proper diet, avoiding sweets and fatty foods, and regular laboratory evaluation. all the responsibilities inside and outside home interfered with performing the health behaviors and with reference to them, women said as being too busy, leading to lack of time, and overexpectations. despite having the knowledge, motivation, and attention to disease prevention, a number of middle - aged women had several responsibilities in their life that made them not to care for their health status duly. as an example, they said that having several children in each age group and supervising them do not leave any opportunity for them to care for themselves. i have more children and have to care them, i am busy, i want to care about myself to some extent but i ca nt. outside working along with the household duties and the responsibilities of the children are considered as a barrier for women in caring for themselves : i do not exercise at all because i do not have any time, i must work at home and also do the outside home tasks. another employed participant said : i ca nt (be) present in the fitness classes at evenings as it wastes my time ; children s extra classes in school and their extra language courses requires that i myself have to take and bring them which interferes with my program and i ca nt do it in the morning as i am employed and have to work. the ideas of the participants showed that the women with better ability of controlling, managing, planning, and decision making in their life affairs can perform the health - promoting behaviors better than others. on the other hand, the passive people, without managing power and with poor decision making, were weaker than others in health - promoting behaviors and we referred to these properties as the life management skills by women. the skills that they were required to have included decision - making, planning, and management skills. all the participants referred to the role of will and power to make decisions in performing the health behaviors under all circumstances. some of the participants had very high strength and felt confident to perform healthy behaviors, for example, a participant who was asked about what caused her have a regular walking program replied : my will ; also i am planning for it, if you are a precise person, you can regulate to do it regardless of any problem. some of the participants expressed low self - confidence in their ability to have a healthy attitude and their commitment to the continuation of that behavior. in this relation, ambition is the first required thing for having a regular exercise program, namely, one must have a strong will, for example, i must have the ambition to say myself go and do your walking until the weather is nice and then do your rest (of the) tasks but i never do so. in addition to these, almost all the participants recognized the important role of planning in the health promotion behaviors and believed that proper planning is required for having a healthy life. a participant who was asked about what helped her to have a regular program replied : i think where there is a will there is a way, every thing must be planned, if so, everyone can reach to everything he wants, in such a manner he can care for himself, his family, and his parents. in comparison with the other skills, management skills, particularly time management, income management, and family costs, are considered as the most effective factors on the women s health - promoting behaviors. in preparing the foods, i consider my health and the family members health, i myself decide what must be purchased, my son or husband does the purchase but i tell them what they must buy ; for instance, when they come home at night they must always buy a packet of milk. the special role of health and prevention costs in the family expenses is not yet recognized and most of the participants avoid spending in this field and do not consider it as a necessary matter, as a participant says : maybe i am guilty, i say to myself that instead of going to laboratory and spending much money i prefer to spend that money in my life requirements ; i know, i make a mistake. according to this study, lack of awareness and not paying attention to healthy lifestyle, lack of time, and multiple roles were the barriers of health - promotion behaviors in the participants. also, having a noncommunicable disease or fear of getting affected by it and empowerment in family management were the internal motivators of a healthy behavior. one of the extracted concepts was the knowledge and awareness of women in the field of nutrition, physical activity, and health responsibility. nutritional knowledge means having information and awareness about the nutritional needs of women in this age group, familiarity with different food groups, their properties, importance of each food group for providing health, and body needs to each one of the food groups. if women had good nutritional knowledge, health - promoting behaviors related to nutrition would be observed in them and vice versa. one of the participants said : i use vegetables and dairy products or at least i try to search and find my used protein in this rows by using grains and cereals and so on and determine my diet on this basis, namely, using a complete plate of salads with different vegetables, which included different grains and vegetables and surely there is dairy in my diet. also, another participant who did not have any proper health - promoting behaviors stated : i do not know what to do and what do eat, i do not have access to anywhere, i know that exercise, proper food, and fruit are good for health but i do not know what to do. knowledge and awareness about the physical exercise and its effects on physical and psychological health is another area of healthy behavior. although most people had more knowledge about the positive effects of exercise on the mental health and human spirit, many of them had not much information about the effects of exercise on the health of cardiovascular and musculoskeletal systems and they recognized the most important effect of exercise as preventing obesity and improving physical fitness. i do exercise and go to fitness classes for almost 20 years, but it is for about 6 years that i exercise continuously because i have found very good effects of it on my mood and body. most of the women considered their daily living activities and household chores and home affairs as the physical activities, and the special programs, such as routine walking and recreational or regular physical activity, were followed by a few of them. most of the women did not have any awareness about screening of breast and cervical cancer. for example, when a participant was asked if she underwent the breast and gynecologic examinations, she replied : no, i never had a problem or pain and i did nt do that. most of the studied women had no proper understanding of preventive methods of disease, screening, and early detection of diseases, and they thought that for referring to doctor and controlling the health, one needs to have clinical symptoms and health problems. but the women themselves confessed that they need to be trained for health - promoting behaviors and stated that spreading awareness and information through the media, offering training packages to homes, and providing training programs in women s weekly religious meetings are the proper training methods. giving importance to health and considering it as a value in life is considered as another effective factor for performing the health - promoting behaviors, but most people do not care to perform health - promoting behaviors even when they know its importance. i know i must do something for my health but in fact, i did nt care about them till now. and a participant who was asked whether she had any knowledge about this matter replied : why not ; nowadays the media gives information about this matter but as i did nt have any special problem and i was healthy, so i did nt care to do it. till now, i never thought of exercise and in reality one does nt think about her aging until a part of her body starts to pain, we feel that only the others are aged. in this study, many of the participants were not aware of the importance of the role of physical factors (proper diet and physical activity and health responsibility) ; especially the young women had an unrealistic optimism that they would never face any important physical problem and this group was so indifferent to the health - promoting behaviors. chronic disease is a long - term disease that can cause changes in the body and also limits the bodily functions. chronic diseases usually need long - term treatment and have difficult recovery process. in some cases, the disease is incurable and there is not any certainty and clear treatment for it. one has to endure it for a long period of his life or until the end of his life, and cope with its complications. so, affliction or fear of affliction to chronic disease in some persons, observing the disease in the families and relations, and also observing the side effects of these diseases caused the women pay more attention to performing the health - promoting behaviors. i got dizziness in my 45 years ; i was tested and found to have high blood fat. since then, i am so careful with my diet. existence of a disease in the participants was another factor that led to health behaviors. also, considering aging, chronic diseases, and avoiding health complications in some people led to performing the desired behavior. i do blood test once a year ; i could nt say that i m afraid but if i could just do something with a little prevention, i do not want my kids to fall in trouble and i do nt want to have their care most participants got more precise in performing the healthy behaviors by observing the illness and its consequences in their surroundings. women had different ideas about cancer ; in some of them the fear of cancer made them pay more attention to screening and in some of them it prevented them from this behavior. as i am afraid of breast cancer, i have visited doctor and went twice to do mammography. while the other participant stated : i know that the breast examination and mammography is so important but i do nt follow them, i am afraid of mammography ; i am afraid that some dangerous thing is diagnosed in my breast ; now i am relaxed by knowing nothing. the rate of awareness and knowledge about the disease discovering methods were effective on the women s attitude about using them. on the other hand, disease, especially heart disease and osteoarthritis, sometimes i decide to walk at least but i leave it (because of) my back and knee pain. the prevalence of chronic diseases in middle - aged and elderly women participants was a motivating factor for considering a proper diet, avoiding sweets and fatty foods, and regular laboratory evaluation. all the responsibilities inside and outside home interfered with performing the health behaviors and with reference to them, women said as being too busy, leading to lack of time, and overexpectations. despite having the knowledge, motivation, and attention to disease prevention, a number of middle - aged women had several responsibilities in their life that made them not to care for their health status duly. as an example, they said that having several children in each age group and supervising them do not leave any opportunity for them to care for themselves. i have more children and have to care them, i am busy, i want to care about myself to some extent but i ca nt. outside working along with the household duties and the responsibilities of the children are considered as a barrier for women in caring for themselves : i do not exercise at all because i do not have any time, i must work at home and also do the outside home tasks. another employed participant said : i ca nt (be) present in the fitness classes at evenings as it wastes my time ; children s extra classes in school and their extra language courses requires that i myself have to take and bring them which interferes with my program and i ca nt do it in the morning as i am employed and have to work. the ideas of the participants showed that the women with better ability of controlling, managing, planning, and decision making in their life affairs can perform the health - promoting behaviors better than others. on the other hand, the passive people, without managing power and with poor decision making, were weaker than others in health - promoting behaviors and we referred to these properties as the life management skills by women. the skills that they were required to have included decision - making, planning, and management skills. all the participants referred to the role of will and power to make decisions in performing the health behaviors under all circumstances. some of the participants had very high strength and felt confident to perform healthy behaviors, for example, a participant who was asked about what caused her have a regular walking program replied : my will ; also i am planning for it, if you are a precise person, you can regulate to do it regardless of any problem. some of the participants expressed low self - confidence in their ability to have a healthy attitude and their commitment to the continuation of that behavior. in this relation, ambition is the first required thing for having a regular exercise program, namely, one must have a strong will, for example, i must have the ambition to say myself go and do your walking until the weather is nice and then do your rest (of the) tasks but i never do so. in addition to these, almost all the participants recognized the important role of planning in the health promotion behaviors and believed that proper planning is required for having a healthy life. a participant who was asked about what helped her to have a regular program replied : i think where there is a will there is a way, every thing must be planned, if so, everyone can reach to everything he wants, in such a manner he can care for himself, his family, and his parents. in comparison with the other skills, management skills, particularly time management, income management, and family costs, are considered as the most effective factors on the women s health - promoting behaviors. in preparing the foods, i consider my health and the family members health, i myself decide what must be purchased, my son or husband does the purchase but i tell them what they must buy ; for instance, when they come home at night they must always buy a packet of milk. the special role of health and prevention costs in the family expenses is not yet recognized and most of the participants avoid spending in this field and do not consider it as a necessary matter, as a participant says : maybe i am guilty, i say to myself that instead of going to laboratory and spending much money i prefer to spend that money in my life requirements ; i know, i make a mistake. according to this study, lack of awareness and not paying attention to healthy lifestyle, lack of time, and multiple roles were the barriers of health - promotion behaviors in the participants. also, having a noncommunicable disease or fear of getting affected by it and empowerment in family management were the internal motivators of a healthy behavior. the obtained results of the present study show that having sufficient knowledge and information for performing healthy behaviors and giving importance to healthy behavior, in the first stage, is a necessary factor for making a healthy lifestyle. people with better nutritional information gave more importance to their health and were aware of the effects of poor nutrition and lack of exercise on their health and also, they had better nutritional behaviors and physical activities compared with others. also, awareness of the disease and screening methods and their importance in early diagnosis of disease were considered as an effective factor in health responsibility and performing a healthy behavior. in providing global strategies for improving the proper nutrition and physical activity in order to control noncommunicable diseases, world health organization has propounded increasing overall awareness and understanding about the effects of diet and physical activity on health and the positive impact of preventive interventions for communities as one of the presented aims and also considers providing accurate and balanced information about the food to the consumers as executive instructions in order for people to have better information and healthy choices. according to the study by brug., examination of the social - psychological determinants in fruit and vegetable usage in adults, six different categories were generally introduced as the most important factors in fruit and vegetable usage in adults, in which the understanding of healthy results arising from fruit and vegetable usage, lack of awareness of healthy results of fruit and vegetable usage, and also lack of awareness of recommended rates for usage are recognized as the most important determinants. in a study carried out to examine the effective factors on the healthy food usage, mcgee. through a qualitative research found that the understanding of people about a healthy diet is influenced by personal and external factors, including the health, concerns, family effects, need, and access to nutritional information, and the participations of this study tended to be trained in the fields of healthy nutrition, food preparing skills, and controlling the food portions. the results of another study showed that people with lower practical knowledge about nutrition had lower consumption of fruits, vegetables, and water, and higher consumption of sausages. although there is a general agreement that nutrition knowledge is an essential factor, it is not a sufficient factor to change the feeding behavior. also, in the field of preventive care and health care, researchers believe that the sufficient knowledge will be effective. several studies have shown that having sufficient information and awareness of symptoms of disease and methods of screening for cancer is one of the important stimuli, and lack of awareness about it is considered as a major obstacle for performing these behaviors.[2126 ] in a qualitative study performed by beeker. for the examination the obstacles of screening for colon cancer in middle - aged men and women, older than 50 years, it was found that the poor information about colon cancer, the potential benefits of screening, little reporting from the doctors or mass media, negative attitude to the screening procedure for colon cancer, and the fear of cancer were some of the barriers to the screening. giving importance to the health was another concept, which appeared from the women s ideas ; also, wang. found that the understanding of healthy behaviors has a positive effect on using healthy foods, such as fruits and vegetables, and also it has a negative effect on using unhealthy foods, such as the sweets and fats. in a study by naderian., there was a significant relationship between the internal barriers (lack of interest, lack of awareness, and physical - mental problems) and the rate of motivation to exercise and also, in this study, lack of awareness was considered as an effective barrier to the shortage of physical activities. but in comparison with the other barriers, lack of awareness is considered as a minimal barrier to physical activities. training people in health centers, the use of mass media, internet, and so on, are considered as strategies to increase knowledge, motivation, and importance of women s health promotion. according to researches, health status is a major stimulus for changing the eating habits or physical activity. some people choose a desired nutritional behavior or do physical activities because of suffering from a chronic disease, such as diabetes, hypertension, or hypercholesterolemia, and they believe that are required to follow the desired behavior for the prevention of these diseases ; also, most of the adults are more likely to make changes in their diet only after diagnosis of a disease. also, mcgee. found that health problems are strong incentives, which are effective in making changes in the food consumption patterns, so that participants believed that diseases, such as hypertension, diabetes, kidney disease, and cardiovascular disease, are effective in influencing their food choices. but on the other hand, osteoporosis, arthritis disease, or heart disease in women were considered as a barrier to participation in physical activity ; in the study by rimmer., physical pain and problems (63/6%) was considered as the most important personal barrier to continue the physical activities. expressed the diseases associated with diabetes as a reason for stopping the regular exercise program. also, the research society of american retirees showed that lack of time, fatigue, health problems, such as arthritis, chronic pain, injury, disability, health and heart problems, are considered as the major barriers to physical activities in the middle - aged women. in this study, women s family responsibilities and their busy schedule at home are introduced as another effective factor for not having a healthy lifestyle. although nowadays women form a big part of workforce, this matter has not changed the expectations from women. women still have a major responsibility of caring family, children, and other family members, in addition to their responsibilities outside the home. this large complex set of responsibilities leaves them with little time for self - care. so, women mentioned lack of time as a barrier to exercise and preparing fresh food. a study in america for examination of the barriers and facilitators of fruit and vegetable consumption among different ethnic communities showed lack of time as the first personal barrier in providing and preparing fresh fruits and foods as they are time - consuming tasks, and other studies have obtained similar results too. the employed women who also must do housekeeping duties face a lot of stress, which can cause negative effects on their health status if they were not supported by the society and family. most of the studies carried out by the researchers consider the lack of time issue for women and show that the time stress and lack of time are the barriers for women that prevent them from engaging in their healthy affairs. so, the multiple responsibilities of women in doing the duties of out of home as an emploee, engaging in household tasks, training children, caring for children and elderly parents, and their other responsibilities at home make them forget to take care of themselves. in addition, women often because of feeling motherly if they prefer their needs to family needs, it will be course feeling selfishness and role conflict. the next effective factor, namely, management and planning in the family, is also important. management at home means managing or optimizing the home affairs at all levels ; meanwhile, women have always played a key role, and a mother, as a manager, must acquire the necessary ability for executing the health - economy issues, improving the internal situation of the family, promoting the relationships between family members, creating economic savings, training children, planning for a healthy diet, and so on. in a qualitative study by parvizi., they introduced a healthy family as a central variable and a precondition for the health of women and also, they introduced the training of life skills, problem solving, time management, and house controlling as a strategy for strengthening of family and women s health. health care providers with a mission and a great role in preserving and promoting the health of women can consider both the women and families as a target group in health - social planning and also in training the life skills as the basis of health trainings that they plan for women. although the findings of the qualitative research can not be generalized to other communities, there are some ways for providing acceptability and objectivity for data and increasing the accuracy of used data, which can help in applying these results for similar communities. by examination of the main categories emerged in this study, we can conclude that the personal abilities of the participants in the health - promoting behaviors are considered as an important determinant factor in health. results of the study show that women were empowered by having skills of family management, planning for life, and decision making for health - promoting behaviors. by considering the fact that the present health system strategy for health promoting of the middle - aged women could not have an all - out support, it is suggested that for designing program for middle -aged women s health, life skills and home management training have to be considered synchronous with healthy lifestyle training.	background : a healthy lifestyle is one of the basic health - promotion strategies. several factors are involved in shaping health - promotion behaviors. the internal barriers are the opinion and feelings that surround the individual and are the reasons that complicate the change of behavior. the aim of this study was to identify internal motivations and barriers effective on the healthy lifestyle in middle - aged iranian women.materials and methods : this was a qualitative study based on content analysis of in - depth semi - structured interviews with 21 middle - aged women in the city of yazd, who were selected using purposeful sampling approach. the interviews continued until data saturation was reached ; and the interviews were audio - recorded and transcribed exactly. the transcripts were analyzed.results:five main themes emerged from the analysis of the interviews : women s knowledge of health - promoting behaviors, importance of health and healthy behavior of women, affliction or fear of affliction to chronic disease and its consequences, responsibilities of women in the family and society, and skills of life management in women.conclusion:the findings suggest that empowering individual participants in health promotion is the most important factor determining their health. thus, designing appropriate programs for education and empowerment of people is essential to promoting health. health policy makers, with knowledge of these factors, can design comprehensive, socialization programs to promote women s health.
despite ghana s progressive abortion law, one of the most liberal in sub - saharan africa, mortality caused by unsafe abortion remains a matter of concern. the country s maternal mortality ratio (mmr) in 2010 was an estimated 350 maternal deaths per 100 000 live births (world health organization. 2012), compared with an average mmr of 240 in the developing world. among the biggest contributors to maternal mortality in the country are complications of unsafe abortions (ghana statistical service. estimates from the 2007 ghana maternal health survey suggest a national abortion rate of 15 abortions per 1000 women of reproductive age (1544). further, 40% of abortions are performed by untrained providers ; lack of training increases the risk of unsafe abortion and, therefore, of danger to a woman s life. evidence suggests that many health care providers are unaware of the abortion law (lithur 2004 ; ipas 2008). studies show that substantial proportions of providers are either unaware of all allowable conditions or believe that it is illegal (morhe. 2007 ; many feel that providing abortions conflicts with their religious values, and view women seeking an abortion with suspicion (aboagye. lack of knowledge of the law, coupled with social and religious stigma, drives the practice underground, resulting in clandestine procedures from untrained providers or attempts at self - inducing an abortion (hill. further, while ghana allows doctors and various non - doctors to provide an abortion, many non - doctors have no clinical knowledge of how to provide one (hessini. 2007). in 2003, the ghanaian government introduced changes in its reproductive health policy, and issued guidelines for the provision of comprehensive abortion care services (cac), within the limits of the law (ghana health service 2005). to ensure the full implementation of this policy, in 2006, the ministry of health, in partnership with a consortium of international health organizations, including ipas, engender health, marie stopes international (msi), the population council, and willows foundation, launched the programme reducing maternal mortality and morbidity (r3 m). the programme, aimed largely at health care providers, sought to increase access to cac to reduce morbidity and mortality caused by unsafe abortion, and to widen access to family planning services to reduce the unwanted pregnancies that lead to abortions in the first place (aboagye. the r3 m programme was initiated in three regions accra, ashanti, and eastern and within these regions, a total of seven districts were chosen (table a1). the timeframe for the programme s first phase was between 2006 and 2008, which was extended to 2009. phase ii was implemented between january 2010 and december 2011, and two additional districts in each r3 m region were added during this time. the consortium provides a mutually reinforcing basket of services to providers, communities and facilities, such as training in abortion techniques and contraceptive services, sensitizing the community and health care providers to client needs, and providing equipment and products to facilities (table a2 lists selected facility level interventions). for instance ipas and the ghana ministry of health have the task of training the providers in cac in the public health facilities, while msi focuses on the private health facilities. engender health provides training on contraceptive counselling and services, while the willows foundation and population council focus on educating the communities, and provide guidance to the programme. in this article, we use a quasi - experimental approach to determine whether the r3 m programme has made a difference in the provision of safe abortion services and postabortion care (pac) in facilities. since the study began in 2011, and the intervention was first implemented in 2006, it was impossible to conduct surveys directly before and after the programme was implemented. the quasi - experimental study design used in this article approximates this design by comparing provision of safe abortion services and pac for the facilities exposed to the programme with those facilities that were not a part of the programme. additionally, we examine the role that providers knowledge of the law, and their attitudes play in influencing the two outcomes, specifically for those providers who have clinical knowledge of abortion provision. the data come from a primary survey administered in 20112012 in selected health facilities to providers of obstetric and gynaecologic services. the survey was designed to compare the attributes of providers who were exposed to the programme with those of providers who were not. the data were collected through face - to - face interviews using a structured questionnaire that sought information on a range of topics, including background information of the respondents and of the facility they practised in ; and respondents training, experience and attitudes on a range of issues. we collected data from 457 providers, who were legally eligible to provide abortions, in 166 facilities that had the capacity to provide some gynaecological services. of them, 116 were doctors, while 341 were non - doctor health care providers, including midwives, nurses and medical assistants. while the sample seems skewed towards mid - level providers, in ghana, the proportion of doctors to other providers is relatively small. we did attempt to over - sample doctors ; however, as an estimated 90% of ghana s doctors live in accra (in greater accra) and kumasi (in ashanti) (brookman - amissah 2004), most of the providers from the other regions are perforce mid - level providers. since the study design was explicitly quasi - experimental, with treatment and control groups, our sample has three analysis groups : one treatment group and two control groups. the treatment group consists of 197 providers in the 64 health facilities located in the seven districts that participated in the first phase of the r3 m programme. the first control group of providers not exposed to the programme consists of a sample of 148 providers drawn from 58 facilities in 17 districts in the same three regions as the treatment group (ashanti, eastern and greater accra). of these 17 districts, 13 were not included in the first phase of the r3 m programme. although the remaining four districts were part of the first phase of r3 m, we selected only facilities located in the 13 districts that did not participate in the first phase of the programme. the second control group that was not exposed to the treatment consists of 112 providers from 44 facilities located in seven districts in the brong ahafo region, which was not part of the r3 m programme. these 44 facilities were chosen because they are located outside of the three regions whose facilities participated in the r3 m programme, and thus would have minimal exposure to it. thus, the rationale for having two control groups [both will be called non - r3 m (n = 260) ] is that non - participating but nearby facilities may still indirectly benefit from geographic proximity. we assume that the second, more distant control group will be much less likely to benefit from the r3 m programme than the first control group. although providers in all non - r3 m facilities may also have been exposed to some cac - related interventions through the programmes of the government and other organizations, these activities would not be as intense as those in the r3 m districts. the main hypothesis this article thus tests is that providers in r3 m districts are more likely than providers in non - r3 m districts to provide safe abortion services and pac. the second hypothesis we test is that providers who have clinical knowledge may still be reluctant to offer it because of unfavourable attitudes and lack of knowledge of the law. we used propensity score analysis because of its effectiveness in simulating an experiment and measuring treatment impacts in non - experimental studies, especially post - test only studies such as this one (jsi research and training institute 2007 ; stuart 2010). the main feature of the technique is that it removes selection bias by balancing the sample on the characteristics that potentially influenced the selection of cases into the treatment group. compared with non - r3 m districts, the districts chosen for the programme were more urbanized, and had more secondary and tertiary facilities, better qualified doctors, and more private facilities. since this situation affected the selection of providers into the programme, we balanced our sample on the following provider - level variables : the type of facility they work in (primary, secondary or tertiary) ; whether the facility is in an urban or rural area ; whether it is in the private or public sector ; their total number of years of practice (< 15 years or 15 years) ; and by medical background (doctors or non - doctors). balancing / adjusting for selection bias was done using a logit model, whose estimates were used to obtain the propensity scores (dehejia and wahba 2002) (table 1). table 1characteristics of r3 m providers compared with non - r3 m providers, by variables used in balancing the sample and obtaining propensity scores, ghana 20112012variablesr3 m facilitiesnon - r3 m facilitiesn% distn% distdemographic distribution, by facility in which they workprovider works in urban or rural setting urban17186.818972.7 rural2613.27127.3 total197100.0260100.0ownership of facility in which provider works public17387.819273.9 private2412.26826.2 total197100.0260100.0level of facility where provider works primary7437.612849.2 secondary / tertiary12362.413250.8 total197100.0260100.0demographic distribution, by provider 's own characteristicsprofessional experience below 15 years11055.813551.9 15 years or more8744.212548.1 total197100.0260100.0provider type doctors (including ob / gyns)5930.05721.9 non - doctors (including midwives)13870.120378.1 total197100.0260100.0notes : all data are unweighted. all estimates for this sample are from information that was current at the time of the survey. total n = 457.p < 0.001, p < 0.05, p < 0.1. characteristics of r3 m providers compared with non - r3 m providers, by variables used in balancing the sample and obtaining propensity scores, ghana 20112012 notes : all data are unweighted. all estimates for this sample are from information that was current at the time of the survey. total n = 457. p < 0.001, p < 0.05, p < 0.1. propensity scores are the predicted probabilities [e^(x) ] that are generated from this model. these scores estimate the probability of each case receiving the treatment (i.e. being in the r3 m programme). we then used propensity score weighting (psw) to estimate the effect of the treatment on the two outcomes : provision of safe abortion services and of pac (imbens and wooldridge 2009). in the psw model, the inverse of the propensity scores are used as weights in an analysis, to estimate the average treatment effect on providers who actually received the treatment, i.e. participated in the r3 m programme (att i.e. the average treatment effect on the treated) (guo and fraser 2010 ; dugoff. 2014). we also estimate the potential average treatment effect (ate) on all providers, including those in the control groups, had they hypothetically been exposed to the programme. for att models, the weights are defined as : (w, x)=w+(1w)e^(x)1e^(x) for ate models, the weights are defined as : (w, x)=we^(x)+1w1e^(x) where w is the indicator for the treatment, and is equal to 1 for those respondents who were exposed to the treatment, and is equal to 0 for those who were not. once the weights had been generated, we checked to see if the selection bias had been removed by performing bivariate tests against the treatment variable on all the variables used to remove selection. since the treatment indicator was non - significant in all these models, we concluded that the selection bias had been successfully removed, and our subsequent models would be balanced (dehejia and wahba 2002). except for the variable on type of provider (doctors or non - doctors) the variables included as controls in the multivariate model are different from the variables included in the selection model. this is by design, and is recommended as best practice for psw models (freedman and berk 2008). we used the propensity score weights in logistic regressions to analyse the effect of the treatment on the two outcome variables. further, because we have nested data (providers nested within facilities), we used robust standard errors to estimate significance, by specifying the facilities as a cluster variable. the outcome variables measure whether the respondent was providing safe abortion services or pac at the time of the survey. the main explanatory variable is whether the provider was exposed to the r3 m treatment in the first phase or not, and it captures the training and benefits given to facilities and providers in the first phase, including training in abortion techniques. the multivariate analyses were conducted for three samples : r3 m providers and all non - r3 m providers (both control groups combined ; sample 1) ; r3 m providers and non - r3 m providers in the regions of accra, ashanti and eastern (sample 2) ; and r3 m providers and non - r3 m providers in the region of brong ahafo (sample 3). although these models test the association between the two outcomes and lack of knowledge of law and abortion attitudes ; in order to gauge the extent to which these two variables act as barriers to service provision, we separately analysed both outcomes for a sub - sample of those who reported having clinical knowledge of abortion provision. that is, they knew at least one of dilation and curettage [d&c ], manual or electric vacuum aspiration [mva / eva ], or medication abortion protocols. compared with the previous models that analyse all potential providers, the focus here is on respondents who are actually currently capable of providing service. this allows us to examine if despite knowing abortion techniques, respondents feel constrained from providing one. due to small cell sizes, this analysis was restricted to the overall sample, and will be referred to as sample 4. the construction of the outcome and explanatory variables, and of the variables used to balance the sample, is described in table a3. table 2 has the percentage distribution of the full sample along selected parameters, and it shows that more providers in the treatment (r3 m) facilities have attributes favourable to safe abortion and pac provision compared with non - r3 m providers. greater proportions of providers exposed to the programme provide safe abortion services (54 vs 13%) and pac (66 vs 33%). greater proportions of programme participants also have been trained in abortion techniques (84 vs 52%) and know more about the abortion law (80 vs 62%). further, r3 m providers have more confidence in their ability to provide safe abortions (77 vs 36%), and feel that their facility supports them, compared with non - r3 m providers (82 vs 37%). table 2characteristics of r3 m and non - r3 m facilities and providers, by selected parameters, ghana 20112012variablesr3 m facilitiesnon - r3 m facilitiesn% distn% distfacility variableswas facility r3 m or non - r3 m in phase 1?64102did facility subsequently become r3 m facility ? yes7876.5 no2423.5 total102100.0region where facility is located ashanti1320.32322.5 brong - ahafo00.04443.1 eastern1929.72019.6 greater accra3250.01514.7 total64100.0102100.0provider variablesnumber of providers in r3m / non - r3 m facilities in phase 1197260distribution by abortion provisionnumber of providers who provide safe abortions in average month no abortions performed8745.819886.8 at least one of either mva / eva or d&c or medication abortion10354.23013.2 total190100.0241100.0number providers who provide pac procedures in average month no abortions performed6634.216166.8 at least one of either mva / eva or d&c or medication abortion12765.88033.2 total193100.0241100.0distribution by knowledge and trainingever trained in abortion methods such as mva / eva, d&c, medication abortion yes16583.813451.5 no3216.212648.5 total197100.0260100.0how well does provider know the abortion law ? answered six or more questions about law correctly15779.716061.5 answered less than six questions about law correctly4020.310038.5 total197100.0260100.0distribution by values, attitudes and perceptionprovider attitude towards abortion provision more favourable15479.018571.4 less favourable4121.07428.6 total195100.0259100.0confidence in ability to provide abortion provider has confidence in own ability to provide abortion15177.49335.8 provider has no confidence in own ability to provide abortion4422.616764.2 total195100.0260100.0provider 's perception of facility support facility is supportive of abortion provision15981.19536.7 facility is not supportive of abortion provision3718.916463.3 total196100.0259100.0demographic distribution, by facility in which they worksize of the facility where provider works (measured by number of beds) 08 beds3316.87027.2 950 beds7035.510842.0 51 + beds9447.77930.7 total197100.0257100.0demographic distribution, by provider 's own characteristicsage of provider below 404221.47127.3 40 and above15478.618972.7 total196100.0260100.0religion of provider catholic3919.86324.2 all other religions and sects15880.219775.8 total197100.0260100.0notes : all data are unweighted. missing values were dropped using listwise deletion. p < 0.001, p < 0.1. all estimates for this sample are from information that was current at the time of the survey. characteristics of r3 m and non - r3 m facilities and providers, by selected parameters, ghana 20112012 notes : all data are unweighted. missing values were dropped using listwise deletion. p < 0.001, p < 0.1. all estimates for this sample are from information that was current at the time of the survey. table 3 shows the results of the multivariate analyses that identify the impact of the r3 m programme on safe abortion provision. the att model, which estimates the actual effect of the r3 m programme, shows an association with safe abortion provision : in sample 1 (r3 m and all non - r3 m providers), r3 m participants have four times the odds of providing safe abortions as non - r3 m providers. according to the ate results, the hypothetical effect of all providers participating in the programme would increase the odds of abortion provision only slightly less, by about 2.5 times (odds ratio (or) = 3.5). much of this is driven by sample 3 : while both att and ate estimates for sample 2 show that r3 m providers have 2.5 times the odds of abortion provision, compared with non r3 m providers, the att analysis for sample 3 shows that r3 m providers have over 15.5 times the odds. if all potential providers in sample 3 had received the treatment, the odds of abortion provision would be over 12.5 times as much. table 3maximum likelihood logit estimates of the odds of a provider providing safe abortion services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012explanatory variablesafe abortionsample 1 : all providers surveyed (n = 412)sample 2 : r3 m providers and non - r3 m providers in accra, ashanti, eastern (n = 303)psw (att) logitpsw (ate) logitpsw (att) logitpsw (ate) logit estimateodds ratiorobust std. (no ' omitted)1.3974.0440.4001.3043.6850.3770.924 2.5190.4050.942 2.5660.389knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly1.0722.9200.5581.300 3.6700.5601.313 3.7180.5961.412 4.1040.602values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.4051.4990.3820.3291.3900.3880.2701.3110.3880.2101.2330.387confidence in own ability to provide abortion (low confidence ' omitted) have confidence2.0467.7350.4701.9937.3400.4492.0257.5780.4872.0587.8300.456facility support (low support for abortion ' omitted) facility supports abortion provision0.7952.2140.4710.875 2.3980.4440.8872.4270.4790.935 2.5460.453religion (other religion / sects ' omitted) catholic0.6810.5060.3870.6810.5060.3720.838 0.4330.4210.831 0.4360.420facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.1040.9020.3630.2920.7470.3810.1240.8840.3790.1570.8540.371 51 + beds0.3030.7390.4390.3280.7200.4450.0880.9160.4470.0840.9190.452provider characteristicsprovider type (doctors ' omitted) non - doctors0.4250.6540.3860.4850.6150.3690.7040.4950.4290.7080.4930.425age (below 40 ' omitted) 40 and above0.7402.0960.4580.7152.0450.4410.963 2.6190.4610.8702.3880.454likelihood ratio chi - square69.91068.62062.13064.020n412412303303explanatory variablesafe abortionsample 3 : r3 m providers and non - r3 m providers in brong ahafo (n = 297)sample 4 : all providers that reported training in at least one method of abortion (n = 280)psw (att) logitpsw (ate) logitpsw (att) logitpsw (ate) logit estimateodds ratiorobust std. errorintercept5.5281.2525.4091.1843.8510.9553.8190.920treatmentr3 m (no ' omitted)2.75115.6600.6962.53412.6060.5991.3293.7760.4671.2343.4360.441knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly0.7352.0860.6740.9762.6530.6551.0102.7450.5561.252 3.4980.570values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.5271.6940.4160.5581.7470.4150.5441.7240.3900.4511.5690.408confidence in own ability to provide abortion (low confidence ' omitted) have confidence1.8856.5890.5131.8126.1200.5151.365 3.9150.5771.294 3.6460.549facility support (low support for abortion ' omitted) facility supports abortion provision0.4611.5860.5660.5541.7400.5480.6471.9090.5400.6952.0040.535religion (other religion / sects ' omitted) catholic0.3170.7280.4640.3330.7170.4420.846 0.4290.3950.796 0.4510.384facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.3160.7290.4590.5220.5940.4910.2530.7770.4260.4520.6360.455 51 + beds0.4610.6310.5150.5810.5590.5230.5140.5980.4890.5170.5960.505provider characteristicsprovider type (doctors ' omitted) non - doctors0.2850.7520.4040.4260.6530.3850.1370.8720.4060.2090.8120.377age (below 40 ' omitted) 40 and above0.8042.2350.6170.8552.3510.5770.9202.5080.4820.9182.5050.470likelihood ratio chi - square61.65058.66041.14042.870n297297280280notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score.p < 0.05, p < 0.01, p < 0.001, p < 0.1. maximum likelihood logit estimates of the odds of a provider providing safe abortion services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012 notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score. p < 0.05, p < 0.01, p < 0.001, p < 0.1. the programme also improves the odds of safe abortion provision for providers with clinical knowledge, with those exposed to the programme being nearly four times as likely to provide a safe abortion (att model), compared with trained providers not exposed to the programme. if all trained providers had hypothetically been exposed to the programme (ate model), they would have been nearly 3.5 times as likely to provide safe abortion, compared with whether they had not received the treatment. according to the att model, better knowledge of the abortion law is only marginally associated with safe abortion provision in sample 1 (p = 0.06), although if all potential providers had been exposed to the treatment, they would have had over 3.5 times the odds of abortion provision. net of the effect of the other variables, it raises the odds of safe abortion provision by 2.7 times (att or = 3.7). however, the att analysis found no independent association between being better informed about the law and actually providing safe abortions in sample 3, which includes the brong ahafo group. holding favourable attitudes toward abortion is not associated with safe abortion provision, although being catholic (compared with other religions) lowers the odds of providing safe abortions by about 57% (att estimates) in sample 2. however, providers confidence in their ability to provide safe abortions is important in all samples, and in sample 1, the att estimates show that ceteris paribus, more confident providers have over 7.5 times the odds of providing safe abortions than less confident ones. the association between the outcome and facility support is only marginal (p = 0.09), though the ate estimates for samples 1 and 2 show that there would be an effect if all providers had been exposed to the programme. for the sub - sample of providers with clinical knowledge of abortion provision (sample 4), better knowledge of the abortion law is only marginally associated (p = 0.07) with the outcome, though hypothetically, trained providers would have about 3.5 times the odds of abortion provision if they knew the law, compared with their peers. even among trained providers though, being catholic reduces odds of abortion provision by 57%, compared with other religions (att estimates). provider confidence continues to be associated with abortion provision, with confident providers having nearly four times the odds of providing abortions compared with less confident ones. exposure to the r3 m programme is associated with pac provision only in sample 1 (att or = 1.97), though there is a hypothetical effect of the programme in both samples 1 and 2. however, as with safe abortion provision, providers with clinical knowledge of abortion provision, who were exposed to the r3 m programme, had over twice the odds of pac provision (att model), compared with those who had not received the treatment. this remains true under the hypothetical scenario (ate model) where all potential providers are exposed to the treatment (table 4). table 4maximum likelihood logit estimates of the odds of a provider providing post abortion care services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012explanatory variablepostabortion caresample 1 : all providers surveyed (n = 428)sample 2 : r3 m providers and non - r3 m providers in accra, ashanti, eastern (n = 320)psw (att) logitpsw (ate) logitpsw (att) logitpsw(ate) logit estimateodds ratiorobust std. (no ' omitted)0.679 1.9710.3230.723 2.0610.3100.5641.7570.3780.717 2.0480.361knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly0.1281.1360.3100.2511.2850.3120.1011.1060.3630.2831.3270.383values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.2280.7960.3420.3460.7070.3300.1160.8900.4360.2440.7830.416confidence in own ability to provide abortion (low confidence ' omitted) have confidence1.5914.9060.3271.5204.5740.3321.4604.3050.3711.4114.1000.376facility support (low support for abortion ' omitted) facility supports abortion provision0.4141.5120.3510.5121.6680.3490.3201.3770.3830.3981.4890.381religion (other religion / sects ' omitted) catholic0.1770.8370.2890.3490.7050.2930.4140.6610.3510.5440.5810.350facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.0690.9330.2860.3390.7120.3060.0820.9210.3130.2340.7910.321 51 + beds0.2441.2760.4290.0060.9940.4430.4201.5220.5360.2801.3230.551provider characteristicsprovider type (doctors ' omitted) non - doctors1.171 0.3100.4811.3900.2490.4600.8370.4330.4650.975 0.3770.460age (below 40 ' omitted) 40 and above0.5541.7410.3270.5211.6830.3260.6451.9060.3540.5581.7480.358likelihood ratio chi - square77.11079.08053.01056.680n428428320320explanatory variablepostabortion caresample 3 : r3 m providers and non - r3 m providers in brong ahafo (n = 299)sample 4 : all providers that reported training in at least one method of abortion (n = 288)psw (att) logitpsw (ate) logitpsw (att) logitpsw(ate) logit estimateodds ratiorobust std. (no ' omitted)0.6561.9270.4610.6001.8210.4550.802 2.2300.3620.797 2.2190.373knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly0.1101.1160.3890.1161.1230.3660.2781.3210.3750.4031.4970.352values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.6200.5380.3680.5760.5620.3600.0440.9570.3450.0630.9390.349confidence in own ability to provide abortion (low confidence ' omitted) have confidence1.8096.1040.4241.7555.7810.4001.087 2.9650.4200.848 2.3360.399facility support (low support for abortion ' omitted) facility supports abortion provision0.6431.9020.4490.6861.9860.4470.0010.9990.4210.1641.1790.430religion (other religion / sects ' omitted) catholic0.0130.9870.3700.1330.8750.3570.2460.7820.3240.4490.6380.320facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.0680.9350.3990.2430.7840.3920.0751.0780.3910.1840.8320.403 51 + beds0.0300.9710.5030.2180.8040.4830.0560.9460.4870.2990.7420.495provider characteristicsprovider type (doctors ' omitted) non - doctors1.7820.1680.6861.8980.1500.6370.8680.4200.5781.108 0.3300.544age (below 40 ' omitted) 40 and above0.4361.5470.4130.4641.5900.3830.7802.1810.4170.7702.1590.398likelihood ratio chi - square84.31087.70037.58037.960n299299288288notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score.p < 0.05, p < 0.01, p < 0.001, p < 0.1. maximum likelihood logit estimates of the odds of a provider providing post abortion care services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012 notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score. p < 0.05, p < 0.01, p < 0.001, p < 0.1. neither knowing the abortion law nor religion nor attitudes toward abortion is associated with the odds of providing pac, though abortion attitudes is weakly associated (p = 0.09) with pac for sample 3. however, as with the outcome of safe abortion provision, providers confidence in their ability to provide abortion does predict pac provision, with the actual odds being between six times as much (in sample 3) and about four times as much (in sample 2) for confident providers compared with less confident ones. unlike the models predicting the provision of safe abortion, being a non - doctor does affect their likelihood of providing pac. not being a doctor lowers the odds of providing pac in all samples, with only the att estimates in sample 2 being marginal (p = 0.07). the att estimates range from 63% lower odds for pac provision for non - doctors in sample 1 to 83% lower odds in sample 3. knowledge of law, provider attitudes, and religion are not associated with pac provision for sample 4. a provider s confidence in his or her own abilities, however, continues to be significantly associated (p = 0.01) with pac provision, with confident trained providers being about thrice as likely to provide pac compared with less confident trained providers (att estimates), though the effect size is smaller than for sample 1 (or = 4.9). the psa is a more conservative technique compared with a standard regression, which often provides spurious associations between variables. however, as with any multivariate technique, we can not account for confounding unobserved characteristics. propensity score weights are also dependent on the functional form of the model that is used to estimate them. we tried various functional forms and used the one that gave us the best balance. table 2 has the percentage distribution of the full sample along selected parameters, and it shows that more providers in the treatment (r3 m) facilities have attributes favourable to safe abortion and pac provision compared with non - r3 m providers. greater proportions of providers exposed to the programme provide safe abortion services (54 vs 13%) and pac (66 vs 33%). greater proportions of programme participants also have been trained in abortion techniques (84 vs 52%) and know more about the abortion law (80 vs 62%). further, r3 m providers have more confidence in their ability to provide safe abortions (77 vs 36%), and feel that their facility supports them, compared with non - r3 m providers (82 vs 37%). table 2characteristics of r3 m and non - r3 m facilities and providers, by selected parameters, ghana 20112012variablesr3 m facilitiesnon - r3 m facilitiesn% distn% distfacility variableswas facility r3 m or non - r3 m in phase 1?64102did facility subsequently become r3 m facility ? yes7876.5 no2423.5 total102100.0region where facility is located ashanti1320.32322.5 brong - ahafo00.04443.1 eastern1929.72019.6 greater accra3250.01514.7 total64100.0102100.0provider variablesnumber of providers in r3m / non - r3 m facilities in phase 1197260distribution by abortion provisionnumber of providers who provide safe abortions in average month no abortions performed8745.819886.8 at least one of either mva / eva or d&c or medication abortion10354.23013.2 total190100.0241100.0number providers who provide pac procedures in average month no abortions performed6634.216166.8 at least one of either mva / eva or d&c or medication abortion12765.88033.2 total193100.0241100.0distribution by knowledge and trainingever trained in abortion methods such as mva / eva, d&c, medication abortion yes16583.813451.5 no3216.212648.5 total197100.0260100.0how well does provider know the abortion law ? answered six or more questions about law correctly15779.716061.5 answered less than six questions about law correctly4020.310038.5 total197100.0260100.0distribution by values, attitudes and perceptionprovider attitude towards abortion provision more favourable15479.018571.4 less favourable4121.07428.6 total195100.0259100.0confidence in ability to provide abortion provider has confidence in own ability to provide abortion15177.49335.8 provider has no confidence in own ability to provide abortion4422.616764.2 total195100.0260100.0provider 's perception of facility support facility is supportive of abortion provision15981.19536.7 facility is not supportive of abortion provision3718.916463.3 total196100.0259100.0demographic distribution, by facility in which they worksize of the facility where provider works (measured by number of beds) 08 beds3316.87027.2 950 beds7035.510842.0 51 + beds9447.77930.7 total197100.0257100.0demographic distribution, by provider 's own characteristicsage of provider below 404221.47127.3 40 and above15478.618972.7 total196100.0260100.0religion of provider catholic3919.86324.2 all other religions and sects15880.219775.8 total197100.0260100.0notes : all data are unweighted. missing values were dropped using listwise deletion. p < 0.001, p < 0.1. all estimates for this sample are from information that was current at the time of the survey. characteristics of r3 m and non - r3 m facilities and providers, by selected parameters, ghana 20112012 notes : all data are unweighted. missing values were dropped using listwise deletion. p < 0.001, p < 0.1. all estimates for this sample are from information that was current at the time of the survey. table 3 shows the results of the multivariate analyses that identify the impact of the r3 m programme on safe abortion provision. the att model, which estimates the actual effect of the r3 m programme, shows an association with safe abortion provision : in sample 1 (r3 m and all non - r3 m providers), r3 m participants have four times the odds of providing safe abortions as non - r3 m providers. according to the ate results, the hypothetical effect of all providers participating in the programme would increase the odds of abortion provision only slightly less, by about 2.5 times (odds ratio (or) = 3.5). much of this is driven by sample 3 : while both att and ate estimates for sample 2 show that r3 m providers have 2.5 times the odds of abortion provision, compared with non r3 m providers, the att analysis for sample 3 shows that r3 m providers have over 15.5 times the odds. if all potential providers in sample 3 had received the treatment, the odds of abortion provision would be over 12.5 times as much. table 3maximum likelihood logit estimates of the odds of a provider providing safe abortion services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012explanatory variablesafe abortionsample 1 : all providers surveyed (n = 412)sample 2 : r3 m providers and non - r3 m providers in accra, ashanti, eastern (n = 303)psw (att) logitpsw (ate) logitpsw (att) logitpsw (ate) logit estimateodds ratiorobust std. (no ' omitted)1.3974.0440.4001.3043.6850.3770.924 2.5190.4050.942 2.5660.389knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly1.0722.9200.5581.300 3.6700.5601.313 3.7180.5961.412 4.1040.602values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.4051.4990.3820.3291.3900.3880.2701.3110.3880.2101.2330.387confidence in own ability to provide abortion (low confidence ' omitted) have confidence2.0467.7350.4701.9937.3400.4492.0257.5780.4872.0587.8300.456facility support (low support for abortion ' omitted) facility supports abortion provision0.7952.2140.4710.875 2.3980.4440.8872.4270.4790.935 2.5460.453religion (other religion / sects ' omitted) catholic0.6810.5060.3870.6810.5060.3720.838 0.4330.4210.831 0.4360.420facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.1040.9020.3630.2920.7470.3810.1240.8840.3790.1570.8540.371 51 + beds0.3030.7390.4390.3280.7200.4450.0880.9160.4470.0840.9190.452provider characteristicsprovider type (doctors ' omitted) non - doctors0.4250.6540.3860.4850.6150.3690.7040.4950.4290.7080.4930.425age (below 40 ' omitted) 40 and above0.7402.0960.4580.7152.0450.4410.963 2.6190.4610.8702.3880.454likelihood ratio chi - square69.91068.62062.13064.020n412412303303explanatory variablesafe abortionsample 3 : r3 m providers and non - r3 m providers in brong ahafo (n = 297)sample 4 : all providers that reported training in at least one method of abortion (n = 280)psw (att) logitpsw (ate) logitpsw (att) logitpsw (ate) logit estimateodds ratiorobust std. error estimateodds ratiorobust std. (no ' omitted)2.75115.6600.6962.53412.6060.5991.3293.7760.4671.2343.4360.441knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly0.7352.0860.6740.9762.6530.6551.0102.7450.5561.252 3.4980.570values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.5271.6940.4160.5581.7470.4150.5441.7240.3900.4511.5690.408confidence in own ability to provide abortion (low confidence ' omitted) have confidence1.8856.5890.5131.8126.1200.5151.365 3.9150.5771.294 3.6460.549facility support (low support for abortion ' omitted) facility supports abortion provision0.4611.5860.5660.5541.7400.5480.6471.9090.5400.6952.0040.535religion (other religion / sects ' omitted) catholic0.3170.7280.4640.3330.7170.4420.846 0.4290.3950.796 0.4510.384facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.3160.7290.4590.5220.5940.4910.2530.7770.4260.4520.6360.455 51 + beds0.4610.6310.5150.5810.5590.5230.5140.5980.4890.5170.5960.505provider characteristicsprovider type (doctors ' omitted) non - doctors0.2850.7520.4040.4260.6530.3850.1370.8720.4060.2090.8120.377age (below 40 ' omitted) 40 and above0.8042.2350.6170.8552.3510.5770.9202.5080.4820.9182.5050.470likelihood ratio chi - square61.65058.66041.14042.870n297297280280notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score.p < 0.05, p < 0.01, p < 0.001, p < 0.1. maximum likelihood logit estimates of the odds of a provider providing safe abortion services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012 notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score. p < 0.05, p < 0.01, p < 0.001, p < 0.1. the programme also improves the odds of safe abortion provision for providers with clinical knowledge, with those exposed to the programme being nearly four times as likely to provide a safe abortion (att model), compared with trained providers not exposed to the programme. if all trained providers had hypothetically been exposed to the programme (ate model), they would have been nearly 3.5 times as likely to provide safe abortion, compared with whether they had not received the treatment. according to the att model, better knowledge of the abortion law is only marginally associated with safe abortion provision in sample 1 (p = 0.06), although if all potential providers had been exposed to the treatment, they would have had over 3.5 times the odds of abortion provision. net of the effect of the other variables, it raises the odds of safe abortion provision by 2.7 times (att or = 3.7). however, the att analysis found no independent association between being better informed about the law and actually providing safe abortions in sample 3, which includes the brong ahafo group. holding favourable attitudes toward abortion is not associated with safe abortion provision, although being catholic (compared with other religions) lowers the odds of providing safe abortions by about 57% (att estimates) in sample 2. however, providers confidence in their ability to provide safe abortions is important in all samples, and in sample 1, the att estimates show that ceteris paribus, more confident providers have over 7.5 times the odds of providing safe abortions than less confident ones. the association between the outcome and facility support is only marginal (p = 0.09), though the ate estimates for samples 1 and 2 show that there would be an effect if all providers had been exposed to the programme. for the sub - sample of providers with clinical knowledge of abortion provision (sample 4), better knowledge of the abortion law is only marginally associated (p = 0.07) with the outcome, though hypothetically, trained providers would have about 3.5 times the odds of abortion provision if they knew the law, compared with their peers. even among trained providers though, being catholic reduces odds of abortion provision by 57%, compared with other religions (att estimates). provider confidence continues to be associated with abortion provision, with confident providers having nearly four times the odds of providing abortions compared with less confident ones. exposure to the r3 m programme is associated with pac provision only in sample 1 (att or = 1.97), though there is a hypothetical effect of the programme in both samples 1 and 2. however, as with safe abortion provision, providers with clinical knowledge of abortion provision, who were exposed to the r3 m programme, had over twice the odds of pac provision (att model), compared with those who had not received the treatment. this remains true under the hypothetical scenario (ate model) where all potential providers are exposed to the treatment (table 4). table 4maximum likelihood logit estimates of the odds of a provider providing post abortion care services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012explanatory variablepostabortion caresample 1 : all providers surveyed (n = 428)sample 2 : r3 m providers and non - r3 m providers in accra, ashanti, eastern (n = 320)psw (att) logitpsw (ate) logitpsw (att) logitpsw(ate) logit estimateodds ratiorobust std. (no ' omitted)0.679 1.9710.3230.723 2.0610.3100.5641.7570.3780.717 2.0480.361knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly0.1281.1360.3100.2511.2850.3120.1011.1060.3630.2831.3270.383values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.2280.7960.3420.3460.7070.3300.1160.8900.4360.2440.7830.416confidence in own ability to provide abortion (low confidence ' omitted) have confidence1.5914.9060.3271.5204.5740.3321.4604.3050.3711.4114.1000.376facility support (low support for abortion ' omitted) facility supports abortion provision0.4141.5120.3510.5121.6680.3490.3201.3770.3830.3981.4890.381religion (other religion / sects ' omitted) catholic0.1770.8370.2890.3490.7050.2930.4140.6610.3510.5440.5810.350facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.0690.9330.2860.3390.7120.3060.0820.9210.3130.2340.7910.321 51 + beds0.2441.2760.4290.0060.9940.4430.4201.5220.5360.2801.3230.551provider characteristicsprovider type (doctors ' omitted) non - doctors1.171 0.3100.4811.3900.2490.4600.8370.4330.4650.975 0.3770.460age (below 40 ' omitted) 40 and above0.5541.7410.3270.5211.6830.3260.6451.9060.3540.5581.7480.358likelihood ratio chi - square77.11079.08053.01056.680n428428320320explanatory variablepostabortion caresample 3 : r3 m providers and non - r3 m providers in brong ahafo (n = 299)sample 4 : all providers that reported training in at least one method of abortion (n = 288)psw (att) logitpsw (ate) logitpsw (att) logitpsw(ate) logit estimateodds ratiorobust std. (no ' omitted)0.6561.9270.4610.6001.8210.4550.802 2.2300.3620.797 2.2190.373knowledgeknowledge of law (answered fewer than six questions correctly ' omitted) answered six or more questions correctly0.1101.1160.3890.1161.1230.3660.2781.3210.3750.4031.4970.352values, attitudes and perceptionattitude toward abortion provision (less favourable ' omitted) more favourable0.6200.5380.3680.5760.5620.3600.0440.9570.3450.0630.9390.349confidence in own ability to provide abortion (low confidence ' omitted) have confidence1.8096.1040.4241.7555.7810.4001.087 2.9650.4200.848 2.3360.399facility support (low support for abortion ' omitted) facility supports abortion provision0.6431.9020.4490.6861.9860.4470.0010.9990.4210.1641.1790.430religion (other religion / sects ' omitted) catholic0.0130.9870.3700.1330.8750.3570.2460.7820.3240.4490.6380.320facility in which they worknumber of beds in facility they practice (08 beds ' omitted) 950 beds0.0680.9350.3990.2430.7840.3920.0751.0780.3910.1840.8320.403 51 + beds0.0300.9710.5030.2180.8040.4830.0560.9460.4870.2990.7420.495provider characteristicsprovider type (doctors ' omitted) non - doctors1.7820.1680.6861.8980.1500.6370.8680.4200.5781.108 0.3300.544age (below 40 ' omitted) 40 and above0.4361.5470.4130.4641.5900.3830.7802.1810.4170.7702.1590.398likelihood ratio chi - square84.31087.70037.58037.960n299299288288notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score.p < 0.05, p < 0.01, p < 0.001, p < 0.1. maximum likelihood logit estimates of the odds of a provider providing post abortion care services, by whether they were part of the r3 m programme or not, and other characteristics, ghana 20112012 notes : components may not sum to totals due to rounding. all estimates for this sample are from information that was current at the time of the survey. observations with missing values on the dependent variable were dropped as were those cases that had a missing or a negative propensity score. p < 0.05, p < 0.01, p < 0.001, p < 0.1. neither knowing the abortion law nor religion nor attitudes toward abortion is associated with the odds of providing pac, though abortion attitudes is weakly associated (p = 0.09) with pac for sample 3. however, as with the outcome of safe abortion provision, providers confidence in their ability to provide abortion does predict pac provision, with the actual odds being between six times as much (in sample 3) and about four times as much (in sample 2) for confident providers compared with less confident ones. unlike the models predicting the provision of safe abortion, being a non - doctor does affect their likelihood of providing pac. not being a doctor lowers the odds of providing pac in all samples, with only the att estimates in sample 2 being marginal (p = 0.07). the att estimates range from 63% lower odds for pac provision for non - doctors in sample 1 to 83% lower odds in sample 3. knowledge of law, provider attitudes, and religion are not associated with pac provision for sample 4. a provider s confidence in his or her own abilities, however, continues to be significantly associated (p = 0.01) with pac provision, with confident trained providers being about thrice as likely to provide pac compared with less confident trained providers (att estimates), though the effect size is smaller than for sample 1 (or = 4.9). the psa is a more conservative technique compared with a standard regression, which often provides spurious associations between variables. however, as with any multivariate technique, we can not account for confounding unobserved characteristics. propensity score weights are also dependent on the functional form of the model that is used to estimate them. we tried various functional forms and used the one that gave us the best balance. the pilot phase of the ghanaian government s r3 m programme in 2006 created a natural experiment to compare facilities selected to participate in the programme with those that were not. we collected data on providers and their provision of safe abortion and pac services to see how they differed according to exposure and geographic proximity to exposure to the r3 m programme. our findings show that, net of all potentially confounding variables, participation in the r3 m programme does have a substantial association with the provision of safe abortion services in all groups, including among those who have clinical knowledge of abortion provision. however, the differences are much stronger between r3 m and non - r3 m providers in brong ahafo, than between the r3 m and non - r3 m providers in the r3 m regions. the stronger association for the former is likely because of geographical distance, which reduces contamination of samples due for instance, to the transfer of providers between facilities. while the r3 m programme is associated with pac provision in samples 1 and 4, there is no association for either samples 2 or 3. while the results for sample 1 show that the programme made a difference in the provision of pac overall, the lack of association for the sub - samples is hardly surprising, since pac is not nearly as controversial as safe abortion and has long been part of essential emergency obstetric care in ghana (government of kenya 1997 ; republic of ghana ministry of health 1997 ; billings. further, while ceteris paribus, we expect doctors to be more likely to provide pac, it is important to involve the mid - level providers in pac provision owing to the paucity of doctors in ghana, especially outside the two biggest cities accra and kumasi (brookman - amissah 2004 ; hessini. 2006). inability to receive life - saving pac, due to lack of trained providers, only exacerbates the problem of maternal mortality. earlier studies have suggested that negative provider attitudes and ignorance of the law may hinder abortion provision (aboagye. our analyses, however, found no such uniform association with the provision of safe abortion services. in the analyses for all providers (samples 13), we found no association for abortion attitudes for any sample ; though being catholic was negatively associated with the outcome for sample 2. while abortion law knowledge was significantly and positively associated with sample 2, the actual (att) association was only marginal in sample 1 ; and it had no association in sample 3. these variables had no independent association with pac provision. to get a clearer picture of the association of these variables with service provision, we separately analysed a sample of only those who were trained in clinical provision of abortion services, since only these providers can potentially provide a safe abortion or pac. as with the other analyses, the analysis for sample 4, showed no association of abortion attitudes towards provision of safe abortion or pac, though being catholic the actual association of abortion law knowledge with safe abortion provision was also only marginal, although it does have the potential to improve service provision in the hypothetical scenario where all providers are exposed to the treatment. a key driver of abortion and pac provision seems to be a provider s confidence in their ability to provide such care. this variable is significantly associated with both outcomes in all samples, though much larger for sample 1 compared with sample 4, indicating that training in clinical provision of abortion services is key to building confidence. while facility support was marginally significant at best in the att models, the ate results from samples 1 and 2 show that it has the potential to make a difference if all providers and their facilities were exposed to the treatment. in sum, our results show that any intervention to improve comprehensive abortion care in ghana should focus on providing training in abortion techniques and on building provider confidence in service provision, in particular for mid - level providers in all regions, due to a greater availability of such providers coupled with a paucity of doctors, who mostly live in the big cities (brookman - amissah 2004 ; hessini. further, although we found no consistent association between better knowledge of legal criteria and abortion provision, this does not mean that providers should remain ignorant of the law. it may matter less for the average provider, but individual providers may refuse to provide abortions simply because they are unaware when women legally qualify for them (morhe. 2007 similarly, interventions that focus on transforming individual providers attitudes, should continue, since it can only help strengthen service provision (hessini. for instance a broader assessment of the programme could include women the clients who avail of cac services. it could also examine the impact of the programme on facilities, such as changes in facility infrastructure, or changes in the number of abortion procedures performed in a facility. both facility and women level outcomes were however outside the scope of this study since our survey focused strictly and narrowly on providers. while studies exist on women s abortion experiences (sundaram. 2012), more research is needed on the impact of the programme on the women who are the intended beneficiaries of such an intervention, and on the impact of the programme on the facilities. future research could also include analyses that simultaneously examine the association between the provider outcomes and facility characteristics, such differences in number of available providers in the facility and quality of infrastructure, using multilevel modelling techniques. this would also help shed greater light on the impact of the r3 m programme. however, such an analysis was also outside the scope of our study owing to lack of facility level data. we hope however that our findings help ghanaian policy makers better assess the success of the current r3 m intervention and related policies, and also aid them in finding solutions to problems of implementation. our findings should also hopefully encourage the expansion of the r3 m programme to cover the entire country, especially the more remote areas, in order that barriers that currently prevent women from accessing safe abortion services and quality pac are removed. removing such barriers would contribute to substantial reductions in the unconscionably high levels of maternal morbidity and mortality that currently prevail in the country (hu. this work was supported by the consortium for research on unsafe abortion in africa, department for international development (dfid), government of the united kingdom, and the dutch ministry of foreign affairs, government of the netherlands.	in 2006, in response to the high maternal mortality, driven largely by unsafe abortions, the government of ghana, in partnership with other organizations, launched the reducing maternal mortality and morbidity (r3 m) programme in seven districts in greater accra, ashanti and eastern, to improve comprehensive abortion care services. this article examines whether this intervention made a difference to the provision of safe abortion services and postabortion care (pac). we also examine the role played by provider attitudes and knowledge of the abortion law, on providers with clinical training in service provision. primary data on health care providers in ghana, collected using a quasi - experimental design, were analysed using propensity score weighting. apart from the treatment group, the sample included two controls : (1) districts in accra, ashanti and eastern, not exposed to the treatment ; and (2) districts from distant brong ahafo, also not exposed to the treatment. the findings show that providers in the treatment group are nearly 16 times as likely to provide safe abortions compared with their peers in brong ahafo, and 2.5 times as likely compared with providers in the other control group. r3 m providers were also different from their peers in providing pac. associations between provider attitudes and knowledge of the law on both outcomes were either non - significant or inconsistent including for providers with clinical knowledge of abortion provision. provider confidence however is strongly associated with service provision. we conclude that the r3 m programme is helping safe abortion provision, with the differences being greater with control groups that are geographically distant, perhaps owing to lower contamination from movement of providers between facilities. increasing provider confidence is key to improving both safe abortion provision and pac.
they help to achieve an accurate diagnosis, build trust with patients, and improve compliance to treatment, overall patient satisfaction, therapeutic outcome, and avoid litigation. the initial and most important step in communication with patients is the establishment of rapport. this may take time, but will definitely decrease the patient 's tension and fear and improve doctor patient relationship, which in turn creates a strong base for further management. rapport is established through a series of verbal and nonverbal means of communication such as smiling, introducing oneself, calling the patient by the preferred name, making eye contact and assuming a welcoming posture, and showing empathy. when communication improves, the risk of malpractice may decrease, but compliance, patient satisfaction, and outcomes improve. moreover, physicians who use their rapport - building skills often experience less stress in their work and are more satisfied. building rapport, as an essential part of physician patient communication and relationship, needs training that emphasises on using the local language and being aware of the cultural attributes of patients. for example, in the ksa, many patients prefer to be called by a nickname that includes their oldest son 's name, but shaking hands with the opposite sex is unacceptable. communication skills have not been part of some of the study curricula in pregraduate medical schools in saudi arabia. furthermore, foreign doctors, even those who are trained in communication skills have difficulty in applying their skills due to language problem and cultural barriers. the objective of this study was to assess the establishment of rapport with patients by physicians working in the primary health care center (phcc), dammam city, ksa and assess patients ' satisfaction with that relationship. a cross - sectional study was carried out at the phcc in dammam, ksa. all physicians and patients at the phcc, dammam, who were more than 15 years of age and were mentally and physically able to communicate in simple arabic about their medical care were included. patients who were < 15 years of age and unable to communicate verbally (e.g., speech disorder and hearing impairment) were excluded. using the automated sample size calculator, the sample size of attendees was estimated at 374 subjects from the phcc with a total population of 9000 patients per month, with a confidence interval of 5%. the sample size of the physicians was a comprehensive sample of all 30 physicians, 27 (90.0%) of whom participated in the study. the patient sample of attendees was selected using a systemic sampling procedure, whereby every seventh patient visiting the phcc, dammam, on two days per week, over a period of 3 weeks was selected. the days were divided into morning and afternoon clinics. on day 1, the females were taken in the morning, and the males in the afternoon and the converse was done on the 2 day of that week. data were collected using a self - administered structured questionnaire completed by the selected physicians and attendees visiting the phcc, dammam, under direct supervision. each positive and negative response was assigned a mark of 1 or 0, respectively. a rapport score was computed as the sum of all positive answers of the rapport questions. score values equal or greater than the median were considered as good, and those below as poor. the rapport score was treated as the dependent variable and the independent variables included age (categorized as young, middle or elderly), gender, years of experience (categorized as short, average, and long), job title (general practitioner, resident, specialist, and consultant), whether speaks arabic or not, and whether the primary language is arabic or not arabic. in addition, physicians were categorized into those whose specialty was of a surgical nature, i.e., whether the management of their patients involved surgical procedures or not. each positive and negative response was assigned a mark of 1 or zero, respectively. the score for satisfaction with rapport was computed as the sum of all positive answers on the rapport questions. score values equal or greater than the median were considered as good, and those bellow it as poor. satisfaction with the rapport score was similarly treated as the dependent variable for which the independent variables included age, gender, level of education, residence, with or without a chronic disease, with or without an appointment, visit type, physicians ' specialty, and language. age was recorded to the nearest year and subjects were categorized as young (< 30 years), middle - aged (3054 years), or elderly (55 years and over) ; level of education was defined as low (illiterate and primary school), intermediate (intermediate and high school), or high(university graduates or postgraduate) ; has or does not has chronic disease, e.g. diabetes mellitus, hypertension, bronchial asthma, and sickle cell anemia ; with or without an appointment ; visit type defined as new or follow - up ; physicians ' specialty categorized as surgical or nonsurgical involving surgical means of patient management ; physicians ' professional status recorded as consultant, specialist or other ; and physicians ' language defined as arabic or non - arabic. physicians ' experience was categorized as short (< 5 years), intermediate (59 years), and long (10 years and over). data were entered into the computer and analyzed using the statistical package for social sciences (spss), version 20.0. the questionnaires ' reliability, as tested by spss, using the split half guttman method, was 67.1% and 79.3% for patients ' and physicians ' questionnaires, respectively. the study proposal was approved by the research committee of the national guard health authority, eastern region, as well as by the local supervisory training committee for family medicine, in the eastern province, ksa. a pilot study involving 20 subjects was carried out to test the logistics of the study, but the data were not included in the main study. a total of 374 attendees participated in the study with a response rate of approximately 92% and 27 physicians participated with a response rate of 90.0%. the attendees ' mean age was 37.12 11.2 years (mean standard deviation [sd ]), with a range of 1675 years. attendees in the young, middle - aged, and elderly groups were 46.8%, 44.4%, and 8.8%, respectively. females and males in the sample were 55.9% and 44.1%, respectively [table 1 ]. attendees residing in dammam made up 75.7% of the total, 19.8% resided in al - khobar and 4.5% in other areas. the majority (59.1%) had average education, and very few were highly educated [table 2 ]. sociodemographic characteristics of study sample in relation to rapport score at primary health care center, dammam, kingdom of saudi arabia, 2013 relation of the study sample attendees satisfaction with sociodemographic and disease - related variables at the primary health care center, dammam, kingdom of saudi arabia, 2013 almost 40.0% of the attendees had chronic illnesses. those who had appointments were 48.1%, while those attending as walk - in were 51.9%. about 75% were seen by physicians with a nonsurgical specialty, and 25.4% were seen by physicians whose specialty was surgical in nature. fourteen percent physicians were consultants, 31.0% were specialists, and 54.5% were of junior status [table 3 ]. physicians characteristics in relation to attendees satisfaction score with the physicians at primary health care center, dammam, kingdom of saudi arabia, 2013 the attendees seen by male and female physicians were 44.9% and 55.1%, respectively. ninety four percent were seen by arabic - speaking physicians and 6.0% were seen by non - arabic speaking physicians. for 23.0%, it was the first visit to the physician, while for 77%, it was a follow - up visit. the physicians ' mean age was 41 9.9 years (mean sd) with a range of 2558 years. young physicians constituted 37.0%, middle - aged physicians 48.1%, and older physicians were 14.8% of the sample. the mother tongue of 88.9% of the physicians was arabic and for 11.1% it was not arabic. the mean years of experience was 14.9 8.4 years (mean sd). physicians with short experience (working years for 16 years) came to 40.7%, 22.2% of the physicians had average experience (712 years), while 37.0% had long experience (more than 12). physicians with booked clinics were 55.6% while those with walk - in clinics were 44.4%. the percentage of physicians with good rapport practice score was 51.9% with a mean rapport score of 71.8% 11 (mean sd), and a range of 47.491.2%. regarding the relation of sociodemographic and clinical characteristics of the study sample physicians with rapport practice score, there were significantly more older physicians than younger ones who had a good rapport score (p < 0.016). significantly more physicians with long experience than those with shorter experience had a good rapport score (p < 0.031, and p < 0.031, respectively). furthermore, significantly more consultant physicians than the rest had a good rapport score (p < 0.043) [table 1 ]. attendees who were satisfied with their physician 's rapport with them amounted to 50.5% while 49.5% were dissatisfied. the mean satisfaction score was 77.7% 16.9 (mean sd) with a range of 20.6100%. significantly more elderly attendees, those with a lower level of education, those with chronic illnesses, and those with fixed appointments were satisfied with physicians ' rapport than other groups (p < 0.0001) [table 2 ]. regarding physicians ' characteristics, significantly more consultant physicians and those with nonsurgical specialties affected attendees ' satisfaction (p < 0.0001) [table 3 ]. logistic regression analysis of the relation of significant factors predicting rapport practice score among study sample physicians showed that the age of the physician was a significant predictor of a good rapport score (p < 0.042) [table 4 ]. logistic regression analysis of variables predicting rapport practice score among study sample physicians at primary health care center, dammam, kingdom of saudi arabia, 2013 moreover, logistic regression analysis of the relation of significant factors predicting satisfaction score among study sample attendees showed that attendees with chronic diseases, those with a prefixed appointments and those seen by consultant physicians, as well as those seen by nonsurgical physicians were significantly satisfied with the attention received (p < 0.001, p < 0.004, p < 0.005, and p < 0.000, respectively)[table 5 ]. logistic regression analysis of variables predicting satisfaction score among study sample attendees at primary health care center, dammam, kingdom of saudi arabia, 2013 in this study, the sample of attendees comprised mainly the young and middle - aged, providing a wide spectrum of ages. the percentages of females and males were almost the same, is roughly representative of the population of saudi arabia. the education of the majority of the study sample attendees average which is similar to the population of saudi arabia as published by the central department of statistics and information, saudi arabia, 2013. the percentage of satisfied attendees was 50.5%, and in a phcc setting, which is considered the main venue for health care provision, and continuous care, this percentage is mediocre, especially since the range of ages sampled is very wide, covering almost all age groups. research has shown good communication skill to be the key to better patient outcomes, and better patient satisfaction. also, patients ' satisfaction appears to be a strong motivation for compliance to treatment and its success. for instance, physicians who are warm, friendly, and understanding demonstrate better rapport with their patients than those who are not. the study revealed that most attendees were seen by physicians who were not consultants, and who displayed little rapport with their patients thus failing to stimulate much attendee satisfaction. the lack of skills for building rapport in saudi arabian hospitals may be explained by the fact that though physicians are of diverse nationalities, religions and cultures, and speak different languages, they all have to deal with the cultural norms, religion, and language of the saudi population. moreover, since these factors influence rapport building the lack of training on communication skills is made all the more obvious. it appears that the ideal circumstances for optimal communication in health care are for both physicians, especially primary care physicians, and patients to speak the same language. besides, the physician is required to identify with and respect different values, beliefs, cultural norms, especially those related to gender and social circumstance. unfortunately, there does not seem to be many programs that train physicians on communication skills, nor are there studies to address this problem in saudi arabia in general and in phccs in particular. elderly attendees and those with little education indicated greater satisfaction than younger and more educated attendees. in addition, those with chronic illnesses and those who had appointments were also better satisfied than others. research has shown that satisfaction was related to patients ' age, psychological morbidity, and most significantly satisfaction with the length of waiting time at the clinic, which was less for patients who had appointments. in addition, patients ' confidence and trust in their physicians increased with the patient 's age. another possible explanation is that patients with chronic illness usually had constant health care at booked clinics, where a good relationship with their caregiver had already been built. this shows the importance of continuity of health care and the fact that sustained continuity of care is associated with improved patient satisfaction, especially in patients with chronic conditions. furthermore, it was found that continuity of care was associated with a lower rate of visits to the emergency department and hospitalization. in addition, continuity of care improves the quality of the patient physician communication and relationship, management of patients with chronic conditions, and patients ' quality of life. this may also explain the dissatisfaction of walk - in attendees or low rapport scores because walk - in patients tend to impose an unusual burden on the physician. a system should, therefore, be developed to deal effectively with the attendance of walk - in patients. visiting a physician with a high professional status was also observed to be positively related to satisfaction with physicians ' empathy, while the gender of the physician and the language had no such effect. it was found that surgeons ' expression of empathy with patients was infrequent, and any social conversation was brief. it has been documented that the specialty physician had an effect on patterns of communication and on patient satisfaction, for the satisfaction of the patients of family physicians was more closely linked to rapport building, psychosocial exchange, and patient - centeredness than they were for other specialties. similar to the satisfaction score, physicians ' practice score for rapport was moderate, only 51.9%. it was found that the older the physician, the better he / she was in establishing rapport. also, more consultants and highly experienced physicians had better rapport scores than others. the reason for this is that the development of a good relationship with patients comes with time and certain skills. this improved communication can only be developed through mutual trust, and respect which promotes the development of a therapeutic relationship. however, rapport and good communication with patients are not skills that are easily acquired. nonetheless, practitioners who are clinically competent, consistent, honest, and committed to their patients are able to further the building of patient trust and improve communication, rapport, and outcomes. though the establishment of rapport is important when dealing with patients, it is not given much attention. it is the responsibility of the health care providers to give more training to improve their skills and practice to create better physician moreover, the study showed that attendees ' satisfaction with physicians ' empathy was not high.	background : establishing rapport is an important step in physician patient communication resulting in a positive effect on patient satisfaction and overall clinical outcomes. however, there is a dearth of studies on the condition of doctor patient relations in saudi arabia. this study was performed to estimate the proportion of physicians who have a good rapport with patients in their practice and the proportion of satisfied attendees.materials and methods : a cross - sectional study was conducted at a primary health care center, dammam, ksa. the data were collected through a structured self - administered questionnaire given to samples of attendees and physicians to estimate patient satisfaction and the practice of rapport by physicians.results:a total of 374 attendees and 27 physicians participated in the study. the percentage of physicians who had good rapport was 51.9%. factors that showed significant relationship with rapport practice were : physician 's age (p = 0.016), physician 's experience (p = 0.043), and professional status (p = 0.031). the attendees satisfied with their physician 's rapport with them were 50.5%. factors that showed significant relationship with satisfaction were : attendee 's age (p < 0.0001), educational level (p < 0.0001), having a chronic illness (p < 0.0001), having appointment (p < 0.0001), physicians ' professional status (p < 0.0001), and a nonsurgical specialty (p < 0.0001).conclusion and recommendation : physicians ' rapport with patients and patients ' satisfaction with physicians ' empathy is not high. training is required to optimize physician patient communication.
smooth muscle contraction plays a fundamental role in regulating the functions of the hollow organs in the body such as the blood vessels, intestines, bladder, airways, and uterus. dysfunctional contraction of the smooth muscles is a key pathological feature of many diseases including hypertension, bladder overactivity (oab), irritable bowel syndrome, erectile dysfunction, and asthma. abnormal uterine contractions may also lead to preterm labor, the latter being responsible for prenatal mortality, neonatal morbidity, and childhood developmental disorders. by and large, smooth muscle hyperactivity disorders involve immense social cost and financial burden to the health services, thus, a considerable effort has been made to understand their etiologies and also to develop drugs with potent smooth muscle relaxant activities. contractions of all smooth muscles absolutely depend on the presence of ca which activates the contractile machineries [2, 3 ]. elevating intracellular ca, which can be achieved by agonists (e.g., acetylcholine, oxytocin, and prostaglandin f2), causes smooth muscle contractions. acetylcholine (ach), the main contractile transmitter in many smooth muscle tissues (e.g., the urinary bladder and gastrointestinal tract), activates muscarinic (m3) receptors and raises intracellular ca levels by activating the gq - phospholipase c- (plc-) inositol triphosphate (ip3) pathway. it is also believed that extracellular ca facilitates an increase in intracellular ca via opening of the voltage - gated ca channels. m2 receptor binding of ach also elevates intracellular ca through a number of controversial mechanisms including inhibition of the production of cyclic amp (camp). in the bladder smooth muscles, where muscarinic receptor is the primary efferent receptor, agonist - induced contraction is largely dependent on ca entry through nifedipine - sensitive channels and activation of the rho - kinase pathway. on the other hand, oxytocin and prostaglandin f2 act on oxytocin and prostaglandin receptors, respectively, and release ca from intracellular stores through stimulation of the gq - plc - ip3 system [810 ]. they may also propagate the influx of extracellular ca through voltage - gated l - channels. in addition, oxytocin may activate nonselective cation channels and ca - activated cl channels leading to depolarization of myometrial cells and, eventually, the opening of voltage - dependent ca channels [2, 11 ]. in light of these observations, drugs that could block the effects of these agonists induce smooth muscle relaxation through some mechanisms that could block or interfere with ca entry. antimuscarinic agents, those that oppose the effects of ach, are effective bladder and intestinal smooth muscle relaxants and are well - known standard therapies for oab and in some forms of gastrointestinal motility disorders. in addition, ca channel blockers (ccbs) are effective oab interventions although they are more commonly used for hypertension and other cardiovascular diseases. ccbs block ca entry by binding to the l - type ca channels in the heart and smooth muscles of the peripheral vasculature, thereby generating vasodilation and eventually lowering blood pressure. oxytocin antagonists, ccbs, prostaglandin synthase inhibitors, and -adrenergic agonists are used as tocolytics (medications to suppress premature labor) by virtue of their influence on lowering intracellular ca levels. -adrenergic agonists, alike some nonsteroidal anti - inflammatory drugs, increase the level of camp which results in the decrease in intracellular ca by stimulating efflux of ca from the cell and also uptake by the sarcoplasmic reticulum. however impressive the above - named agents are in managing abnormal smooth muscle contractions, their efficacy and application are limited due to some reported drug - induced side effects. in fact, the application of these compounds may exacerbate the diseases albeit only in extreme cases. antimuscarinic drugs, although effective in inhibiting bladder and intestinal contractility, also influence normal contractility thus affecting normal voiding and excretion functions. moreover, muscarinic m3 receptors are found in the salivary glands thus severe dry mouth is expected with the use of antimuscarinic agents. the standard tocolytics, although efficacious in arresting preterm labor, also produce serious maternal and cardiovascular or adverse fetal side effects [15, 16 ]. altogether, these findings indicate that there is a necessity to develop other smooth muscle relaxants, preferably those that act on a different mechanism. another way to counteract defective smooth muscle contractility is to enhance repolarizing (potassium [k ]) currents. k channels are abundantly expressed in smooth muscles where they play an important role in determining and regulating the excitability of the cell by acting as an excitability brake. a number of k channel openers have been developed, and they showed promise in preclinical and clinical studies for a variety of smooth muscle hyperactivity disorders. among them are the openers of the atp sensitive k (katp) channels and ca - activated k (kca) channels. however, as k channels are ubiquitously expressed in virtually all cell types, it has been thought that k channel openers may not show tissue selectivity. indeed, the most investigated katp channel openers pinacidil and cromakalim effectively abolished unwanted bladder contractions without affecting normal voiding. however, they also exhibited limited bladder selectivity and influenced cardiovascular functions [1923 ]. at least two noncollaborating laboratories have reported the synthesis of novel k channel openers which they thought as effective interventions for smooth muscle hyperactivity disorders, particularly in oab. butera and colleagues first reported the production of benzofuroindole analogues in their continued effort to develop potent bladder relaxants with minimal hemodynamic effects. these benzofuroindole compounds were produced by manipulating the structure of the benzopyran - based antihypertensive and prototype katp channel opener celikalim. initial structural modifications of celikalim accidentally led to the production of the fisher - indole product 10h - benzofuro[3,2-b]indole (compound 7) (figure 1), a derivative which displayed not only potent bladder relaxant effects in in vitro screenings, but also high bladder (versus aorta) selectivity. on the other hand, another group produced benzofuroindole compounds by overlaying compound 7 (see above), with bms-204352, a prototypical opener of one type of kca channels, the large conductance ca - activated k (bkca) channel. one of the derivatives, compound 22, (4-chloro-7-trifluoromethyl-10h - benzofuro[3,2-b]indole-1-carboxylic acid) (figure 1) demonstrated in vitro inhibition of bladder contractions without influencing contractility of the blood vessels. multiple analyses showed that the above - mentioned benzofuroindole compounds were potent activators of the bkca channels [2426 ]. bkca channels, relative to other k channel types, have more superior biophysical, molecular, and pharmacological properties making them more appealing targets to achieve smooth muscle relaxation (see below). in the succeeding sections, the attractive features of benzofuroindole compounds as smooth muscle relaxants are described, as well as some of the concerns that need to be addressed if they were used clinically as antispasmodics or tocolytics. as stated above, the first ever synthesized benzofuroindole analogue (compound 7) was derived from a katp channel opener. thus, it came as a surprise when the compound potently activated the bkca channels. accordingly, the effects of compound 7 were readily reversed by the specific bkca channel blocker iberiotoxin, but not by glyburide (a selective katp channel blocker). furthermore, voltage clamp studies on isolated rat bladder myocytes showed that the compound caused an iberiotoxin - sensitive increase in hyperpolarizing current, further intensifying its bkca channel - potentiating properties. on the other hand, gormemis. compound 22, along with other novel benzofuroindole derivatives, was shown to effectively potentiate cloned bkca channels expressed in xenopus laevis oocytes. the ionic currents caused by compound 22 were blocked by the peptide bkca channel blocker charybdotoxin indicating selective activation of the bkca channels. further electrophysiological characterizations of one the potent derivatives, compound 8 (7-trifluoromethyl-10h - benzofuro[3,2-b]indole-1-carboxylic acid), showed that it highly activated cloned bkca channels from the extracellular side independent of subunits and regardless of the presence of intracellular ca (for a review on bkca channel structure, see figure 2). in addition, it activated native bkca channels from rat hippocampus pyramidal neurons, a finding which might have important clinical roles. but just how remarkable is it when a compound is an opener of the bkca channels ? some excellent reviews on the structure, pharmacology, functions (figure 2), and the potentiality of bkca channels as novel therapeutic targets have been made [29, 30 ]. structurally, bkca channels are composed of two different subunits : the pore - forming subunit and the auxiliary subunits. although channels formed only by four subunits can be functional, subunits alter the biophysical and pharmacological properties of homomeric channels, including ca and voltage sensitivity and gating kinetics [28, 3134 ]. these characteristics of bkca channels make them appealing targets, and their activators potent therapies for many diseases : (1) abundant distribution like other k channel types, (2) high conductance (~200 ps) even at low probability of opening, thus facilitating more efficient k efflux and membrane hyperpolarization (relaxation), (3) high sensitivity to both intracellular ca concentrations and voltage, (4) ca independence, that is, bkca channels can open even in the absence of ca and the ca and membrane potential dependence of the channels are independent of each other [29, 30 ]. a number of bkca channel openers, derived from natural products and from synthetic chemistry, have been developed and reported (e.g., dehydrosoyasaponin - i, maxikdiol, ns1619, bms-204352, 17-estradiol, ethylbromidetamoxifen, pimaric acid, and epoxyeicosatrienoic acids [3538 ]. these substances, however, differ in properties and in some respects, mechanisms of action. for instance, dehydrosoyasaponin - i and 17-estradiol may require subunits for optimum channel potentiation [31, 39 ], while some compounds (e.g., dehydrosoyasaponin - i and 17-estradiol) may act only on the intracellular side of the channel by being highly impermeable. unexpectedly, given the potentiality of bkca channel openers as future interventions in many disease states, it is surprising that only four bkca channel openers have entered clinical development (ns-8, ta-1702, bms-223131, and bms-204352). to the best of our knowledge, clinical trials for the bkca channel openers ns8, bms204352, and ta-1702 have been discontinued, while only one drug candidate, andolast (for the treatment of asthma), remains in the early phase of clinical development. in the view of garcia., there is still much validation required for bkca channel openers to progress as future smooth muscle relaxants. novel bkca channel openers must show appropriate potency and selectivity, efficacy in preclinical disease models, and, most of all, lesser toxicity. aside from remarkably potentiating bkca channels from rat bladder myocytes, benzofuroindole compounds developed by butera and colleagues were also shown to be highly bladder selective with aorta / bladder ic50 ratios ranging from 8- to 46-fold. this was ascertained through organ bath studies with isolated rat bladder and aortic rings. in their studies, compound 15 showed to be the most bladder selective (ic50 ratio aorta / bladder = 46). the structure - activity relationships for these compounds have been reported and reviewed [14, 24 ]. by looking at the structures of compounds 7, 14, 15 and those of the other highly bladder selective derivatives (compounds 22, 23, and 24), bladder specificity could be attributed to the imbedded 5,5 ring system that is fairly tolerant of structural modifications (figure 3). meanwhile, dela pea. also disclosed the bladder (versus aorta) selectivity profile of compound 22, the benzofuroindole analogue synthesized by gormemis and colleagues. in their multiple screenings, compound 22 or ldd175 displayed 20-fold selectivity for the rat bladder compared with the aorta (when emax values are compared). what is more, compound 22 did not have any significant vasorelaxant activity. in vivo screenings in the spontaneously hypertensive rat (shr), an animal model of hypertension also showed that compound 22 did not alter the rat 's hemodynamic activities. in addition, the same group demonstrated that oral administration of compound 22 reduced voiding frequency and lengthened void intervals in shr, a putative animal model of oab. it is noteworthy that these effects were seen only in the shr and not in the normotensive strain, the wistar kyoto rats, a finding that might have significant clinical implications. in certain disease states such as oab, a major drawback of current pharmacotherapies as well as those drugs in development, is their ability to affect cardiovascular activities. katp channel openers, compounds first developed for oab, also activated katp channels in the heart and peripheral blood vessels and brought hemodynamic side effects. for this reason, the development of katp drugs for oab has been abandoned in recent years. the focus has been shifted to other k channel openers, such as bkca and the recently identified kcnq channels openers. compared with katp channels, bkca channels are less expressed in the heart tissue [29, 30 ] but are abundant in the bladder smooth muscles and also in neuronal tissues. with regard to their expression in neuronal tissues, bkca activators could then impact oab whether the underlying etiology is either neurogenic or myogenic in nature. in fact, it is proposed that targeting the neuronal channels could minimize cardiovascular side effects, although it might also lead to the emergence of unwanted neuronal side effects. bkca channels are abundantly distributed in many smooth muscle types such as gastrointestinal smooth muscles and the uterus [4547 ]. as not much is known about the effects of benzofuroindole compounds in other tissues, an ongoing effort has been made to investigate this matter. compound 22 also displayed potent inhibition of both spontaneous and agonist - induced contractions of the ileum and uterus. ec50 values of the relaxant effects of compound 22 are shown in table 1. while compound 22 did not have any intrinsic relaxant activity in the bladder (suggesting its lack of effect on myogenic contractions), it significantly inhibited spontaneous contractions of the ileum and the uterus. however, the effect of compound 22 was more demonstrated in ach - induced contractions of the bladder versus ileum and uterus. the detrusor was not responsive to the effects of compound 22 in electric field stimulation- (efs-) induced contractions. conversely, the relaxative effect of compound 22 in efs - induced ileal contraction was comparable to that induced by ach. in bladder and ileum strips, however, compound 22 was not as potent as atropine, an antimuscarinic drug, in inhibiting ach - induced contractions [26, 43 ]. in the uterus, although compound 22 relaxed oxytocin, prostaglandin f2, and ach - induced contractions, its effects were not as potent as those of standard tocolytics (note, however, the similarity in the degree of potency between diclofenac and compound 22 in inhibiting pgf2-induced contractions). nevertheless, the effects of compound 22 were all consistent with the activation of bkca channels, as evidenced by the counter effects of bkca channel blockers iberiotoxin or penitrem a [26, 43, 44 ] on the relaxative effects of compound 22 in high k- (20 mm kcl-) induced contractions (table 1). other ion channels or contractile machineries may also be at play considering the effects of compound 22 in high - k- (6080 mm kcl-) induced contractions [26, 43, 44, 48, 49 ]. taken together, these findings indicate that in addition to bladder - relaxant activities, compound 22 also influences intestinal and uterine contractions. although k channel openers hold promise as effective and safer alternatives to many smooth muscle relaxants that are used today, most k channel openers in development have not lived up to our expectation. a majority of katp channel openers lacked selectivity and brought unwanted side effects (e.g., cardiovascular) considered worse than those wrought by standard antispasmodics or tocolytics. thus, the development of katp channel openers has been discontinued, and the interest was shifted to developing other k channel openers that are as efficacious as standard smooth muscle relaxants, but with significantly better side effect profile. recent years have seen great advances in our understanding of the structure and function of existing k channels. due to unmet expectations with katp channel openers, newer channels or associated channel proteins have been characterized as potential drug targets. as stated above, among those explored were the bkca channels. while the role of bkca channels in the cns is complex and still an area of active academic research, there appears to be a consensus on the contribution of these channels to the regulation of smooth muscle tone. in this paper, we reviewed the contribution of benzofuroindole derivatives in the field that searches for ideal compounds for oab (or for other pathophysiologic conditions). we have stated the remarkable profiles of these compounds if considered as future oab drug treatment. some investigators, however, showed the potency of a certain benzofuroindole analogue (compound 22) to relax ileum and uterine contractions, indicating that like other k channel openers, selectivity is still an area of concern with benzofuroindole compounds. however, it is still too early to conclude that benzofuroindole analogues have no place in the roll of alternative or safer smooth muscle relaxants. more investigations are required to better understand their mechanics and characteristics and ultimately to address their lack of selectivity. the potency of various benzofuroindole compounds in smooth muscle types can be compared and from there we may discover the drugs ' most appropriate clinical application. finally, the worth of bkca channel openers as better smooth muscle relaxants is just proven theoretically but not in clinical practice. not much is known about the physiology of bkca channels and their activators in the disease state, thus, whether or not a bkca channel opener will be found to have therapeutic utility will depend on the appropriate counterbalance of bkca channel activation versus other excitatory inputs. moreover, some reported bkca channel openers do not satisfy some of the criteria set in clinical tests to prove their worth as effective smooth muscle relaxants (for review see). this explains, in part, the slow pace in the development of bkca channel openers as smooth muscle hyperactivity interventions. however, as molecular biology and drug development techniques are getting more and more advanced, it is plausible that, in the next few years, concerns that limit the potential use of bkca channel openers (especially those that are recently characterized) will be resolved. therefore, the road ahead may be tedious for benzofuroindole compounds, but there is still optimism with regard to their potential use as effective smooth muscle relaxants.	at least two laboratories have independently reported the synthesis of benzofuroindole compounds having potential therapeutic implications in many disease states including those that involve smooth muscle hyperactivity. through a series of in vitro screenings, they demonstrated the efficacy (and selectivity) of these compounds to potentiate large conductance calcium- (ca2 + -) activated k+ (bkca) channels, by far, the most characterized of all ca2 + -dependent k+ channels. interestingly, promising benzofuroindole derivatives such as compound 7 (10h - benzo[4,5]furo[3,2-b]indole) and compound 22 (4-chloro-7-trifluoromethyl-10h - benzo[4,5]furo[3,2-b]indole-1-carboxylic acid) both exhibited high bladder (versus aorta) selectivity, making them attractive alternative treatments for bladder overactivity. in recent reports, compound 22 (ldd175 or tbic) also showed inhibition of ileum and uterine contractions, indicating multiple target tissues, which is not surprising as bkca channels are ubiquitously expressed in the animal and human tissues. in this paper, the authors discuss the value of benzofuroindole compounds and the challenges that need to be overcome if they were considered as smooth muscle relaxants.
we report a 50-year - old female patient with a stage 2 idiopathic macular hole that closed spontaneously. the stage 2 idiopathic macular hole closed spontaneously in 6 weeks with a lamellar defect in the outer retina due to the formation of the bridging retinal tissue, but without any evidence of the common mechanisms of spontaneous closure such as posterior vitreous detachment or epiretinal membrane formation. an idiopathic macular hole was identified as a unique clinical entity more than 100 years ago. most macular holes occur as an age - related primary idiopathic condition, unrelated to other ocular problems or antecedent events. the hallmark inciting event of an idiopathic macular hole formation gass [3, 4 ] classified macular holes into 4 stages. stage 1, also known as impending macular holes, stages 24 include full - thickness macular holes, which are further divided into smaller holes (< 400 m in diameter (stage 2)), holes larger than 400 m in diameter (stage 3) and with a complete posterior vitreous detachment (stage 4). it has been clinically established that stage 1, impending macular holes, have a 50% chance for spontaneous closure with the resolution of symptoms. however, spontaneous resolution with hole closure and a restoration of the normal foveal contour is very rare in full - thickness macular holes (stages 24). it occurs in 24% of the eyes [6, 7 ] ; therefore, these cases are usually treated surgically by pars plana vitrectomy, with or without internal limiting of the membrane peeling. we report a 50-year - old female patient with a stage 2 idiopathic macular hole that closed spontaneously. she also had a lamellar defect in the outer retina due to the formation of the bridging retinal tissue, but without any evidence of the common mechanisms of spontaneous closure such as posterior vitreous detachment or epiretinal membrane formation. the patient presented to us in may 2013 with a right eye vision diminution, which started 1 month ago. on examination, the best - corrected visual acuity was 20/125 in the right eye and 20/30 in the left eye. slit lamp examination revealed a clear cornea and a pupil that was normal in size and reacting to light ; early cataract in both eyes was also detected. the posterior segment showed the presence of a full thickness centric macular hole (stage 2) in the right eye. further examination with the amsler grid test demonstrated central metamorphopsia in the right eye. in the other eye, the fundus, the disc and the retinal vasculature appeared healthy ; the macula was within normal limits. optical coherence tomography (oct) was performed for the right eye, which revealed a full - thickness macular hole (stage 2) with cystic spaces suggesting an accumulation of secondary vitreous fluid and no posterior vitreous detachment (fig. 1). various treatment options, including conservative management, were discussed with the patient. the best corrected visual acuity was checked and was 20/40 on the snellens chart for the right eye and 20/30 for the left eye. fundus examination of the right eye revealed no evidence of a macular hole, and the watzke - allen test was negative. oct revealed the closure of the macular hole with the resolution of the cystic spaces and bridging of the inner retinal layers with lamellar defects without any posterior vitreous detachment or glial tissue proliferation (fig. the follow - up after 12 weeks showed persistent outer lamellar defects on the oct ; visual acuity improved marginally to 20/30 (fig. in our patient, macular hole closure was confirmed by oct examination, showing a sensory retina bridging over the former area of the macular hole with small outer layer defects. the bridging of the retinal tissue allowed the resolution of the cystoid spaces by preventing the influx of vitreous fluid into intraretinal spaces and therefore leading to a spontaneous closure of the macular hole. four explanations have been proposed for the spontaneous resolution of a macular hole : (1) complete detachment of the posterior hyaloid from the foveal area leading to a release of traction ; (2) cell proliferation at the base of the hole ; (3) formation of a contractile epiretinal membrane resulting in shrinkage and closure of the hole, and (4) bridging of the retinal tissue across the hole. out of the 4 mechanisms mentioned above, posterior vitreous detachment and epiretinal membrane formation may or may not always be evident in patients with spontaneous closure of a macular hole, but the bridging of the sensory retina and the smaller size of the macular hole appear to be the most consistently reported findings for the spontaneous closure of macular holes [12, 13 ]. as to the origin of the bridging of the retinal tissue, proliferation of the retinal postmitotic neurosensory cells has been proposed, but could not be identified ; proliferation of glial or retinal pigment epithelial cells has also been suggested. since the concept of cell proliferation as a mechanism of macular hole closure is still speculative, the exact mechanism of how the spontaneous macular hole closure with maintained normal retinal structure occurs is still unclear. none of the authors have financial or proprietary interests in any material or method mentioned.	objectivewe report a 50-year - old female patient with a stage 2 idiopathic macular hole that closed spontaneously.methodthe case is presented on the basis of an observational case report.resultsthe stage 2 idiopathic macular hole closed spontaneously in 6 weeks with a lamellar defect in the outer retina due to the formation of the bridging retinal tissue, but without any evidence of the common mechanisms of spontaneous closure such as posterior vitreous detachment or epiretinal membrane formation.
a variety of techniques have been introduced for making orthotopic neobladder after simple and radical cystectomy for benign, invasive or recurrent bladder tumors, such as ileum, colon and sigmoid. nonetheless, a controversy exists and there are scarce reports in the literature on the use of gastric pouch for this purpose. some are against it (1), some advocate its application for certain and specific patients (2), and some others believe that the use of bowel has some limitations including excessive mucus production, metabolic acidosis, stone formation, infection, perforation and malignancy (3). there are some advantages for using a pouch of stomach for cystoplasty and gastric neobladder for example : reimplantation of ureter to this segment is easer, because of its thickness. because stomach is beyond the field of radiotherapy and free of adhesion. stomach is an organ of choice for diversion during chronic renal failure (crf). in the current study we used gastric pouch for augmentation cystoplasty in transitional cell carcinoma (tcc) of bladder for the first time in khorasan province (4), also benign diseases of bladder like severe contracted bladder due to tuberculosis and failed vesicovaginal fistula (5). in addition, cases of gastric neobladder for malignant disease for malignant of bladder were studied. the aim of this retrospective study was to report our experience, mostly on gastric neobladder (not gastrocystoplasty) for benign and malignant bladder diseases, its complications, outcomes and follow - up results. 1996, seven cases of gastric pouch were performed in the urology department of qaem educational hospital, mashhad, iran. five cases of invasive bladder tumor, aged between 40 - 70 (mean : 61.6) years were selected for radical cystectomy after performing 1 - 3 times transuretheral resection of bladder tumor (turbt). all patients underwent staging before cystectomy including turbt, histopathologic studies, bimanual exam under anesthesia, abdominal ct, chest x ray and renal and liver tests. 1, a.a., a 62-year - old male ; tcc of bladder, stage b2, grade 2. 2, m.b., a 62-year - old female ; tcc of bladder, stage b2, grade 3. 3, m.m.k., a 51-year - old male ; recurrent multiple papillary tumor, involving trigone and lateral walls, stage a, grade 2 (history of repeated turbt in a short period of time). 4, g.a., a 70-year - old male with tcc of bladder, stage b2, grade 2. 5, a.a.k., a 63-year - old male with tcc of bladder, stage b2, grade2. all patients underwent cystourethroscopy (including evaluation of urethra) and biopsy from suspicious areas was performed ; however, no in situ carcinoma was diagnosed in any case. patients no. 6 and 7 had urinary tuberculosis (tb) with positive results for urine culture. 6 was a 72-year - old man who had irritative symptoms plus gross painless hematuria and nearly true urinary incontinence. his ivp (intra venous pyelography) showed ureterovesical junction (uvj) stenosis with a severely contracted (thimble) bladder. after eight weeks of anti - tb therapy because of a 1.7 mg/100 creatinine level (normal : 0.5 - 1.5 mg/100), he underwent gastric neobladder. 7 was a 40-year - old man from afghanistan with a right hydronephrotic kidney with 5% function on dmsa and grade three reflux on the left side in voiding cystourethrography (vcug). because of left hydroureteronephrosis, percutaneous nephrostomy (pcn) was performed on the left side. creatinine decreased from 5.1 mg/100 ml to 4.2 mg/100 ml and nephrostography showed a left - sided stenosis. it consisted of liquid food as well as bisacodyl, 2 tablets a day, enema at night with normal saline, plus metronidazol 2 tablets every 8 hours. patients were npo (nil per os meaning nothing through mouth) from 12 hours before the procedure and received broad spectrum antibiotics intraparentally. routine exploration of intra - abdominal organs was performed as well as bladder mobility evaluations. radical cystectomy was performed for patients no. 2, 3, 4 and 5. regional lymphadenectomy was performed for patients no. 2 and 3, but for patients no. 4 and 5, frozen section of a suspicious lymph node had negative results, therefore lymphadenectomy was not performed. 3, 4 and 5 who were male, total bladder, urachus, seminal vesicles, prostatic urethra, up to the prostatic apex were removed leaving a stump from the membranous urethra. all ureters were cut from 2 cm from the bladder, except for patient no. 1, in whom only partial cystectomy with 2 cm free margin was performed. for patient 2 (female case), anterior pelvic exenteration with hysterectomy, oopherectomy and salpingectomy were performed leaving only a partial section of vagina and urethral stump. 7 whom had a thimble bladder. by marking a triangle on the fundus of the stomach with methylene blue, a 15 cm long base (in our first experience) and then 20 cm long (after the first case) far from antrum to the fundus was selected. gastric wedge was brought to the pelvis by passing it through the transverse mesocolon and small bowel mesentery in a straight direction (figures 1, 2). in case the gastric wedge was anastomosed to the bivalve bladder, after partial cystectomy. in patients 2 and 3, the gastric pouch was anastomosed to the urethra, and then the two ureters in each case were anastomosed to the new bladder. two 8fr feeding tubes were put into the ureters as a stent and a three way 22fr foley catheter inside the bladder (a d.j. because of a small flap and a short pedicle, a distal part of the flap was tubularized around a 22 three way catheter and was anastomosed to the membraneous urethra by six sutures with 2 - 0 chromic catgut. at the end of the operation the bladder was filled with saline solution to evaluate neobladder volume and also urinary leakage. the operation time for patients 1 to 7 were 3, 4.52, 5.5, 6, 5, 6 and 10 hours, respectively (mean : 325 minutes). intravenous cimetidine 200 mg four times a day, was administered after the operation for 4 - 5 weeks. ureteral catheter was removed one week after the operation, and hemovac drain one day later (if the amount of secretion was less than 30 ml). bladder irrigation was performed continuously for the first and second patients, but for patients 3 to 7, it was stopped. however, after getting more experience in benign conditions (t.b.), bladder irrigation was stopped. foley catheter was removed four weeks after the operation, after retrograde cystography to assess leakage. all patients started walking from the third post - op day except patient no. 5, who experienced a longer operation time and post - op tachyarrhythmia. routine exploration of intra - abdominal organs was performed as well as bladder mobility evaluations. radical cystectomy was performed for patients no. 2, 3, 4 and 5. regional lymphadenectomy was performed for patients no. 2 and 3, but for patients no. 4 and 5, frozen section of a suspicious lymph node had negative results, therefore lymphadenectomy was not performed. 3, 4 and 5 who were male, total bladder, urachus, seminal vesicles, prostatic urethra, up to the prostatic apex were removed leaving a stump from the membranous urethra. all ureters were cut from 2 cm from the bladder, except for patient no. 1, in whom only partial cystectomy with 2 cm free margin was performed. for patient 2 (female case), anterior pelvic exenteration with hysterectomy, oopherectomy and salpingectomy were performed leaving only a partial section of vagina and urethral stump. 7 whom had a thimble bladder. by marking a triangle on the fundus of the stomach with methylene blue, a 15 cm long base (in our first experience) and then 20 cm long (after the first case) far from antrum to the fundus was selected. gastric wedge was brought to the pelvis by passing it through the transverse mesocolon and small bowel mesentery in a straight direction (figures 1, 2). the gastric wedge was anastomosed to the bivalve bladder, after partial cystectomy. in patients 2 and 3, the gastric pouch was anastomosed to the urethra, and then the two ureters in each case were anastomosed to the new bladder. two 8fr feeding tubes were put into the ureters as a stent and a three way 22fr foley catheter inside the bladder (a d.j. 4 and 5, because of a small flap and a short pedicle, a distal part of the flap was tubularized around a 22 three way catheter and was anastomosed to the membraneous urethra by six sutures with 2 - 0 chromic catgut. at the end of the operation the bladder was filled with saline solution to evaluate neobladder volume and also urinary leakage. the operation time for patients 1 to 7 were 3, 4.52, 5.5, 6, 5, 6 and 10 hours, respectively (mean : 325 minutes). intravenous cimetidine 200 mg four times a day, was administered after the operation for 4 - 5 weeks. ureteral catheter was removed one week after the operation, and hemovac drain one day later (if the amount of secretion was less than 30 ml). bladder irrigation was performed continuously for the first and second patients, but for patients 3 to 7, it was stopped. however, after getting more experience in benign conditions (t.b.), bladder irrigation was stopped. foley catheter was removed four weeks after the operation, after retrograde cystography to assess leakage. all patients started walking from the third post - op day except patient no. 5, who experienced a longer operation time and post - op tachyarrhythmia. fortunately, rare complications occurred from such massive and pr operations. expected complications were summarized in table 1. all patients were followed up by renal and bladder us (ultrasonography), ivp (intravenous pyelography) and metabolic assay by measuring na, k, bicarbonate, blood and urine ph, and in one case measuring serum gastrin. 2, 5, 6 and 7 were living in the current city and had a more precise follow - up compared to patients no. 2 and 4 who lived in other cities. our follow - up period was 5 - 36 (mean : 20.2) months. in these cases we had one dysuria hematuria syndrome (which is more common in pediatric patients), who had undergone gastrocystoplasty after failed vesicovaginal and contracted bladder. in this case, we prescribed h2 blocker, continuously. as we had no urodynamic facility at that time, only one case was sent to a higher advanced center for this purpose and the results are presented, but their bladders capacity and residual urine were measured. its results are as follows : the neobladder volume was 400 ml in patients 1, 2, 4 and 5 but in cases 3, 6 and 7, were 310, 100 and 350 ml, respectively. final follow - up for patients no. 1, 3 and 5 were 36, 24 and 5 months, respectively, and all were in a good condition until that time. in patient 7 was alive with end stage renal disease and awaiting renal transplantation up to writing the article (we have no waiting list for iranian patients but as a law, a foreigner must introduce a relative live donor from his or her country). one of our patients was a woman who received a urethral catheter due to urinary urge incontinence. the rest of our patients had acceptable levels of urinary continence (figures 3 - 12). 2, 5, 6 and 7 were living in the current city and had a more precise follow - up compared to patients no. 2 and 4 who lived in other cities. our follow - up period was 5 - 36 (mean : 20.2) months. in these cases we had one dysuria hematuria syndrome (which is more common in pediatric patients), who had undergone gastrocystoplasty after failed vesicovaginal and contracted bladder. in this case as we had no urodynamic facility at that time, only one case was sent to a higher advanced center for this purpose and the results are presented, but their bladders capacity and residual urine were measured. its results are as follows : the neobladder volume was 400 ml in patients 1, 2, 4 and 5 but in cases 3, 6 and 7, were 310, 100 and 350 ml, respectively. final follow - up for patients no. 1, 3 and 5 were 36, 24 and 5 months, respectively, and all were in a good condition until that time. in patient 7 was alive with end stage renal disease and awaiting renal transplantation up to writing the article (we have no waiting list for iranian patients but as a law, a foreigner must introduce a relative live donor from his or her country). one of our patients was a woman who received a urethral catheter due to urinary urge incontinence. the rest of our patients had acceptable levels of urinary continence (figures 3 - 12). sinaiko performed the first gastric neobladder in 1956 after partial cystectomy in a patient with bladder tumor (7) ; then leong used a gastric pouch for augmentation after tuberculosis cystitis and bladder tumor (8). since then a few articles were published on advantages and disadvantages of this type of procedure. all authors believe that it is a good segment for bladder replacement in boys (9), as well as adults after cystectomy for benign (5) and malignant diseases (10). however, it is still controversial weather to use it as a neobladder in adults after radical cystectomy for bladder tumors. believed that adult gastric neobladder is associated with poor urodynamic parameters and high incontinence rates (1). on the other hand, koraitim. reported that stomach is an ideal and suitable segment for bladder augmentation and/or creating a neobladder (10). compared to other segments of the gastrointestinal tract, stomach has a thicker wall, which makes it easy to perform antireflux ureteral anastomosis. furthermore, in patients with invasive bladder tumor who receive radiotherapy, stomach is far from the field of radiotherapy compared to other parts of the gastrointestinal tract. moreover, it makes less mucus production, so suprapubic catheter in gastric neobladder is not necessary (5) ; and the last but not least, stomach is impermeable to most ions especially chloride, which is transported in urine (10) ; thus, it has a protective effect against acidosis in patients with renal impairment. one of the most common complications of gastric neobladder or gastrocystoplasty is perforation of the pouch. studies showed that perforation in gastric pouch is negligible compared to ileal, ileocaecal or sigmoid ones. therefore, it is an ideal segment in patients with concomitant renal failure and invasive bladder tumors. our mean operative time was 325 minutes (180 - 600) compared to 365 (300 - 450) minutes in wang. study with more than 20 years of experience on gastric replacement therapy (3). there are many intraoperative and postoperative complications such as blood loss, hematemesis, intestinal adhesion, increased serum gastric and cardiac failure (one case, due to anesthetic problem, is not in this series). santucci. performed seven gastric neobladders and concluded that gastric pouch has a smaller capacity with greater incontinence rates (2). they performed urodynamic study after an average of 9.1 months and reported the volume of neobladder as 311 ml. lin. performed eight gastric neobladders (malignancy in seven and undiversion in one) with an average follow - up of 9.1 months. they reported that gastric neobladder capacity reduced to a mean of 309 ml and concluded that routine use of gastric neobladder in adults is not recommended (1). capacity of neobladder is an important factor for incontinence and involuntary contraction (11). the final capacity of gastric neobladder can be determined six months (11) and one and a half years after the operation (12). wang. reported their experiences on nine cases of gastric neobladder by performing it after laparoscopic radical cystectomy, showing a good capacity of pouch and continence (3). gastric neobladder is an ideal procedure after cystectomy for bladder benign and malignant diseases. to prevent ureteral stenosis, we should have adopted to do tension free ureteral reimplant ; however, it is more challenging compared to the rate of continence and is totally related to volume of neobladder and surface of the wedge. when the volume and surface of the gastric wedge is high, the rate of continence is greater. nevertheless, we recommend investigating more patients with longer and better follow - up including urodynamic study and gastrin measurement.	background : there is a controversy regarding the use of gastric pouch for benign and malignant bladder diseases.objectives:the aim of this retrospective study was to report our experience, mostly on gastric neobladder (not gastrocystoplasty) for benign and malignant bladder diseases, its complications, outcomes and follow - up results.materials and methods : in this retrospective case series, we described our experience on seven gastric pouches (2 gastrocystoplasty and 5 gastric neobladders).results : postoperative complications were rare. continence was defined as bladder capacity over 400 ml. their follow - up period ranged from five months up to writing the article. one of the studied cases is still alive and awaiting renal transplantation.conclusions:gastric pouch is a suitable segment for bladder cystoplasty and neobladder. continence is mostly related to the capacity of pouch.
endometrial cancer is the commonest gynecological cancer in the developed world. over the years, most accepted structural, immunohistological, and molecular correlations have been linked with epidemiological and outcome data to arrive at a histological model of endometrial cancer.1,2 most endometrial cancers are early stage, low grade, and of endometrioid histology (type i), with a 5-year survival of greater than 85%.4 uterine serous cancer (usc)3 and serous endometrial intraepithelial carcinoma (eic) (also known as minimal uterine serous cancer [musc ]) comprise a highly aggressive histologic type of endometrial cancer (type ii). although type ii cancer represents less than 10% of all endometrial cancers, it accounts for more than 50% of relapses and deaths attributed to endometrial carcinoma.57 type ii cancer is common in older patients, is associated with an atrophic endometrium, and is frequently associated with the tumor protein (p)53 gene mutation. the presence of a p53 mutation helps in the immunohistochemical diagnosis of usc / serous eic. serous eic is found in the background of usc, and hence is considered a precursor of usc.8,9 recent studies show that serous eic is not only the likely precursor of usc but in its pure form, demonstrates a stage - dependent potential for aggressive behavior similar to that of usc.10,11 thus, pure serous eic appears to be a form of minimal usc and has extrauterine disease at presentation in 33% to 45% of cases.10,11 however, pathological and clinical studies of serous eic are limited to a few reports. this case series is an effort to correlate pathological staging with clinical outcome and propose management guidance for pure serous eic. we identified cases of pure eic and usc treated in our institute between 2009 and 2012, from the surgical and histopathology database. only cases with pure eic were included in the series, based on final histopathology. the cases excluded were comprised of all other types of endometrial cancer, including papillary serous cancer and any stage other than stage 1a serous eic. surgical and histopathology reports were reviewed using the oxford university hospital database (case notes) and patient records. the pathology reports and all hematoxylin and eosin (he) slides from endometrial curetting / aspiration biopsy, subsequent hysterectomy specimen, and staging material were reviewed. available results of peritoneal fluid cytology, pelvic nodes, omental biopsy, and uterine histology were noted. this paper also reviews the english language literature for studies on serous eic / musc. a medline (pubmed) search of the relevant literature published in the english language over the 30-year period between january 1980 and february 2013 was performed. endometrial cancer were combined and then searched for the keywords serous endometrial cancer, serous intraepithelial cancer, minimal serous endometrial cancer, and minimal uterine serous cancer. additional publications were identified via cross - referencing from reference lists within publications retrieved from the medline search. as this malignancy is extremely rare and there is paucity of published data, all peer - reviewed case series and reviews and related case reports with adequate number of patients were included in the review. follow - up information (range 328 months) was obtained through the oxford university trust case notes and for cases referred from outside institutions, by contacting the referring clinicians. the pathology reports and all hematoxylin and eosin (he) slides from endometrial curetting / aspiration biopsy, subsequent hysterectomy specimen, and staging material were reviewed. available results of peritoneal fluid cytology, pelvic nodes, omental biopsy, and uterine histology were noted. this paper also reviews the english language literature for studies on serous eic / musc. a medline (pubmed) search of the relevant literature published in the english language over the 30-year period between january 1980 and february 2013 was performed. endometrial cancer were combined and then searched for the keywords serous endometrial cancer, serous intraepithelial cancer, minimal serous endometrial cancer, and minimal uterine serous cancer. additional publications were identified via cross - referencing from reference lists within publications retrieved from the medline search. as this malignancy is extremely rare and there is paucity of published data, all peer - reviewed case series and reviews and related case reports with adequate number of patients were included in the review. follow - up information (range 328 months) was obtained through the oxford university trust case notes and for cases referred from outside institutions, by contacting the referring clinicians. the average age at diagnosis in our group of patients was 72 years (range 6479 years). all the patients in the selected group presented with postmenopausal bleeding, and the initial investigations included pelvic ultrasound scans and endometrial biopsy in all five. three patients (patients 1, 2, and 5) also had a diagnostic hysteroscopy, during which biopsies were taken. the other two patients (patients 3 and 4) were diagnosed by pipelle endometrial biopsy and endometrial polypectomy. clinically, patient 1 had an atrophic endometrium with an isolated polyp as well as an abnormal cervical smear with glandular cells. in patients 2, 3, and 5, an initial diagnosis of serous cancer was made on multiple endometrial polyps, and patient 4 had a thickened endometrium of 6 mm, without a polyp. one patient (patient 5) had received hormone replacement therapy for 2 years at the age of 55. patients 2, 3, 4, and 5 were on treatment for hypertension ; patient 3 had bowel cancer 23 years previously. all five patients had ultrasound scan, which showed endometrial thickness varying from 4 mm to 8 mm, and four out of five patients had endometrial polyps suspected. preoperative imaging confirmed the absence of metastatic disease, and the radiological diagnosis for all five cases was stage 1a. a preoperative cancer antigen or carbohydrate antigen (ca) 125 was available in three patients, and these were within normal range. the four patients who had complete surgical staging performed revealed no extrauterine disease (table 2). the gross appearance of the endometrial cavity showed no significant difference between the patients except for biopsy - related changes, and three out of five uterine specimens had small (12 cm) intramural fibroids. histopathological examination following surgery showed that patients 14 had serous eic in an endometrial polyp (figure 1), in a background of atrophic endometrium. patient 5 had foci of serous eic with a polypoid endometrium and high grade nuclear atypia (figures 2 and 3). four patients had pelvic lymphadenectomy done, with an average node count of 19.5 (range 16 to 25), of which none were positive for disease. three out of five cases had immunohistochemical staining for p53, which exhibited strong reactivity (figure 4). the mean duration of follow up in our series was 16.6 months (range 4 to 30). patient 1 had 30 months follow up, with distal recurrence in the right lung at 10 months from initial surgery. she was treated with a right pneumonectomy at 10 months postsurgery and presented with widespread disease at 20 months. patients 3 and 5 are alive and well, 20 months and 4 months, respectively, after surgery. there are no obvious identifiable risk factors, such as nulliparity, late menopause, hormone replacement therapy, and obesity, linked to the development of serous eic.12 according to one review, there is no link between breast and ovarian cancer syndromes and serous eic.13 there are no proven strategies or lifestyle modifications that can reduce the overall risk of developing serous eic. in this case series, all five patients had postmenopausal bleeding as the presenting symptom, and none of them had any breast or ovarian cancer association. recent studies demonstrate that serous endometrial cancer is found with a background of eic and on its own, as pure serous eic.10,11 thus, pure serous eic appears to be a form of minimal usc. the diagnosis of pure serous eic can be difficult, particularly in biopsy specimens, as the lesion can be very focal and small, involving surface epithelium, and portions of endometrial glands. although it can present in the background of serous endometrial cancer, mixed endometrioid, or sometimes with clear cell cancers,10,11,14,15 pure serous eic is recognized histologically by markedly atypical nuclei, sometimes with prominent nucleoli, many mitotic figures, and apoptotic bodies. various studies describe serous eic as being confined to an endometrial polyp,7,11,16,17 which was the initial presentation in four out of five patients in this series. immunohistochemical stains, such as p53 and ck7, as well as estrogen receptor status have been described in literature to identify these tumors.14,18 three out of five specimens had p53 overexpression with negative estrogen receptor status. in contrast to endometrioid carcinomas, serous eic / musc presents in an atrophic endometrium,8,9 and this was clearly demonstrated in four out of five specimens in our series. routine pelvic ultrasound scan is useful during the initial diagnosis, as most serous eic presents with endometrial polyps, but staging a computed tomography (ct), scan is advisable due to its unusual behavior and presence of extrauterine disease at presentation.19,20 it is evident that at presentation, type ii uterine cancer has an increased an probability of being of advanced stage compared with type i.9,13,19,21,22 even when these tumors are of early stage at initial presentation, they can recur soon after complete surgical staging. recent literature on early stage serous uterine / type ii cancer suggests an excellent prognosis with adequate surgical staging.10,19,20,23,24 serous eic is considered to be the precursor of serous uterine cancer, hence, complete staging for the treatment of serous eic may be recommended. all except one patient in this series had complete staging, confirmed to be stage 1a on final histopathological examination. there are no specific case series or randomized trials investigating pure serous eic, hence, it is vital to manage this early stage malignant disease aggressively with complete staging, even if the lesion is limited to a polyp. currently there is no consensus on the use of systemic adjuvant treatment or pelvic irradiation in early stage serous eic,4,25,26 but with the emerging evidence of unusual sites of recurrence and aggressive tumor behavior, it may be appropriate to revisit adaptation of adjuvant treatment options, particularly since there are no proven tumor markers to detect recurrent disease. systemic treatment with chemotherapy may also be adopted, as distant recurrent disease was one of the recognized patterns of recurrence of disease.5,7,21,22,2426 there are no obvious identifiable risk factors, such as nulliparity, late menopause, hormone replacement therapy, and obesity, linked to the development of serous eic.12 according to one review, there is no link between breast and ovarian cancer syndromes and serous eic.13 there are no proven strategies or lifestyle modifications that can reduce the overall risk of developing serous eic. in this case series, all five patients had postmenopausal bleeding as the presenting symptom, and none of them had any breast or ovarian cancer association. recent studies demonstrate that serous endometrial cancer is found with a background of eic and on its own, as pure serous eic.10,11 thus, pure serous eic appears to be a form of minimal usc. the diagnosis of pure serous eic can be difficult, particularly in biopsy specimens, as the lesion can be very focal and small, involving surface epithelium, and portions of endometrial glands. although it can present in the background of serous endometrial cancer, mixed endometrioid, or sometimes with clear cell cancers,10,11,14,15 pure serous eic is recognized histologically by markedly atypical nuclei, sometimes with prominent nucleoli, many mitotic figures, and apoptotic bodies. various studies describe serous eic as being confined to an endometrial polyp,7,11,16,17 which was the initial presentation in four out of five patients in this series. immunohistochemical stains, such as p53 and ck7, as well as estrogen receptor status have been described in literature to identify these tumors.14,18 three out of five specimens had p53 overexpression with negative estrogen receptor status. in contrast to endometrioid carcinomas, serous eic / musc presents in an atrophic endometrium,8,9 and this was clearly demonstrated in four out of five specimens in our series. routine pelvic ultrasound scan is useful during the initial diagnosis, as most serous eic presents with endometrial polyps, but staging a computed tomography (ct), scan is advisable due to its unusual behavior and presence of extrauterine disease at presentation.19,20 it is evident that at presentation, type ii uterine cancer has an increased an probability of being of advanced stage compared with type i.9,13,19,21,22 even when these tumors are of early stage at initial presentation, they can recur soon after complete surgical staging. recent literature on early stage serous uterine / type ii cancer suggests an excellent prognosis with adequate surgical staging.10,19,20,23,24 serous eic is considered to be the precursor of serous uterine cancer, hence, complete staging for the treatment of serous eic may be recommended. all except one patient in this series had complete staging, confirmed to be stage 1a on final histopathological examination. there are no specific case series or randomized trials investigating pure serous eic, hence, it is vital to manage this early stage malignant disease aggressively with complete staging, even if the lesion is limited to a polyp. currently there is no consensus on the use of systemic adjuvant treatment or pelvic irradiation in early stage serous eic,4,25,26 but with the emerging evidence of unusual sites of recurrence and aggressive tumor behavior, it may be appropriate to revisit adaptation of adjuvant treatment options, particularly since there are no proven tumor markers to detect recurrent disease. systemic treatment with chemotherapy may also be adopted, as distant recurrent disease was one of the recognized patterns of recurrence of disease.5,7,21,22,2426 diagnosis of pure serous eic can be challenging. optimum surgical staging might be essential for best outcomes. future multicenter trials, given the rarity of the entity, using adjuvant treatment in the management of pure eic, would possibly help improve outcomes.	backgroundminimal uterine serous cancer (musc) or serous endometrial intraepithelial carcinoma (eic) has been described by many different names since 1998. there have been very few cases reported in literature since eic / musc was recognized as a separate entity. the world health organization (who) classification favors the term serous eic. although serous eic is confined to the uterine endometrium at initial histology diagnosis, a significant number of patients could have distal metastasis at diagnosis, without symptoms. serous eic is considered as being the precursor of uterine serous cancer (usc), but pure serous eic also has an aggressive behavior similar to usc. it is therefore prudent to have an accurate diagnosis and appropriate surgical staging. there are very few published articles in literature that discuss the pure form of serous eic. the aim of this series is to share our experience and review evidence for optimum management of serous eic.patients and methodswe report a series of five women treated in our institute in the last 3 years. we reviewed the relevant literature on serous eic and various management strategies, to recommend best clinical practice.conclusionpure serous eic is a difficult histopathological diagnosis, which requires ancillary immunohistochemical staining. it can have an aggressive clinical behavior with early recurrence and poor survival. optimum surgical staging, with appropriate adjuvant treatment, should be discussed when treating these patients.
tiling arrays are also gaining popularity for detecting novel transcripts in sequenced genomes. in this context, sequence annotation is an essential step in understanding the genome and the transcriptome of a species. large - scale comparisons of prokaryotic and eukaryotic genomes reveal thousands of conserved regions obtained by homology or synteny. these regions might be protein - coding sequences (1) or non - coding rna genes (2,3,4). annotation by comparative analysis typically involves two steps : first aligning the sequences, then analysing the multiple alignment to detect an evolutionary pattern that is representative of the selection pressure. this idea is exploited in exoniphy (5) for exon detection, in rnaz (6) or evofold (7) for structural rna prediction or in qrna (8), that implements both a coding and an non - coding model for rnas. in this computational protocol this task, however, is difficult because sequence similarity is often reduced at the nucleic level. regarding protein coding genes, nucleic acid sequences exhibit a much larger sequence heterogeneity compared to their encoded amino acid sequences due to the redundancy of the genetic code. it is well known that the combination of nucleic acid and amino acid sequence information leads to improved alignments (9,10). so pure - sequence - based multiple alignment tools perform poorly on low - homology datasets of structural rnas (11). in this article, we present the magnolia website, whose objective is to provide an advanced tool for aligning nucleic acid sequences. the idea is to get rid of the dichotomy between aligning and predicting the function. if we assume that sequences are either protein - coding or structural rnas, it is possible to incorporate some functional information into the alignment algorithm to improve the result. the multiple alignment can then be used as a starting point to refine the comparative analysis or to carry out further predictions, such as motif finding or phylogeny reconstruction. first, it tries to predict the function of the sequences according to the substitution pattern between sequences. second, a multiple alignment is built based on the putative function of the sequences. if the sequences are recognized as coding sequences, then the multiple alignment uses the amino acid sequences. if the sequences are recognized to contain a conserved secondary structure, then the mutiple alignment takes into consideration long - range base pair interactions. magnolia includes three specific modules : protea for protein coding sequences, carnac and gardenia for structural rnas. here the idea is that the selection pressure tends to preserve the encoded amino acid sequence, and it is possible to identifies coding sequences by looking for a global conservation of common reading frames. the method first identifies best potential reading frames from each pair of sequences, and then incorporates this information into a frame graph from which a coding significancy score is calculated. by doing so, it also predicts the associated reading frame for each sequence. if the sequences are classified as protein - coding sequences, then the multiple alignment of nucleic acid sequences is built from the hypothetical amino acid sequences using clustalw (13), dialign2 (10) or t - coffee (14)., the selection pressure tends to preserve the secondary structure of the molecule, and mutations should retain the ability to form base pairs into energetically favorable stems. carnac is able to recover a conserved secondary structure from a set of unaligned sequences. (16,17), that fold and align several sequences at the same time, for example. the algorithm uses a sankoff - based dynamic programing approach to identify conserved strutures for all pairs of sequences. then all pairwise foldings are combined into a graph - theoretical structure called the stem graph. only frequent common stems that correspond to highly connected subgraphs in the stem graph are retained. the method takes into account both the nucleic sequence and the putative common secondary structure predicted by carnac. it relies on the dynamic programming algorithm for pairwise comparison proposed in ref. (18). rna sequences are encoded as arc - annotated sequences, and a multiple alignment for a set of arc - annotated sequences is a nested common supersequence. the edit scheme incorporates evolutionary operations concerning free bases (base substitution, base deletion) and base pairs (arc - mismatch, arc - removing, arc - breaking, arc - altering), originally defined in ref. the method starts with constructing all pairwise alignments to determine the degree of similarity for each pair of sequences. pairwise alignment of supersequences rely on the same algorithm as pairwise alignments for arc - annotated sequences. this is made possible because supersequences can be viewed as a nested arc - annotated sequences on an extended alphabet. the score of one node is its sp (sum - of - pairs) score. lastly, the space search of the dynamic programming alignment is pruned using constraints coming from the carnac output. magnolia requires as input data a set of rna or dna sequences in the standard fasta format. it can be stored in a file to be uploaded to the server, or pasted directly in the text box. the job assigned a unique identifier that can be used to retrieve results for one week. if input sequences are annotated as coding sequences, then two multiple alignments are displayed. the first alignment is built on the putative amino acid sequences obtained by virtual translation using the predicted reading frame, and the second alignment is the corresponding alignment on nucleic acid sequences obtained by reverse translation, allowing for frameshifts. codons in the nucleic acid sequences are put in color : two base triplets coding for the same amino acid bear the same color. figure 2 shows an example of magnolia output obtained with a family of protein - coding sequences. if input sequences are annotated as structural rna genes, then a multiple alignment taking into consideration the primary structure accompanied by the secondary structure is displayed. concerning the secondary structure, base pairings are indicated in bracket - dot format : each base - pair is represented by a pair of matching brackets and unpaired bases are represented by dots. the lack of pseudoknots in the secondary structure ensures that this notation defines a unique folding. figure 3 shows an example of output obtained with a family of non - coding rnas. for each sequence, the individual putative secondary structure is also provided in five formats : ct, jpeg, ps, bracket - dot format and as a list of constrained base pairings. jpeg and ps files are automatically produced from the ct file using the naview layout program (22). all pairings are correct, and the multiple alignment is consistent with the reference alignment available in rfam - rf00005 (21). all pairings are correct, and the multiple alignment is consistent with the reference alignment available in rfam - rf00005 (21). some data sets are not identified as coding rnas nor as non - coding rnas. the first possibility is that the sequences might have an alternative function, such as untranslated regions in messenger rnas, promoter elements, etc. the evolutionary signal is too weak and the sequences do not exhibit any significant mutational bias towards any model. this limitation is harmless for practical purposes, because standard multiple sequence alignment tools usually yield good results on high - identity data sets. so when the average identity percentage is greater than 90%, the server outputs a warning message and provides a default multiple alignment constructed directly with clustalw on the initial data set. two such examples are the cis - acting regulatory element from the human rhinoviruses, that is located in the open reading frame of the capsid proteins [rfam rf00220 (21) ], or the hepatitis c stem - loop vii structure found in the coding region of the rna - dependent rna polymerase gene ns5b [rfam rf000468 (21) ]. magnolia requires as input data a set of rna or dna sequences in the standard fasta format. it can be stored in a file to be uploaded to the server, or pasted directly in the text box. the job assigned a unique identifier that can be used to retrieve results for one week. if input sequences are annotated as coding sequences, then two multiple alignments are displayed. the first alignment is built on the putative amino acid sequences obtained by virtual translation using the predicted reading frame, and the second alignment is the corresponding alignment on nucleic acid sequences obtained by reverse translation, allowing for frameshifts. codons in the nucleic acid sequences are put in color : two base triplets coding for the same amino acid bear the same color. figure 2 shows an example of magnolia output obtained with a family of protein - coding sequences. if input sequences are annotated as structural rna genes, then a multiple alignment taking into consideration the primary structure accompanied by the secondary structure is displayed. concerning the secondary structure, base pairings are indicated in bracket - dot format : each base - pair is represented by a pair of matching brackets and unpaired bases are represented by dots. the lack of pseudoknots in the secondary structure ensures that this notation defines a unique folding. figure 3 shows an example of output obtained with a family of non - coding rnas. for each sequence, the individual putative secondary structure is also provided in five formats : ct, jpeg, ps, bracket - dot format and as a list of constrained base pairings. jpeg and ps files are automatically produced from the ct file using the naview layout program (22). all pairings are correct, and the multiple alignment is consistent with the reference alignment available in rfam - rf00005 (21). all pairings are correct, and the multiple alignment is consistent with the reference alignment available in rfam - rf00005 (21). some data sets are not identified as coding rnas nor as non - coding rnas. the first possibility is that the sequences might have an alternative function, such as untranslated regions in messenger rnas, promoter elements, etc. the evolutionary signal is too weak and the sequences do not exhibit any significant mutational bias towards any model. but this limitation is harmless for practical purposes, because standard multiple sequence alignment tools usually yield good results on high - identity data sets. so when the average identity percentage is greater than 90%, the server outputs a warning message and provides a default multiple alignment constructed directly with clustalw on the initial data set. two such examples are the cis - acting regulatory element from the human rhinoviruses, that is located in the open reading frame of the capsid proteins [rfam rf00220 (21) ], or the hepatitis c stem - loop vii structure found in the coding region of the rna - dependent rna polymerase gene ns5b [rfam rf000468 (21) ]. in such cases, magnolia releases two multiple alignments. we evaluate the accuracy of the method on two large data sets : pandit (20) and bralibase ii (11). pandit is a registry of families of homologous protein domains, accompanied by curated rna sequence alignments. bralibase ii is a set of non - coding rna families that has been used to establish a benchmark of multiple sequence alignment programs upon structural rnas. it is composed of four families : group ii introns, 5s rrna, trna and u5 spliceosomal rna. for each family 6122 (94.3%) families are correctly classified as coding sequences, among them more than 99% with the correct reading frame predicted for each sequence. less than 3% of the families are classified as structural rna. to estimate the quality of the alignments, we used the sum - of - pairs score (sps) of the baliscore software (23). the sps is calculated such that it increases with the number of sequences correctly aligned. we compared magnolia with clustalw, t - coffee and dialign2 on the same nucleic acid sequences. the x - axis represents the average identity percentage and the y - axis represents the sps value. for magnolia, we tried all possible combinations concerning the alignment tool in the amino acid alignment step : clustalw, t - coffee and dialign2. for dialign2, we selected the appropriate option translation of nucleotide diagonals into peptide diagonals when comparing the nucleic acid sequences. the x - axis represents the average identity percentage and the y - axis represents the sps value. for magnolia, we tried all possible combinations concerning the alignment tool in the amino acid alignment step : clustalw, t - coffee and dialign2. for dialign2, we selected the appropriate option translation of nucleotide diagonals into peptide diagonals when comparing the nucleic acid sequences. this benchmark contains 388 alignments, that are classified into high, medium and low identity data sets. magnolia failed to identify a structural evolutionary pattern for 20% of them and falsely assigned a protein coding function for 7% of them. following ref. (11), we use the structure conservation index (sci) to assess the accuracy of alignments. this score provides a measure of the conserved secondary structure information contained within the alignment. results for magnolia are reported in figure 5, together with results for other alignment tools used in the benchmark. we also evaluated the accuracy of the secondary structure found by magnolia and compared it to two recent structural alignment programs : murlet (16) and mlocarna (17). for each software and for each identity class, we computed the percentage of correct base pairings amongst the set of predicted base pairings. furthermore, the total runtime is more than 12 times faster for magnolia than for the two other methods (< 20 min for the whole data set, compared to more than 4 hours). the x - axis represents the average identity percentage and the y - axis represents the sci value. magnolia appears to be the closest curve to the reference curve for identity percentage ranging from 40% to 80%. for higher identity percentages, table 1.accuracy percentage for magnolia, murlet and mlocarna for reference secondary structures of bralibase iiidentity classlowmediumhighmagnolia72.0%76.3%87.0%murlet78.1%76.2%77.8%mlocarna68%71.1%78.9% magnolia alignments on bralibase ii. the x - axis represents the average identity percentage and the y - axis represents the sci value. magnolia appears to be the closest curve to the reference curve for identity percentage ranging from 40% to 80%. for higher identity percentages, 6122 (94.3%) families are correctly classified as coding sequences, among them more than 99% with the correct reading frame predicted for each sequence. less than 3% of the families are classified as structural rna. to estimate the quality of the alignments, we used the sum - of - pairs score (sps) of the baliscore software (23). the sps is calculated such that it increases with the number of sequences correctly aligned. we compared magnolia with clustalw, t - coffee and dialign2 on the same nucleic acid sequences. the x - axis represents the average identity percentage and the y - axis represents the sps value. for magnolia, we tried all possible combinations concerning the alignment tool in the amino acid alignment step : clustalw, t - coffee and dialign2. for dialign2, we selected the appropriate option translation of nucleotide diagonals into peptide diagonals when comparing the nucleic acid sequences. the x - axis represents the average identity percentage and the y - axis represents the sps value. for magnolia, we tried all possible combinations concerning the alignment tool in the amino acid alignment step : clustalw, t - coffee and dialign2. for dialign2, we selected the appropriate option translation of nucleotide diagonals into peptide diagonals when comparing the nucleic acid sequences. this benchmark contains 388 alignments, that are classified into high, medium and low identity data sets. magnolia failed to identify a structural evolutionary pattern for 20% of them and falsely assigned a protein coding function for 7% of them. following ref. (11), we use the structure conservation index (sci) to assess the accuracy of alignments. this score provides a measure of the conserved secondary structure information contained within the alignment. results for magnolia are reported in figure 5, together with results for other alignment tools used in the benchmark. we also evaluated the accuracy of the secondary structure found by magnolia and compared it to two recent structural alignment programs : murlet (16) and mlocarna (17). for each software and for each identity class, we computed the percentage of correct base pairings amongst the set of predicted base pairings. furthermore, the total runtime is more than 12 times faster for magnolia than for the two other methods (< 20 min for the whole data set, compared to more than 4 hours). the x - axis represents the average identity percentage and the y - axis represents the sci value. magnolia appears to be the closest curve to the reference curve for identity percentage ranging from 40% to 80%. for higher identity percentages, table 1.accuracy percentage for magnolia, murlet and mlocarna for reference secondary structures of bralibase iiidentity classlowmediumhighmagnolia72.0%76.3%87.0%murlet78.1%76.2%77.8%mlocarna68%71.1%78.9% magnolia alignments on bralibase ii. the x - axis represents the average identity percentage and the y - axis represents the sci value. magnolia appears to be the closest curve to the reference curve for identity percentage ranging from 40% to 80%. for higher identity percentages,	magnolia is a new software for multiple alignment of nucleic acid sequences, which are recognized to be hard to align. the idea is that the multiple alignment process should be improved by taking into account the putative function of the sequences. in this perspective, magnolia is especially designed for sequences that are intended to be either protein - coding or structural rnas. it extracts information from the similarities and differences in the data, and searches for a specific evolutionary pattern between sequences before aligning them. the alignment step then incorporates this information to achieve higher accuracy. the website is available at http://bioinfo.lifl.fr/magnolia.
organic thin films have attracted considerable interest in the recent past, most notably due to new manufacturing steps and the possibility to create ultrathin and lightweight devices with extreme bending stability, that are very promising in the field of organic electronics. through observing current and projected environmental problems, the aspect of biodegradability has progressively risen in importance and might now be the most crucial argument in pursuing research in the field of organic semiconductors. quinacridone (qa, c20h12n2o2) and other high - performance organic pigments have particularly been in the focus of attention, attributable to the formation of intermolecular hydrogen bonds. quinacridone, also known as linear trans - quinacridone, has a molecular mass of 312.32 amu and was first synthesized 1935 by liebermann. the discovery of its polymorphism and more efficient synthesis methods years later consequently led to simpler synthesis routes and has provoked considerable interest in the scientific community ever since. current literature research unveils a formidable amount of works elucidating how adding various kinds of solubilizing groups to the quinacridone molecule gave rise to a number of new applications, most notably as photodetectors and both donor and acceptor molecules in organic solar cells. their general insolubility, in turn, makes physical vapor deposition the method of choice for manufacturing thin layers, which is in the focus of the present publication. it is well - known that preparation parameters such as substrate temperature, substrate conditions, base pressure, and deposition rate determine the morphology and hence the electronic structure and optical properties of thin films grown by vapor deposition. despite recent reports of air - stable quinacridone field - effect transistors with relatively high carrier mobilities of 0.2 cm/(v s), there is still a lack of knowledge concerning the kinetics of vacuum deposition and film formation on industrially relevant silicon dioxide substrates. comparable investigations on the kinetics of adsorption, layer growth and desorption exist for a number of organic molecules (e.g., pentacene on sio2 and rubicene on sio2), while the focus has only recently shifted to h - bonded semiconductors. in this work, we focus on the growth and desorption behavior of quinacridone on / from sio2 substrates under ultrahigh vacuum conditions and report multiple collision - induced thermal decomposition processes both within the knudsen evaporation cells and on the substrate surface. thermally induced cracking leads to a formation of multiple, typically undefined fragmentation products that may influence, e.g., transistor characteristics. it is therefore of high importance to study molecular decomposition and the underlying driving forces on a chemical level and also to evaluate its occurrence and magnitude upon variation of the deposition methods. complementing the in situ methods of thermal desorption spectroscopy (tds) and auger electron spectroscopy (aes), ex - situ analysis by atomic force microscopy (afm), specular x - ray diffraction (xrd), grazing incidence x - ray diffraction (gixd), and raman spectroscopy were used to address this issue. for all of our experiments sublimation purified quinacridone, provided by tokyo chemical industry with a purity of > 99%, was used. typically, the material was deposited, after proper outgassing, i.e., the evaporation of smaller and more volatile impurity molecules during the heating process, via physical vapor deposition from a stainless steel knudsen cell heated to about 620 k. the inner diameter of the cell was 7 mm, the length was 20 mm, and the effusion hole diameter was 1 mm (see insert in figure 1). for several experiments it was necessary to switch to a glass knudsen cell or other glass evaporation sources, explained in more detail below. as substrate material, silicon dioxide with a nominal thickness of 150 nm, that has been thermally grown on 0.67 mm thick si(001) wafers (siegert wafer gmbh), was used. the 1 1 cm large substrates were mounted onto a stainless steel heating plate via four tantalum clamps. attached to the steel plate are two tantalum wires for resistive heating and a ni / nicr thermocouple to complete the feedback loop, necessary for thermal desorption measurements. the deposition process was conducted with a typical deposition rate of 105 ng/(mincm), equivalent to 0.7 nm / min and controlled by a quartz microbalance, which could be placed intermittently at the sample position. deposition and in situ characterization were carried out in an ultrahigh vacuum system housing a pfeiffer qms 200 mass spectrometer and a staib instruments auger electron analyzer. the base pressure of the unbaked uhv chamber was typically 6 10 mbar. furthermore, the whole sample stage was ln2 cooled to 200 k during film deposition, if not stated otherwise. an analysis of the chemical composition of the untreated substrate surface via aes revealed silicon and oxygen peaks originating from the substrate, with a slight carbon contamination on top. an auger analysis of vapor deposited quinacridone films showed the expected carbon, nitrogen and oxygen signals. unfortunately, it was not possible to perform continuous auger surveillance during different stages of the film growth process, due to the destructive behavior of the electron beam on the organic films. after material deposition the samples were placed in front of a quadrupole mass spectrometer (qms) tuned to typical cracking masses of quinacridone, i.e., either to 128 or 76 amu. continuous heating with a heating rate of 1 k / s subsequently caused material desorption and made for a convenient way to study film characteristics, growth, and desorption behavior as well as desorption order and activation energies for desorption. in this context, we have to emphasize that the sample temperature, as measured at the back side of the silicon wafer, is not the same as on the sio2 surface, due to the bad heat conductivity in silicon and sio2, as well as due to a thermal bridge at the interface between the si wafer and the stainless steel heating plate. however, a temperature correction can be made by comparing the multilayer desorption peak from the sample with that from the metal clamps, the temperature of which can be considered to be equal to the measured temperature. more details on the conducted temperature correction can be found elsewhere. when analyzing td spectra of organic molecules with a qms, cracking of the molecules in the ionization chamber of the spectrometer is unavoidable. the measured cracking masses should not be mixed up with possible decomposition products entering the ionization chamber of the qms after desorption from the surface. ex - situ studies of film morphologies and structures were done utilizing an atomic force microscope (nanosurf easyscan 2) in tapping mode employing ppp - nclr-50 silicon tips from nanosensors. specular x - ray diffraction (sxrd) measurements have been performed on a panalytical empyrean diffractometer fitted with a cu sealed tube and a multilayer mirror (= 1.54) on the primary side. the secondary side was equipped with a slit system and intensities were recorded using a panalytical pixcel detector in 1d mode. grazing incidence x - ray diffraction (gixd) measurements were conducted at the kmc-2 beamline at bessy ii (berlin, germany) using x - rays with a wavelength of 1.00 and a 2d cross - wire detector (bruker). an incident angle of i = 0.13 was chosen to enhance the scattered intensities of the adsorbate. the micro - raman experiments were performed with a hecd - laser excitation wavelength of 325 nm on a labram hr-800 (horiba jobin yvon) raman - spectrometer using gratings of 2400 lines / mm and providing a spectral resolution of 3.92 cm.the detection system was a liquid - nitrogen - cooled ccd spectrometer. initially, thermal desorption experiments were carried out after physical vapor deposition of quinacridone on carbon covered sio2 from a metal knudsen cell. the thermal desorption spectra of a 4.25 nm thick quinacridone film yielded a single desorption peak at about 500 k (denoted, see red line in figure 1) when the qms was tuned to a cracking mass of 128 amu. the cracking fragment at 128 amu was chosen because it is very prominent in the quinacridone cracking pattern and showed a reasonably good signal - to - noise ratio. experimental limitations did not allow detecting the main mass of quinacridone or any other fragments higher than 200 amu. if there were only a single molecular species present on the surface, as one might assume from the red curve in figure 1, then the choice of cracking mass should have no significant influence and spectra for different cracking masses should be comparable. however, surprisingly, when tuning the qms to the cracking mass 76 amu, a second peak at about 420 k (denoted -peak, black line in figure 1) appeared, in addition to the expected peak at 500 k. this points toward the presence of a second, weakly bonded type of molecules with a different cracking pattern and, hence, a different desorption behavior. indeed, a full mass scan between 35 and 150 amu during desorption of the and -peak, respectively, clearly shows different cracking patterns (figure 2). therefore, we can deduce the existence of two distinct and chemically different thin - film species on the surface, despite the fact that only purified quinacridone was present in the knudsen cell. the -peak and its cracking pattern are in good agreement with literature data of quinacridone, and we therefore assume that it corresponds to thermal desorption of pure quinacridone. this assumption is supported by several spectroscopic analyses, as will be shown below. combining the cracking pattern of the -peak with specular x - ray diffraction, raman spectroscopy and gixd allowed us to match this peak with indigo (in, c16h10n2o2). indeed, multiple spectroscopic observations of a film corresponding to the -peak agree reasonably well with data from an earlier publication describing the growth and desorption behavior of pure indigo films that were grown on sio2 under identical conditions. however, the spectroscopic data hint at the existence of an additional type of molecules in this film, which we could identify to be carbazole (ca, c12h9n). thermal desorption spectra for cracking masses of 76 amu (black) and 128 amu (red) of a 4.25 nm thick film which was grown on carbon covered silicon dioxide by evaporation of quinacridone from a stainless steel cell. adsorption temperature tad = 200 k, deposition rate a = 0.7 nm / min, heating rate = 1 k / s. the small peaks at 310, 340, and 380 k stem from desorption from the ta mounting clamps. at this point, the question arises whether indigo is generated on the silicon dioxide surface during heating of a pure quinacridone film or whether this type of molecules is already effusing from the stainless steel knudsen cell. to address this issue, we have performed the following experiments : after the growth of a thin film with the described physical vapor deposition from the stainless steel knudsen cell, a second sample holder with an identical si / sio2 wafer, held at room temperature, was put in front of the primary sample. heating this primary sample to 425 k exclusively caused the molecules corresponding to the -peak to desorb while the more strongly bonded quinacridone remained. the employed geometry (1.0 cm distance between the two 1 cm large samples, assuming a cosine desorption distribution) allowed about 1/3 of the desorbed molecules to readsorb on the stationary sample, which turned out to be a convenient way to create films with solely weakly bonded indigo and/or other decomposition molecules (-peak). after breaking the vacuum this stationary sample was installed on the heatable sample holder and a tds was performed as soon as uhv conditions were reached. the desorption spectrum of this film was similar in shape to the original -peak, demonstrating that this molecular species remains unchanged during the described readsorption and subsequent desorption. applying a similar experimental procedure, material corresponding to the -peak was deposited on the stationary sample and a tds was performed after venting and re - evacuation. in this case, solely the -peak appeared in the desorption spectrum, confirming that the -peak is not a result of quinacridone decomposition during sample heating. thus, the indigo observed in the desorption spectrum (figure 1) must have originated from the stainless steel knudsen cell. indeed, a full mass spectrum between 35 and 200 amu of the deposition flux leaving the metal knudsen cell (see figure 3) confirmed the existence of prominent cracking fragments from both indigo (-peak) and quinacridone (-peak) molecules. thermal decomposition (pyrolysis), dissociation and isomerization processes involving collisions of organic molecules with hot surfaces are commonly observed and well characterized in the field of organic chemistry. apparently, quinacridone seems to be particularly prone to such decomposition processes, due to its high sublimation temperature. however, only sparse reports exist involving cracking and/or restructuring of chemical bonds with the subsequent formation of new and stable molecules for quinacridone - based and other h - bonded materials. haucke. described a smooth and homogeneous transition from indigo to epindolidione in the vapor phase, if the former is heated to 733 k under vacuum conditions. berg. observed decreasing mobilities for quinacridone organic field - effect transistors grown in vacuum after repeated temperature gradient sublimation preparation circles. after having verified that a decomposition process takes place in the stainless steel knudsen cell the question arises as to the quantitative ratio of the effusing molecules, corresponding to the - and -peak. despite the fact that both species exhibit common cracking masses (e.g., 76 amu), one has to be aware that the intensities of the different desorption peaks depend on the detailed measurement conditions and no statements can be made on the actual distribution and relative amount of the molecules on the samples. to address this issue, we have performed afm measurements on films prepared with the method described above, consisting either of material corresponding to the or -peak, respectively. evaluation of the cross sections of the island - like films allowed a quantitative determination of the effusion rates. a flux ratio of 63 5% indigo to 37 5% quinacridone was obtained. a detailed description of the calibration procedure for quinacridone decomposition in the stainless steel knudsen cell will be given in a future publication. all given mean thickness values in this publication correspond to the total amount of adsorbed material, hence including both molecules. the molecular ratio given above should therefore be taken into account if the two types of molecules were to be analyzed separately. accumulated mass spectra (cracking spectra) of the desorption flux in the range of 350450 k (-peak) and 470530 k (-peak). a 3 nm thick film, prepared by evaporation of quinacridone from a stainless steel knudsen cell was used for this experiment. heating rate : 1 k / s, adsorption temperature : 200 k. cracking spectrum of the stainless steel knudsen cell deposition flux. knudsen cell temperature : 650 k. in a first attempt to quantify and describe the chemical cracking process and to confirm the coexistence of different types of molecules on the surface, despite the fact that only purified quinacridone is present in the knudsen cell, the deposition process was repeated with various knudsen cells and different evaporation sources. in order to check the purity of the quinacridone source material and to, therefore, rule out possible contaminations, a 18 nm thick quinacridone film was grown on a silicon dioxide sample via langmuir evaporation from an open quartz glass tube (see inset in figure 4) in a separate vacuum chamber. the sample was then again installed in the uhv chamber and analyzed via thermal desorption. as shown in figure 4, only the -peak appears in the desorption spectrum, indicating that the employed material is indeed pure and that no material decomposition occurs in that case. in the case of langmuir evaporation (or free evaporation) the sublimed molecules can be deposited at the substrate surface without any further collision in between. contrary, in the evaporation from a knudsen cell the sublimed molecules hit many times the inner cell wall before they leave the small effusion hole. for our particular stainless steel knudsen cell dimensions, a distance of 5 cm between sample and knudsen cell orifice and a typical deposition flux of 105 ng/(min cm) we calculate that a sublimed particle hits the inner walls about 400 times before escaping through the effusion hole. this corroborates our assumption of a thermally activated decomposition process within the metal knudsen cell. as a next step, it comes to answer the question whether the decomposition of quinacridone occurs due to a possible catalytic behavior of stainless steel or whether it can be reproduced in other knudsen - like evaporation cells. to this end this cell featured a diameter of about 10 mm with a 3 mm wide nozzle. wiring of the resistive heating filament (0.5 mm thick ta - wires) around the cell was done in different ways, either by focusing on the nozzle area (inset in figure 5a) or on the backside of the cell (inset in figure 5b). to discuss differences in the deposition behavior of this glass cell compared to a typical metal knudsen cell, one needs to first recall the physical processes happening within such a cell. clearly, the limited wiring of the heating filament, combined with the poor heat conductivity of quartz glass lead to a strongly varying temperature distribution along the long cell axis, contrary to the required uniform heating of an ideal knudsen source. in the first case, the nozzle area is, due to its smaller cross - section, significantly hotter than the residual cell walls. due to the missing wiring at the backside we can, in turn, assume, that the back wall, where all the material leaving a knudsen cell in a straight path typically comes from, is significantly colder in comparison and does therefore not contribute to any material deposition whatsoever. instead, all molecules that eventually leave the cell in a direction where they can reach the sample surface need to have at least undergone one collision under low angle with the higher temperature cell wall in the nozzle area. actually, the temperature of the nozzle is much higher than needed to just evaporate the material. therefore, the thermally activated cracking process of quinacridone molecules can be expected to occur more likely in such an excessively hot nozzle. indeed, deposition from a glass knudsen cell featuring dense wiring near the nozzle area resulted in the complete cracking of every single quinacridone molecule, hence only the -peak being visible in the tds spectrum (figure 5a). on the contrary, the usage of a glass knudsen cell with a colder nozzle and a heated backside again showed partial cracking, comparable to results from a metal cell, and different molecular species were once again present on the sample (figure 5b). thermal desorption spectrum for an 18 nm thick quinacridone film deposited from a glass evaporation cell (langmuir evaporation). substrate temperature : 300 k. thermal desorption spectra for quinacridone films deposited from a glass knudsen - type cell with dense wiring near the nozzle (a) and after removal of said wiring and increased heating at the backside (b). the nominal film thicknesses are 5 nm in plot (a) and 2.5 nm in plot (b). adsorption temperature : 200 k. in order to take a closer look at the morphologies of the obtained films and for a possible morphological confirmation of the separate types of molecules on the surface, we used ex - situ atomic force microscopy. all images were taken in tapping mode to not damage the sensitive organic films and plotted using derived data. in order to distinguish quinacridone islands from islands consisting of other molecules, it was necessary to create samples with either just quinacridone or just the decomposed molecules present. samples with no quinacridone molecules adsorbed, i.e., samples with indigo and possible other decomposition products, were prepared by using the glass knudsen cell described above with enhanced heating filament wiring near its opening (figure 5a). additionally, similar films were prepared via partial material desorption (-peak) from one sio2 sample and subsequent adsorption onto another sample, as described above. both methods produced comparable films and will therefore not be distinguished in the following. moreover, all samples above a certain thickness that were produced by either of these methods displayed a deep blue color visible to the bare eye. pure quinacridone samples (with a bright pink color) could be manufactured by using the original metal knudsen cell and heating the substrate to 425 k during the adsorption process, resulting in an immediate desorption of the weakly bonded smaller molecules. figure 6 shows afm images of three thick films on silicon dioxide with either all types of molecules (a), pure quinacridone (b) or solely indigo and other decomposed molecules (c) being present. all films did not show significant dewetting during venting (confirmed by tds measurements) and ostwald ripening or other morphology changes after storage under atmospheric conditions for at least 90 days, independent of molecule type and thickness. a comparison of surface morphologies for films of 5, 60, and 120 nm mean film thickness, as measured by afm immediately after exposure to air and after 24 h, showed no changes in island size, shape, or number. this suggests that molecules within multilayer islands are immobile on the surface and show a stable configuration once they are incorporated into their respective bulk crystal structures. figure 6a shows a 42.5 nm thick film that was deposited from the metal knudsen cell. therefore, multiple types of molecules have been adsorbed and subsist on the surface. a multitude of islands is apparent, either round or slightly elongated, with heights up to 200 nm (cross - section figure 6b). samples grown from the special glass deposition cell or via substrate heating, as depicted in figures 6c - e, show similarly shaped islands with comparable mean heights. interestingly, for the pure films we could not observe the elongated islands as found for the mixed film. initially we hoped to be able to correlate the two different island morphologies to the different types of molecules. while this might still be true, the different experimental conditions for the individual film fabrication (e.g., 425 k substrate temperature during quinacridone deposition, high nozzle temperature during indigo film preparation) might be responsible for this failure. (a) afm micrograph (8 m 8 m) of a quinacridone film containing additional cracking molecules on silicon dioxide deposited from a metal knudsen cell at 200 k substrate temperature ; nominal thickness : 42.5 nm (b) cross section along the line marked by the black arrow in (a). (c) afm micrograph (8 m 8 m) of a 85 nm thick film of the smaller cracking molecules, assumed to be mainly indigo, on silicon dioxide. the film was deposited from a glass knudsen cell with enhanced heating near the opening ; substrate deposition temperature : 200 k, deposition rate : 0.7 nm / min. (d) cross section along the line marked by the black arrow in (c). (e) afm micrograph (8 m 8 m) of a quinacridone film on silicon dioxide deposited from a metal knudsen cell at a substrate temperature of 425 k. for this film, material equivalent to a nominal thickness of 170 nm was deposited onto the sample, but partial desorption caused the resulting layer to be significantly thinner (estimated thickness : 63 nm) ; deposition rate : 0.7 nm / min. (f) cross section along the line marked by the black arrow in (e). quinacridone is a molecule with a planar conformation that crystallizes within the space group p21/c with two molecules per unit cell. in the literature, up to seven different polymorph structures of linear trans - however, only the and -polymorphs (not to be mixed with the and desorption peaks) are commercially important due to -quinacridone transitioning either fully or partially into -quinacridone at elevated temperatures. from the crystal bulk structure it is apparent, that each molecule is connected to two neighboring molecules in the and -phase and to four neighboring molecules in the -phase via intermolecular hydrogen bonds between the carbonyl and imino groups. these comparatively strong bonds allow for a high thermal stability and a high melting point. full crystallographic data of the bulk structure has only sparsely been published due to difficulties of crystal growth in solution and due to the typically insufficient quality of vapor deposited crystals. in our own experiments we have investigated the crystallographic properties of two differently prepared films grown on sio2 with a thickness of 63 nm (sample 1) and 85 nm (sample 2), respectively, by using both specular and grazing - incidence x - ray diffraction. a specular scan of the first sample, grown from a metal knudsen cell at a substrate temperature of 425 k, showed multiple out - of - plane reflections for scattering vectors qz between 0.3 and 2.2 (figure 7). a comparison with calculated diffraction data from powder cell measurements matches the peaks at 0.42 and 0.84 with (002) and (004) orientations of the quinacridone -polymorph structure and peaks at 0.44 and 0.89 with (001) and (002) crystal orientations of the -polymorph. therefore, quinacridone molecules seem to exclusively arrange in crystallographic orientations where the (00l) planes are parallel to the substrate. interestingly, there exist clear additional reflections which could not be attributed to any quinacridone net planes. namely, reflections at 1.38, 1.42, and 1.63 are indications of the presence of p - sexiphenyl (6p, c36h26) and/or other oligophenylenes. however, there is no evidence of such molecules in the thermal desorption or mass spectra. we believe that the occurrence of 6p is due to additional thermal decomposition and reaction of the impinging molecules at the relatively high substrate temperature of 425 k. during the growth process of this sample quinacridone molecules as well as all the described decomposition products in the knudsen cell (indigo, carbazole) are present in the deposition flux. however, the latter can not form a stable phase at a substrate temperature of 425 k and either instantly desorb or undergo further thermal decomposition. from a chemical point of view, both the break - off of co and nh3 molecules is quite likely, leading to a possible explanation of the formation of oligophenylenes from indigo, carbazole, and quinacridone molecules. the newly formed molecules have to be thermally stable on the surface at elevated temperatures, restricting the possible oligophenylenes to p - sexiphenyl and larger molecules. conducted similar experiments with 105 nm thick quinacridone films grown by vacuum sublimation on sio2 substrates. xrd peaks corresponding to -(001), -(002), -(002), and -(004) orientations were found, in accordance with our own experiments. in both phases molecules are orientated almost perpendicular to the substrate with their long axis tilted by only 10 and 20 with respect to the surface normal. furthermore, it was shown that the relative peak distribution changes with increasing substrate temperature for the simple reason that the -phase is metastable and that it can transform into a -polymorph at elevated temperatures. the -polymorph was found to be favored for thin films with 11 nm nominal thickness. therefore, we assign to an interface - near substrate induced thin film phase while the -polymorph is dominant within the bulk phase. additionally, sytnyk. recorded xrd patterns of quinacridone micro- and nanocrystals synthesized at various temperatures, and observed molecular arrangements that correspond to the and -polymorph throughout the measured temperature range. the second film (sample 2) was prepared by using the special glass cell mentioned above with increased heating near the nozzle for the deposition process where desorption hinted at indigo. indeed, a specular xrd scan (shown in figure 7) clearly links the peaks at 0.76 and 1.87 to indigo corresponding to orientations where the (100) and (210) planes, respectively, are parallel to the substrate. however, for this film again a special reflection appears in the xrd (0.66) which can not be attributed to indigo. extensive literature search showed that this reflection could be matched with a multitude of possible quinacridone cracking products, of which the (020) orientation of carbazole seems most likely if we take into account the entirety of the available spectroscopic data. specular x - ray diffraction pattern for a bare si / sio2 substrate (black), a 63 nm thick quinacridone film with additional 6p reflections (sample 1, pink, compare to figure 6e) and for a 85 nm thick film consisting of indigo and carbazole (sample 2, blue, compare to figure 6c). data are vertically shifted for better visibility. preferred orientation of quinacridone molecules packing within the - and -polymorph with the (00l)-planes parallel to the substrate. in order to provide support for the conclusions drawn from the specular x - ray diffraction data and to obtain additional information on the in - plane molecule arrangement we have carried out grazing incidence x - ray diffraction at bessy ii on the same samples as described before. figure 9 shows two - dimensional gixd images with an incidence wavelength of 1 and under an incidence angle of 0.13. numerous in - plane and out - of - plane reflections confirm the existence of a variety of molecular species and additionally crystalline phases with different crystallographic orientations. the superimposed white lines represent the highest intensity reflections of quinacridone and p - sexiphenyl (top) as well as indigo and carbazole (bottom) taken from literature values (csd - codes qnacrd06, qnacrd07, zzzntq01, indigo01, indigo02, and crbzol01). 2d - gixd patterns of a 63 nm thick quinacridone film (sample 1, top) and a 85 nm thick film solely consisting of cracking molecules (sample 2, bottom). the white lines depict debye scherrer rings of selected net planes of quinacridone (solid), p - sexiphenyl (dashed), indigo (solid), and carbazole (dashed) ; for detailed information see table 1. to interpret the results and features observed so far and for more information about the chemical composition of the thin organic films, raman spectroscopy was employed on the two samples that were used for afm, specular xrd and gixd investigations before (compare figures 6, 7, and 9). both samples were irradiated with monochromatic light with a wavelength of 325 nm yielding a multitude of scattering peaks, as shown in figure 10. we stress that the absolute peak intensities of the raman modes in figure 10 are arbitrary values that depend on the molecular orientations within the samples. we did not elaborate this finding in further detail, as our focus is here on a qualitative sample analysis. the quinacridone sample which was grown at a high substrate temperature (sample 1, pink) showed strong features at wavenumbers between 1550 and 1650 cm and certain peaks in the area of 1350 cm that agree reasonably well with our own powder measurements and literature values of quinacridone (see figure 11 and table 2). differences between thin film and powder measurements may, in this case, be explained by (a), the presence or dominant behavior of different polymorphs or (b), an increased amount of intermolecular hydrogen bonds within the thin film compared to powder samples. additional peaks at 1221 and 1279 cm agree very well with p - sexiphenyl raman excitations. the second sample (sample 2, blue) shows a thoroughly different fingerprint with multiple excitations, due to no quinacridone but rather indigo and possible additional cracking molecules being present. features at 1227, 1370, 1474, 1565, 1571, and 1610 cm as well as additional smaller peaks, can be matched with raman excitations of indigo powder (see figure 11 and table 2). certain peaks, most notably at 662, 1029, and 1122 cm, are neither originating from quinacridone, nor from indigo vibrations, again corroborating our claim of the existence of at least one additional cracking molecule. raman spectra of a bare si / sio2 substrate (black), a 63 nm thick quinacridone film (sample 1, pink), and a 85 nm thick film consisting of indigo and carbazole (sample 2, blue) upon irradiation with a wavelength of 325 nm. raman spectra of quinacridone (pink) and indigo (blue) powder samples upon irradiation with a wavelength of 325 nm. in this work, we describe some idiosyncrasies in the context of the preparation of thin quinacridone films upon employing knudsen cell deposition in ultrahigh vacuum. since quinacridone exhibits a comparably high sublimation temperature, due to the existing hydrogen bonds, this causes partial decomposition in the knudsen cell during evaporation, which impedes the formation of pure quinacridone films. we have used a variety of spectroscopies to unravel the thermally activated fragmentation and decomposition in the knudsen cell by analyzing the effusion flux and the composition and structure of the deposited film. by using thermal desorption spectroscopy we could unambiguously demonstrate that at least two different species were deposited on a silicon dioxide substrate upon evaporation of quinacridone from a stainless steel knudsen cell. in addition to quinacridone which desorbed at about 500 k (-peak), a second desorption peak (-peak) appeared already at about 420 k, which could be attributed to indigo. with special experimental methods we were able to prepare films which exclusively consist of molecules corresponding to either the -peak or the -peak, respectively. this was realized by desorbing and readsorbing molecules corresponding to the or the -peak onto a second sample positioned opposite to the primary sample. additionally, it was possible to prepare similar films with only one molecular species on the surface by either using a special knudsen - like glass cell or by quinacridone deposition at elevated substrate temperatures. the latter films were subsequently analyzed by atomic force microscopy, specular x - ray diffraction, grazing - incidence x - ray diffraction, and raman spectroscopy. however, additional decomposition products were found which could not be observed in the desorption spectra. in the film corresponding to the -peak carbazole was detected in addition to indigo, while the film corresponding to the -peak displayed p - sexiphenyl, most likely created through further thermal decomposition processes at the hot substrate surface, in addition to quinacridone. pure quinacridone films could be prepared only upon using a langmuir evaporation source (free evaporation). all films were stable and showed no significant morphology changes during venting and after storage in air for at least 90 days. our study highlights that these findings are of relevance for choosing the proper deposition techniques for organic molecules with high sublimation temperature, in particular for hydrogen - bonded molecules, which attain increasing importance in the context of organic electronics.	thin films of quinacridone deposited by physical vapor deposition on silicon dioxide were investigated by thermal desorption spectroscopy (tds), mass spectrometry (ms), atomic force microscopy (afm), specular and grazing incidence x - ray diffraction (xrd, gixd), and raman spectroscopy. using a stainless steel knudsen cell did not allow the preparation of a pure quinacridone film. tds and ms unambiguously showed that in addition to quinacridone, desorbing at about 500 k (-peak), significant amounts of indigo desorbed at about 420 k (-peak). the existence of these two species on the surface was verified by xrd, gixd, and raman spectroscopy. the latter spectroscopies revealed that additional species are contained in the films, not detected by tds. in the film mainly composed of indigo a species was identified which we tentatively attribute to carbazole. the film consisting of mainly quinacridone contained in addition p - sexiphenyl. the reason for the various decomposition species effusing from the metal knudsen cell is the comparably high sublimation temperature of the hydrogen bonded quinacridone. with special experimental methods and by using glass knudsen - type cells we were able to prepare films which exclusively consist of molecules either corresponding to the -peak or the -peak. these findings are of relevance for choosing the proper deposition techniques in the preparation of quinacridone films in the context of organic electronic devices.
the synapsin (syn) family consists of three neuronal phosphoproteins encoded in mammals by distinct genes (i.e., syni, ii, and iii). although the biology of syni and ii has been extensively studied, the functions of syniii are still largely uncharacterized. in vitro studies show that syniii is involved in axonal elongation and growth - cone formation during early neurodevelopment (feng., 2002 ; ferreira., 2000). accordingly, syniii is the earliest expressed syn isoform during development (porton. moreover, single - nucleotide polymorphisms in syniii have been linked to neurodevelopmental disorders (i.e., schizophrenia ; chen., 2009). similar to the other family members, syniii is a substrate for protein kinases (pks) (cesca., 2010). remarkably, many pathways that are essential for the migration and lamination of cortical neurons during brain development involve pks and lead to the phosphorylation of specific substrates (ayala., 2007). thus, the developmental expression of syniii, its role in neuronal developmental processes in vitro, and its phosphorylation profile suggest that it may be a downstream effector in neuronal migration. here, we showed that syniii is involved in neocortical development in vivo ; specifically, both the knockdown (kd) of syniii and its genetic deletion lead to defective radial migration and orientation of layer ii / iii pyramidal neurons (pns). proper development requires syniii phosphorylation by cyclin - dependent kinase-5 (cdk5), putting syniii downstream of the semaphorin-3a (sema3a)-signaling cascade. to investigate the role of syniii in cortical development, we first confirmed its expression in the rat brain cortex at developmental stages (supplemental results ; figures s1a subsequently, we designed two short - hairpin (sh)rnas against syniii but not syni / ii (figures s2a and s2b)to examine the effects of syniii kd on the radial migration of newly generated cortical pns in vivo. using in utero electroporation (iue) at embryonic day 17 (e17), we expressed active shrnas (shrna no. 1 and shrna no. 2) or a control scrambled shrna vector (shrnascr ; figures s2a and s2b) in a subpopulation of neural progenitors that would normally migrate to layer ii / iii of the somatosensory cortex (dal maschio., 2012). we analyzed the radial migration of layer ii / iii pns derived from shrna progenitors at e21 and postnatal day 7 (p7) (the first and last time points during the peak of syniii expression, respectively) as well as at p14 (the first time point at which syniii expression begins to be endogenously downregulated ; see figures s1a and s1b). at e21, control shrnascr cells were primarily found in the cortical plate (cp) and intermediate zone (iz), whereas only few cells remained in the ventricular zone / subventricular zone (vz / svz) (figures 1a and 1b). the migrating pns located at the iz expressed syniii at e21 (figures s1 g and s1h). interestingly, we observed a significant delay in radial migration when either shrna no. 1 or no. 2 was electroporated (figures 1a and 1b). as the effect on migration was larger in shrna no. 1 experiments, we performed all subsequent experiments with this construct (shrna onward). at p7, almost all shrnascr cells reached cortical layer ii / iii, whereas many shrna cells were misplaced in deep layers (figures 1c and 1i). the severity of misplacement was proportional to the severity of syniii downregulation (supplemental results ; figure s2e). to investigate whether the effect of syniii kd was long lasting, we examined neuronal migration at p14, when endogenous expression of syniii is low (see figures s1a and s1b). we found that syniii shrna cortices exhibited ectopic cells at p7 (figures 1c, 1h, and 1i) and at p14 (figures s3d and s3e). syniii downregulation did not affect neuronal identity, proliferation, or radial glia (rg) scaffold organization (supplemental results ; figures s2c, s2d, and s2f). cell migration, orientation, and polarization share molecular mechanisms, and often the same proteins are involved in all three processes (chen., 2008 ; polleux., 2000 to further explore the role of syniii in cortical development in vivo, we investigated whether its kd affected the orientation of pns in the layer ii / iii. control cells were typically characterized by an upright (ur) orientation with a pyramid - shaped cell body, one thick apical dendrite directed toward the pial surface, and one axon pointing to the ventricle. interestingly, 20% of syniii shrna pns were misoriented in layer ii / iii (figure 1d ; polleux., 2000), 10% of pns were inverted (in), and 10% of pns were horizontal (hor) (figures 1f and 1 g). similar results were obtained at p14 (figures s3a s3c). given the common mechanisms involved in migration and orientation (chen., 2008), we expected a larger effect on the orientation of ectopic cells. indeed, 1/3 of ectopic cells depleted of syniii were in (figures 1e and 1h1j). when we divided the cortical region where the ectopic cells were located (from layer iv to layer vi) in two equal parts, we found that, whereas 65% of the ectopic cells were located in the upper half, the in ectopic cells were evenly distributed in the two halves, suggesting that the cortical location and neuronal orientation were not causally linked (figure 1k). moreover, when we immunostained against the -tubulin centrosomal marker, we found that the centrosome colocalized with the apical dendrite regardless of cell orientation in both shrnascr and syniii shrna cells (figure s4a). whereas syniii regulated the morphological maturation of pns both in the axonal and dendritic compartments, it did not affect the basic electrophysiological proprieties of pns (supplemental results ; figures s3f s3l and s4c s4e). finally, we investigated whether syniii kd also affected the orientation and migration of pns in deep cortical layers (i.e., iv / v) by performing iue at earlier embryonic stages (e15.5). in p7 syniii shrna - transfected brains, we found migration defects and significant cell misorientation similar to that observed in superficial layers (figure s4b). to exclude off - target effects (de paula., 2007), we generated a shrna - resistant syniii cdna with four silent mutations in the shrna target sequence (rsyniii ; figure 2a). after confirming rsyniii insensitivity to shrna (figure 2b), we performed rescue experiments with rsyniii / shrna co - expression in e17 embryos. the co - expression of rsyniii and shrna at a ratio of 0.5:1 rescued the delay in radial migration at e21 (figures 2c and 2d) as well as the number and orientation of ectopic cells (figures 2e2 g) ; however, the phenotype of hor and in cells in the cp at p7 was not rescued (figures 2h and 2i). it is possible that the lack of rescue in the orientation phenotype in the cp was due to the concentration gradient of syniii expression across the cortex, which shows the highest levels in the cp (figures s1c and s1f). indeed, by increasing the syniii / shrna ratio to 1:1, we achieved complete rescue of the orientation defect of layer ii / iii pns (figures 2h and 2i). although syniii knockout (ko) mice display behavioral defects and changes in synaptic transmission, no gross brain abnormalities have been described (feng., 2002 ; porton., nevertheless, no study has specifically investigated whether subtler developmental defects occur in the syniii ko somatosensory cortex. to address this issue, we first performed immunostaining with layer - specific markers (i.e., cux1, layer ii / iii / iv ; ctip2, layer v ; foxp2, layer vi) in somatosensory cortex slices of syniii ko and control littermates. we did not observe any overt abnormality in cortical layering at p7 in syniii ko mice (figure s5). thus, we considered two possible scenarios : (1) given the high homology among syn isoforms, other members of the family, such as syni, which is also expressed at embryonic ages, may compensate for syniii s absence during development and (2) immunostaining with layer - specific markers may not be sufficiently sensitive to detect mild migration / orientation defects. to investigate possibility (1), we performed co - electroporation of syni with syniii shrna and assessed the migration / orientation defect in p7 mouse slices. we found that syni could not rescue syniii shrna effects, indicating that syni can not compensate for loss of syniii - specific functions during development (figures 3a3d). to investigate possibility (2), we performed iue with a pcaggs - ires - tdtomato (tom) vector to visualize migrating pns in e15.5 syniii ko and wild - type (wt) mice. in agreement with the cortical phenotype of shrna - electroporated animals, we observed a misorientation of an 20% of pns at the level of the cp in all ko brains (figures 3e3 g). interestingly, in contrast to controls, in two out of seven ko animals analyzed, we observed pns in ectopic positions, with these cells occasionally exhibiting in orientation (figures 3e and 3h). while rescuing orientation defects in the cp (figures 2h and 2i), the co - transfection of shrna rsyniii and shrna at a ratio 1:1 also led to ectopic cells in deep cortical layers (figures s6a and s6b) ; however, these cells did not exhibit orientation defects (figure s6c). given the tight temporal regulation of syniii expression, we hypothesized that not only downregulation / deletion but also overexpression of syniii could interfere with proper radial migration of pns. to test this hypothesis pns expressing either tom or syniii properly migrated through the cortex toward the cp (figures 4a and 4b). nonetheless, a significant percentage of syniii - overexpressing cells were misplaced at p7 (figures 4c and 4f4h). the effect of syniii on neuronal migration was long - lasting, as syniii overexpression also caused the appearance of ectopic cells at p14 (figures s6 g and s6h). thus, syniii overexpression, kd, and ko all affected migration and resulted in the ectopic placement of cells. however, syniii - overexpressing cells were all properly oriented (figures 4c4h and s6d s6h). these effects could be due to subcellular mislocalization of syniii upon overexpression in vivo, as we demonstrated in neuronal cultures (figure s6i). similar to the phenotype found for syniii kd during development, cdk5 ko leads to a delay in radial migration and misoriented pns (ohshima., 2007). by analyzing the amino acid sequence of syniii, we observed that it carries a conserved consensus sequence for cdk5 phosphorylation, with ser predicted to be the target (songyang., 1996). moreover, we found that cdk5 was expressed in rat cortices during development as early as e15 (figures 5a and 5b) and that cdk5 and syniii expression are highly correlated at perinatal ages (figure 5c). interestingly, we found that syniii and cdk5 co - immunoprecipitated from lysates of rat primary cortical neurons grown for 4 days in vitro (div) (figure 5d), demonstrating that syniii and cdk5 physically interact. syniii and the neuron - specific cdk5 activator p35 consistently colocalized in developing neurons in vitro (figure 5e). to test whether syniii is indeed phosphorylated by cdk5 we first generated a non - phosphorylatable syniii mutant in which ser was replaced with an ala. when immunoprecipitated and dephosphorylated syniii or its s404a mutant was phosphorylated in vitro by active p35/cdk5 complex (figure 5 g), we observed that syniii was phosphorylated by cdk5, whereas phosphorylation was significantly decreased by 31% in the s404a syniii mutant (figures 5 g and 5h). this result indicates that ser represents almost 1/3 of the total cdk5-mediated syniii phosphorylation. to test whether cdk5 phosphorylates syniii in vivo, we performed liquid chromatography tandem mass spectrometry (lc - ms / ms) on endogenous syniii from rat brains at p1, the peak of syniii expression. in figure 5i, the predicted m / z values of fragment ions are shown in blue or red, indicating identified b- and y - type ions, respectively. finally, we generated a phosphorylation - state - specific antibody recognizing phospho - ser in syniii. after testing its specificity for phospho - ser (figure s7b), we analyzed the phosphorylation of syniii on ser in vivo. we found that the absolute amount of phosphorylated syniii present in the cortex plateaued at p1p7 and then dropped to undetectable levels at p16p35. however, the fraction of phospho - syniii to total syniii was high at very early stages of development (e15e18) and steadily decreased thereafter to become undetectable at p16 (figures 5j and 5k). to test whether syniii is indeed a downstream effector of cdk5 during development in vivo, we generated a pseudo - phosphorylated syniii mutant by replacing ser with an asp (d), which mimics the negative charge of the phosphate group (rsyniii s404d). we next verified that both s404a and s404d rsyniii expression and subcellular distribution were similar to that of the wt protein (figures s7c s7e). when expressed alone, rsyniii s404a caused a delay in radial migration at e21 similar to that observed in the syniii kd experiments (figures 6a and 6b), indicating that this mutant may act in a dominant - negative manner on endogenous syniii. interestingly, when overexpressed at p7, rsyniii s404a resulted in the appearance of ectopic cells without orientation defects (figures 6c, 6e, and 6f), suggesting that the presence of the endogenous syniii is sufficient to guarantee proper neuronal orientation. consistent with the dominant - negative effect of the mutant on migration, when s404a rsyniii was co - expressed with shrna at a 0.5:1 or 1:1 ratio (as was done for wt rsyniii ; figures 2h and 2i), it did not rescue the shrna - induced migration defects at either e21 (figures 6a and 6b) or p7 (figures 6c and 6e). moreover, the co - transfection of s404a rsyniii with shrna at either ratio did not rescue the orientation defects in pns located in the cp (figure 6d) or in ectopic pns (figure 6f). conversely, when co - electroporated with shrna at a 0.5:1 ratio, s404d rsyniii significantly rescued the delay in radial migration caused by syniii kd at e21 (figures s7f and s7 g). cdk5 is a downstream effector in the sema3a pathway, and alterations in this pathway at various levels all cause a phenotype similar to syniii kd in the cortex (polleux., 2000 ; behar., 1996). to test the possibility that syniii acts downstream of the sema3a - signaling cascade, we investigated syniii phosphorylation by cdk5 in 4-div cortical neurons after stimulation with 1 nm sema3a. we observed that syniii was phosphorylated on ser only 5 min after the stimulus, and its phosphorylation significantly increased after 20 and 30 min in comparison to vehicle - treated cells (figures 7a and 7b). we next tested the causal involvement of syniii in the sema3a pathway in vivo. first, we investigated the expression of the sema3a receptor neuropilin 1 (np1) (chen., 2008) in rat brain cortices. np1 expression was highly similar to that of syniii during development, with a maximum expression between e21 and p7, followed by a sharp decrease immediately thereafter (figures 7c7e). moreover, as was seen for p35, we observed a colocalization of syniii and np1 in developing neurons in vitro (figure 7f), suggesting that these three molecules may indeed be part of the same signaling cascade. next, to investigate the sema3a / cdk5/syniii pathway in vivo, we electroporated a shrna against np1 in rat embryos (chen., 2008). as expected (chen., 2008), we observed a very strong effect on the radial migration of layer ii / iii pns at p7, with only 35% of pns reaching the cp (figures 7 g and 7h). however, when we co - expressed s404d rsyniii and np1 shrna, we significantly rescued radial migration (figures 7 g and 7h), with 50% more pns reaching the cp in comparison to np1 shrna - electroporated brains. during cortical development, post - mitotic pns migrate away from the vz along rg fibers toward the surface of the cp. on reaching the superficial layer, pns stop migrating, detach from rg fibers, and morphologically differentiate. in this study, we showed that syniii, the syn isoform that is most precociously expressed, plays a key role in cortical development. kd, ko, or upregulation of syniii caused defects in radial migration, with the appearance of ectopic cells at later stages. moreover, syniii kd / ko caused significant orientation defects in ectopic pns as well as in pns that fully completed their radial migration. interestingly, in misoriented pns, the centrosome was properly located on the side of the apical dendrite. if centrosome localization ahead of the nucleus in the direction of migration is necessary for neuronal migration (solecki., 2009), the question arises of how most of the syniii - kd - misoriented neurons completed their migration. it is possible that syniii - kd neurons migrate along glial fibers when the leading process is directed to the pial surface and stop migrating when they change orientation. conceivably, re - orientation of migrating pns occurs several times during migration, and consistent with the higher levels of syniii shrna in ectopic pns the more syniii is kd, the more migrating cells will re - orientate, resulting in a slower migration and ultimate ectopic positioning. conversely, pns expressing lower levels of syniii shrna will reach the cp, detach from rg fibers, and, without the fiber s restriction, acquire all possible degrees of orientation before morphological maturation. syniii kd causes the described phenotypes through a cell - autonomous mechanism, as confirmed by an intact rg scaffold in shrna - transfected brains. the migration and orientation defects involved the sema3a - signaling cascade ; indeed, upon sema3a stimulation, syniii is phosphorylated by cdk5. as shown by syniii phospho mutant experiments, / cdk5 signaling to control neuronal migration and orientation (figure 7i). given that syniii kd caused similar defects when performed in neuronal progenitors of both layer ii / iii and layer iv / v, and considering that cdk5 activity is essential for migration and dendrite development of layer v pns (ohshima., 2007), we hypothesize that the mechanism by which syniii regulates proper cortical organization is generalizable to the development of all cortical layers. future studies should investigate whether syniii also regulates migration in other brain areas, as previously described for cdk5 (jessberger., 2008). although many factors have been identified in recent years to be involved in cortical migration, we are still far from understanding the entire framework, and many actors await identification. among these, syniii was a good candidate. first, the emergence of the syn3 gene correlates with the appearance of the telencephalic region during evolution (kao., 1999). second, we demonstrated that syniii is highly expressed and phosphorylated on ser during development and has a well - defined spatial expression along cortical layers at distinct developmental stages. finally, in vitro data suggest a role for syniii in neuronal development (ferreira. in particular, a tight regulation of syniii expression is necessary for proper cortical development. indeed, syniii expression peaks in a defined temporal window, beyond which it is detrimental for cortical pn development, as proven by the following facts : (1) both syniii kd and overexpression caused defects in radial migration and in the morphological maturation of pns ; (2) a shrna - insensitive rsyniii completely rescued the kd phenotypes only when co - expressed with shrna at optimal levels ; and (3) the cortical phenotype caused by syniii kd during early development was irreversible and persisted even when the expression of the protein was endogenously downregulated at later developmental stages, such as p14 (also see the supplemental discussion). data from the literature indicated no major brain abnormalities in syniii ko mice (feng., 2002), whereas our data demonstrated that acute kd of syniii by rnai profoundly affected cortical development. interestingly, many other cases have been described where acute kd of a specific protein in a subset of neurons by iue leads to strong defects in brain development, whereas the constitutive ko of the same protein does not lead to an apparent phenotype (e.g., dcx, corbo., 2003 ; p75, lee., 1992 versus zuccaro., 2014 ; gabab receptor, schuler., 2001 versus bony., 2013) two main reasons could explain this finding : (1) the chronic and widespread depletion of the protein in ko mice could trigger redundancy or compensatory mechanisms by other proteins that overcome the deficits in brain development and/or (2) subtle developmental impairments in ko animals may be simply overlooked due to the complexity of the in vivo system and/or the lack of adequate methods to study mild defects. here, we provided evidence for both possibilities. indeed, marking developing neurons with a fluorescent reporter via iue in syniii ko mice allowed us to examine their cortical development with an unprecedented focus on neuronal migration and orientation. using this technique allowed us to conclude that syniii ko animals display the same phenotype as observed in shrna - transfected brains, although to a milder extent. because of the high homology between syniii and syni / ii, we hypothesized that they could compensate for the loss of syniii and that this compensation could result in a mild developmental phenotype in ko animals. nevertheless, we showed that synii is not significantly expressed at perinatal ages in the cortex and that the overexpression of syni did not rescue syniii shrna - mediated effects. it is possible that the differences between syni and syniii in terms of their amino acid sequence, location of phosphorylation sites, and spatio - temporal expression profile could account for their lack of involvement in neuronal migration and orientation. in conclusion, we hypothesize that proteins other than syni and ii may compensate for syniii loss in ko animals, and we propose the use of iue for expressing fluorescent reporters as a means to uncover subtle developmental phenotypes in ko animals. our data consistently show that syniii kd leads to a peculiar effect in the orientation of neurons located in the cp and also in the ectopic cells. interestingly, the orientation defects in cortical neurons described in the literature are primarily associated with the faulty expression of genes involved in the sema3a - signaling pathway (behar., 1996 ;, 1998, 2000 ; sasaki., 2002 ; shelly., sema3a is a secreted protein well known for its role as a guidance signal upon binding to its receptor np1. then, fyn activation promotes cdk5 binding to plexin a2 and eventually activates cdk5 by phosphorylating it on tyr (figure 7i). we demonstrated that syniii physically interacts with cdk5 and is a substrate for the kinase by showing that (1) syniii and cdk5 are both expressed at perinatal ages in the cortex and colocalize in developing neurons in culture ; (2) syniii and cdk5 co - immunoprecipitate ; and (3) syniii is phosphorylated by cdk5 both in vitro and in vivo. the rescue experiments performed with phospho mutants in vivo proved that ser phosphorylation has a biological significance and that the phenotype we observed in syniii - null pns is at least partially due to a missing step in cdk5 signaling. moreover, we showed that ser phosphorylation increased upon sema3a stimulation in vitro and that the pseudo - phosphorylated mutant s404d rsyniii partially compensated for the loss of sema3a signaling caused by np1 kd in vivo. thus, our data demonstrate that syniii is part of the sema3a pathway (figure 7i ; also see the supplemental discussion). little is known regarding syniii interactors. in general, syn family members can bind to synaptic vesicles (svs), actin (benfenati., 1989a, b, 1992), and tubulin (petrucci and morrow, 1991) through the highly conserved a, c, and e domains (candiani. indeed, syniii also binds svs and regulates their trafficking within nerve terminals (kao., are requisites for membrane rearrangements during cell migration (itofusa and kamiguchi, 2011), syniii may participate in radial migration by the latter processes (figure 7i). the phenotypic features of syniii - kd or -overexpressing pns support this hypothesis : delayed migration, ectopic positioning, and morphologically impaired leading processes are typical of neurons that lose their contact with rg fibers (elias., 2007 ; gupta., 2003 ; sanada., 2004 ; valiente., 2011). the dysregulation of syniii activity could indeed interfere with the cytoskeletal dynamics and membrane rearrangements involved in the formation and disassembly of cell adhesions and, consequently, interactions between migrating neurons and rg fibers (rivas and hatten, 1995 ; also see the supplemental discussion). in this study, we showed that syniii acts downstream sema3a and regulates the radial migration and orientation of pns. neuronal migration is fundamental for the development of a fully functional brain, as demonstrated by the implication of defective neuronal migration in a number of neurodevelopmental disorders (ayala., 2007). indeed, sema3a - signaling dysregulation in brains from individuals with schizophrenia (eastwood., 2003) and a genetic linkage between syniii mutations and schizophrenia have been reported (hall., 2007 ; lachman., 2005 ; porton., moreover, as we observed in syniii kd / ko animal model brains, reduced syniii expression (porton and wetsel, 2007), abnormal cytoarchitecture, and dendritic aberrations (arnold., 1991 ; benes., 1991 ; glantz., 2000 ; mirnics., 2000 ; rajkowska., 1998 ; rosoklija., 2000) interestingly, the altered directionality of axonal elongation in syniii - kd neurons that we describe in the present study is consistent with the disruption of axonal fiber connectivity in schizophrenic patients (zalesky., 2011). finally, syniii ko mice exhibit abnormal sensory motor gating in response to prepulse inhibition (geyer., 2001), similar to what is observed in patients with schizophrenia and in mouse models of schizophrenia. thus, the clarification of the role of syniii in the sema3a pathway could be important in shedding light on the pathogenesis of neurodevelopmental diseases and further increase our knowledge of the complex mechanisms that regulate cortical development in health and disease. for detailed cloning procedures and list of used primers, see the supplemental experimental procedures. cos7 cells were cultured in dulbecco s mem (dmem) (gibco) supplemented with 10% fetal calf serum (gibco), 1% l - glutamine, 100 u / ml penicillin, and 100 g / ml streptomycin (biowhittaker) and maintained at 37c in a 5% co2 humidified atmosphere. primary cultures of dissociated cortical neurons were prepared from e18 rats and maintained in neurobasal medium supplemented with 2% b-27, 0.5 mm glutamine, 50 u / ml penicillin, and 50 g / ml streptomycin (invitrogen). the neurons were maintained at 37c in a 5% co2 humidified atmosphere. for stimulation experiments, the neurons were washed with neurobasal medium and incubated for 30 min at 37c. msema3a - fc (1 nm ; r&d systems) was added to the medium and, after incubation, the cells were lysed and processed for western blot. total cell lysates were obtained from cos7 cells 48 hr after transfection or from cortical neuron cultures at 4 div. for detailed procedures, reagents, and a list of antibodies, following transient transfection, cos7 cells were lysed and syniii and s404 syniii were immunoprecipitated and dephosphorylated. the samples were then subjected to in vitro phosphorylation. for detailed procedures and reagents, syniii ko mice were generated by homologous recombination (feng., 2002) and backcrossed onto the c57bl/6j background for over ten generations. all of the experiments were performed in accordance with the guidelines established by the european communities council (directive 2010/63/eu of september 22nd, 2010) and were approved by the italian ministry of health. slice histology and immunostaining were performed as previously described (bony., the images were acquired using a confocal laser - scanning microscope (tcs sp5 ; leica microsystems) or with an epifluorescence microscope equipped with neurolucida (microbrightfield) software. for detailed procedures on acquisition and analysis, see the supplemental experimental procedures. tryptic digests were performed as previously described (ballif., 2006), and extracted peptides were subjected to lc - ms / ms analysis in a linear ion trap - orbitrap mass spectrometer, as previously described (ballif., 2008 ; buel., if not indicated otherwise in the legend, the numbers in parentheses on the histograms indicate the number of processed animals. statistical analysis between control and experimental groups was performed with student s t test or, in the case of more than two experimental groups, with one - way anova followed by post hoc multiple comparisons. for non - normally distributed data, anova on ranks test and non - parametric post hoc comparisons were used. l.e.p. participated in the design of the experiments and performed the molecular biology and biochemistry experiments, iue, immunocytochemistry, immunohistochemistry, image acquisition, and data analysis. l.e.p. also wrote the manuscript and made the figures. performed the in vitro phosphorylation, co - ip experiment, and neuronal cell culture experiments.	summarysynapsin iii (syniii) is a phosphoprotein that is highly expressed at early stages of neuronal development. whereas in vitro evidence suggests a role for syniii in neuronal differentiation, in vivo evidence is lacking. here, we demonstrate that in vivo downregulation of syniii expression affects neuronal migration and orientation. by contrast, syniii overexpression affects neuronal migration, but not orientation. we identify a cyclin - dependent kinase-5 (cdk5) phosphorylation site on syniii and use phosphomutant rescue experiments to demonstrate its role in syniii function. finally, we show that syniii phosphorylation at the cdk5 site is induced by activation of the semaphorin-3a (sema3a) pathway, which is implicated in migration and orientation of cortical pyramidal neurons (pns) and is known to activate cdk5. thus, fine - tuning of syniii expression and phosphorylation by cdk5 activation through sema3a activity is essential for proper neuronal migration and orientation.
erdheim - chester disease (ecd) is a rare non - langerhans - cell histiocytosis of unknown etiology involving infiltration of bones and various organs by lipid - laden histiocytes with foamy or eosinophilic cytoplasm. lipid - laden histiocytes produce xanthogranulomas, which can be found in long tubular bones, skin, lung, heart, kidney, retroperitoneal space, orbit, and pituitary gland. the mean age of the diseased population is 53 years, and there is a slight predominance of males. most patients have skeletal involvement at the time of diagnosis ; extra - skeletal involvement occurs in approximately 50% of patients. the bone involvement is constant and includes symmetric osteosclerosis of long bones, especially of the low extremities. first described by erdheim and chester in 1930, fewer than 500 cases of ecd have been reported in the published literature. in korea, the first case of ecd was described in 1999, and 15 cases have since been reported. herein, we report on a unique case of ecd with bone marrow, axial skeleton, and lymph node involvement. in march 2009, a 76-year - old man with a 20-year history of hypertension was referred to smg - snu boramae medical center with exertional dyspnea. despite treatment with antihypertensive medication, white blood cell, hemoglobin, and platelet counts were 3,180 cell / mm, 7.4 g / dl, and 65,000/mm, respectively. serum and urine protein electrophoresis, immunoelectrophoresis, and immunofixation electrophoresis of serum were normal. serum blood urea nitrogen and creatinine levels were 43 and 2.3 mg / dl, respectively. there was heavy proteinuria in 24-hour urine protein, and his 24-hour urine protein was 1,816 mg / day. thyroid - stimulating hormone and free thyroxine levels were 16.63 iu / ml and 0.71 ng / ml, respectively. small - sized kidneys and mild splenomegaly (13.2 cm) were observed by abdominal ultrasonography. cardiac contraction was normal with an ejection fraction of 61.8% and a pattern of mild diastolic dysfunction was observed by echocardiography. bone marrow examination showed cellularity of 60%, which was slightly hypercellular for the patient s age, with slightly increased foamy histiocytosis (fig. immunohistochemical stains were negative for acid - fast bacilli, oil red o, and periodic acid schiff, eliminating the likelihood of infectious causes. we diagnosed the patient with anemia of chronic disease, cytopenia related to chronic illness, chronic renal failure due to hypertension, and hypothyroidism. the patient s complete blood count was still compatible with anemia of chronic disease accompanied by mild thrombocytopenia, which was not significantly different from what was observed at the initial visit 6 months earlier. echocardiography showed normal systolic function, with an ejection fraction of 67%, and mild diastolic dysfunction, which was not different from what had been observed previously. coronary angiography showed 50% stenosis in the left anterior descending branch of the left coronary artery and right coronary artery. the patient was treated with diuretics, which resulted in improvement of the dyspnea and generalized edema. the patient was discharged without coronary intervention and with plans to continue medical therapy and follow - up in the nephrology and cardiology departments. in may 2011, white blood cell, hemoglobin, and platelet counts were 5,190 cell / mm, 7.2 g / dl, and 72,000/mm. serum blood urea nitrogen and creatinine levels were 92 mg / dl and 6.4 mg / dl, respectively. when the bone marrow examination was repeated, cellularity was slightly decreased and histiocyte infiltration was increased when compared to the previous examination. the patient s renal function had deteriorated to chronic renal failure stage v. hemodialysis was initiated, and the patient s symptoms improved. in january 2014, the patient presented with low back pain that had originally started 2 years earlier and had recently become aggravated. on physical examination, the patient had multiple enlarged lymph nodes in both axillary areas and did not show any signs of fever or weight loss. the results of liver function tests, lipid profiles, and tumor markers (-fetoprotein, 1.66 ng / ml ; carcinoembryonic antigen, 1.82 ng / ml ; cancer antigen [ca]-19 - 9, 3.44 u / ml ; ca-125, 10.7 u / ml) were normal. compression fractures of the t5, l3, and l4 vertebral bodies were identified by magnetic resonance imaging of the spine (fig. in particular, the l3 vertebral body showed extensive bone marrow edema and paravertebral soft tissue swelling, with an anterior bulging contour suggestive of a pathologic etiology such as myeloma or metastases. involvement of the metaphysis and diaphysis, with relative sparing of the epiphysis, was also observed. f - fluorodeoxyglucose positron emission tomography showed diffuse hypermetabolic activity in the t5 and l3 vertebral bodies, the right proximal humerus, and both axillary lymph nodes. technetium-99 m bone scintigraphy showed increased uptake in t5 - 6, t7 - 8, t12, and l3 - 5 and both tibia. plain radiography of the humerus showed sclerotic lesions in both anatomical neck areas of the humerus. compression fractures were observed by a computed tomography (ct) scan of the patient s chest in t5, t7 - 8, t12, and l3 - 4 spines. in addition, ct imaging identified cardiomegaly and multiple prominent lymph nodes in both axillae. the ct scan of the abdomen was unremarkable, other than the observation of relatively small sized kidneys and mild splenomegaly (14.4 cm). histopathological examination of the bone biopsy revealed marrow fibrosis and granulated tissue, with infiltration of lipid - laden histiocytes. 4), which were found to be positive for cd68 and negative for s100 and cd1a. there were no mutations in braf v600e mutation - specific antibody stains of the bone and the axillary lymph node. in addition, mutation v600e of braf was not detected by direct dna sequencing of braf gene exon 15. bone marrow biopsy was also performed, which showed the same results as the bone and axillary lymph node biopsy. with the suspicion that bone marrow involvement of ecd had been present since march 2009, we reviewed the results of the bone marrow biopsy performed in march 2009. immunohistochemical stain of the bone marrow biopsy examined in march 2009 showed that the infiltrating histiocytes were positive for cd68 (fig., a diagnosis of ecd with involvement of the bone, bone marrow, and axillary lymph nodes was made. we also suspected ecd involvement of the heart and kidney ; however, this could not be accurately confirmed without tissue analysis. ecd is a rare, non - langerhans cell, histiocytic, infiltrative disorder primarily involving the peripheral long bones. it can also involve other organ systems, including the musculoskeletal, central nervous, cardiac, pulmonary, and renal systems. the pathological features of ecd are diffuse xanthogranulomatous infiltration of large foamy histiocytes, rare touton - like giant cells, and medullary fibrosis. in immunohistochemical staining, the tumor cells stain positive for cd68 and negative for cd1a and s100. the most commonly involved bones in ecd are the femur, tibia, and fibula ; the ulna, radius, and humerus are less frequently involved. ecd involved bones are characterized by bilateral and symmetric sclerosis of the metaphyseal regions of the long bones, while the epiphysis is spared. approximately half of all patients have extra - skeletal manifestations, which can include retroperitoneal fibrosis, orbital infiltration, interstitial lung disease, bilateral adrenal involvement, testis infiltration, and involvement of the breast, the central nervous system, or cardiovascular systems. the clinical presentation of ecd ranges from asymptomatic to various and multiple symptoms related to the involved organs. the clinical details of the largest number of reported cases to date were described retrospectively by veyssier - belot. and included 52 previously reported cases and seven additional cases. in this report, patients with ecd most commonly presented with juxtaarticular bone pain, usually of the knees and ankles. it is very unusual for the lymph nodes, liver, spleen, or axial skeletons to be affected in ecd ; however, these areas are frequently affected in langerhans cell histiocytosis and rosai - dorfman disease (rdd). in the case presented herein, the patient presented with an unusual pattern of involvement of bone marrow, axial skeleton, and lymph nodes. involvement of lymph nodes was first documented by sheu. in a report on a 50-year - old white male who presented with hypogonadism and diabetes insipidus. because the clinical characteristics of ecd are largely unknown and it is difficult to diagnose, it is important to be clinically critical. diagnosis of ecd is based on the pathologic evaluation of the involved tissue in combination with the clinical context. several differential diagnoses are generally possible based on the presenting features, resulting in the necessity of a battery of investigations. it is important to consider a number of differential diagnoses, including metabolic and endocrine disorders, malignancies, multiple sclerosis, and thyroid abnormalities. langerhans cell histiocytosis presents with an infiltrate of histiocytes, which have a characteristic reniform nuclear morphology and express the markers of langerhans cells, including cd1a, s100, and langerin. in contrast, ecd tumor cells lack central nuclear grooves and birbeck granules that are typical of langerhans cell histiocytosis. the primary differential diagnosis should also include rdd, which presents as a benign disorder with histiocyte accumulation in the lymph nodes and occasionally in the skin. the pathology of rdd is typified by prominent emperipolesis and a mixed infiltrate with histiocytes that express s100. ecd tumor cells have a similar immunohistochemical staining pattern to juvenile xanthogranuloma (jxg) cells. jxg cells express factors viiia and cd68 and are negative for cd1a, langerin, and s100. however, the clinical presentation and age of our patient were not compatible with jxg. systemic jxg occurs mainly in young children, whereas ecd is a disease of middle - aged and elderly adults, with a mean age at diagnosis of 53 years (range, 21 to 77 years). jxg is usually benign and self - limiting and bone involvement is more likely to be focal, and lytic. in contrast, ecd is often a fatal disorder, with death occurring in more than 50% of patients. death often occurs as a result of cardiac or respiratory failure resulting from heart or lung involvement. systemic steroids, various cytotoxic agents, radiation therapy, and autologous stem cell transplantation have all been used in treatment of patients with this condition. glucocorticoids are reserved for patients who can not tolerate more aggressive systemic therapies and for patients with very mild symptoms who wish to avoid the potential side effects associated with more aggressive therapies. a recent study reported that interferon- is a valuable therapy in treatment of ecd patients. if interferon- fails, imatinib mesylate has been suggested as an experimental alternative therapeutic regimen by some investigators. in addition, it has been suggested that treatment with a braf inhibitor could be a useful therapy, as patients with multisystemic and refractory ecd and the braf v600e gene mutations have demonstrated partial clinical responses following treatment with the braf inhibitor. in summary, our patient underwent a bone marrow examination for diagnosis in march 2009 because of cytopenia in the peripheral blood. although bone marrow examination revealed increased foamy histiocytosis, we failed to diagnose ecd initially because there was no bone lesion or any other symptoms that might be suspicious of ecd. in january 2014, the patient was re - admitted to the hospital because multiple bone lesions were newly detected and cytopenia could still be detected in the peripheral blood examination. we performed bone marrow examination, bone biopsy, and lymph node biopsy for diagnosis of ecd. in addition, we reviewed the bone marrow results examined in march 2009 with additional immunohistochemical stain, and we were finally able to confirm the diagnosis of ecd. however, axial skeleton and lymph node involvement were detected 5 years after the initial presentation. unlike other cases of ecd, our patient developed slow progression during a period of more than 5 years. we thought that the cause of this slow progression may be that despite having bone marrow involvement, there was no vital organ dysfunction. ecd presenting with cytopenia resulting from bone marrow involvement, axial skeletal lesions rather than peripheral bone lesions, and involvement of lymph nodes have been rarely described in the literature. thus, we report on a rare case of ecd with axial skeleton, lymph node, and bone marrow involvement.	erdheim - chester disease is a rare non - langerhans - cell histiocytosis with bone and organ involvement. a 76-year - old man presented with low back pain and a history of visits for exertional dyspnea. we diagnosed him with anemia of chronic disease, cytopenia related to chronic illness, chronic renal failure due to hypertension, and hypothyroidism. however, we could not determine a definite cause or explanation for the cytopenia. multiple osteosclerotic axial skeleton lesions and axillary lymph node enlargement were detected by computed tomography. bone marrow biopsy revealed histiocytic infiltration, which was cd68-positive and cd1a - negative. this report describes an unusual presentation of erdheim - chester disease involving the bone marrow, axial skeleton, and lymph nodes.
kitaibelii (winter savory) were collected in the period may - june, 2005, in the region of mountain zlatibor, serbia, as labelled. the voucher specimen of the collected plant material was confirmed by biljana boin and deposited at the herbarium of the department of pharmacognosy, faculty of medicine, university of novi sad. all chemicals and reagents were of analytical reagent grade and were purchased from sigma chemical co. (st louis, mq, usa), aldrich chemical co. (steineheim, germany) and " zorka " (abac, serbia).. " vital ", oil and vegetable fat factory (vrbas, serbia). commercial preparation of liposomes (pro - lipo s, with 30% soybean phosphatidylcholine) was obtained from lucas - meyer, hamburg, germany. the dried aerial parts (moisture content 3.72%) of winter savory were ground in a grinder (dlfu bhler miag laboratory disc mill, germany) with a 2 mm in diameter mesh. this material (100 g) was macerated with 70 % methanol in water (2 2500 ml) at room temperature for 2 24 h. the obtained extract was concentrated under reduced pressure to remove methanol, and then extracted sequentially with petroleum ether (2 200 ml), chloroform (2 200 ml), ethyl acetate (2 200 ml) and n - butanol (2 200 ml). the petroleum ether, chloroform, ethyl acetate, n - butanol and remaining water extract were evaporated to dryness under reduced pressure at 40c with a water bath. the yields, average of triplicate analysis, of the extracts were : petroleum ether, m = 0.580.029 g ; chloroform, m=0.490.024 g ; ethyl acetate, m=0.550.028 g ; n - butanol, m=2.130.106 g and water, m=6.030.30 g. hydroxyl radicals were obtained during a fenton reaction by mixing 200 l 0.3 m 5,5-dimethyl-1- pyroline - n - oxide (dmpo), 200 l 10 mm h2o2, 200 l 10 mm fe and 10 l of methanol (blank). the influence of different types of extract on the formation and transformation of hydroxyl radicals was investigated by adding the petroleum ether, chloroform, ethyl acetate, n - butanol and water extracts in the fenton reaction system at the range of concentrations 0.10.5 mg / ml (probe). the esr spectra were recorded on a bruker 300e, after 5 min, with the following spectrometer settings : field modulation 100 khz, modulation amplitude 0.512 g, receiver gain 210, time constant 81.92 ms, conversion time 163.84 ms, centre field 3440.00 g, sweep width 100.00 g, x - band frequency 9.64 ghz, power 20 mw, temperature 23c. the antioxidant activity (aaoh) of the extracts was defined as : aaoh = 100 (ho - hx)/ho [% ] where ho and hx are the height of the second peak in the esr spectrum of the dmpo - oh spin adduct of the blank and probe, respectively. oxidation was investigated in a solution containing 1 ml of sunflower oil, 0.0018 g acva, 0.0274 g of -phenyl - n - tert - butylnitrone (pbn) and 380 l of various concentrations of methanolic solutions of the investigated extracts (final concentrations were in range 15 mg / ml). liposome suspension was prepared by adding 1 g of commercial liposomes to 10 ml water and incubated with stirring at 37c for 1h. oxidation was investigated in the solution containing 1 ml of liposome suspension, 0.0019 g aaph, 0.0274 g of pbn and 380 l of various concentrations of methanolic solutions of the investigated extracts (final concentrations were in the range 15 mg / ml). lipid oxidation in both systems was measured by following the formation of peroxyl radicals with esr spin trapping method. the esr spectra of the formed pbn - peroxyl radical spin adducts were recorded with the following spectrometer settings : field modulation 100 khz, modulation amplitude 1.021 g, receiver gain 510, time constant 20.48 ms, conversion time 327.68 ms, centre field 3440.00 g, sweep width 100.00 g, x - band frequency 9.64 ghz, power 20 mw, temperature 23c. the antioxidant activity value (aaloo) of the extract was defined as : aaloo=100(ho - hx)/ho [% ], where ho and hx are the height of the second peak in the esr spectrum of the pbn - ool spin adduct obtained in the absence and in presence of the investigated extracts, respectively. splitting constants were calculated from computer - generated second derivatives of the spectra, after optimizing signal - to - noise ratios, and were verified by computer simulations. for these investigations the disc diffusion method and microbroth dilution methods were applied. from the primary isolation medium 23 colonies of investigated bacteria were taken by flamed loop, suspended in mueller - hinton or sabouraud broth, and they were incubated at 37c. the suspension for inoculation the number of cells in 1 ml of suspension for inoculation measured by the mcfarland nephelometer was 110 cfu / ml. a volume of 1 ml of this suspension was homogenized with 9 ml of melted (45c) mueller - hinton or sabouraud dextrose agar and poured into petri dishes. for screening, sterile 6 mm discs (himedia, mumbai, india) were impregnated with 10 l of 100 mg / ml of satureja montana l. subsp. after incubation for 2448 hours in the thermostat, inhibition zone diameters, zi, (including disc) were measured and expressed in mm. the presence of the inhibition zone indicates the activity of tested extracts against bacteria or yeasts. the minimum inhibitory concentration (mic) was reported as the lowest concentration of the extracts capable of inhibiting the growth of the bacterium tested. the mic was determined by the broth macrodilution test using an inoculum of 110 cfu / ml. final concentrations of the investigated extracts were 6.25, 12.5, 25, 50, 75 and 100 mg / ml. both tests were done in three replications. pseudomonas aeruginosa (atcc 27853, gram negative), escherichia coli (atcc 25922, gram negative), staphylococcus aureus (atcc 25923, gram positive), sarcina lutea (atcc 9341, gram positive), bacillus cereus (atcc 10707, gram positive), saccharomyces cerevisiae (112, hefebank weihenstephan) and candida pseudotropicalis (clinical isolate) microorganism strains were employed for the determination of antimicrobial activity. penicillin (10 iu / disc) and amoxicillin (25 g / disc) were used as reference standards as obtained from bioanalyse co., ltd., ankara, turkey. in parallel with the antibacterial investigation of satureja montana l. subsp. kitaibelii extracts, pure solvent was tested too, and it did not exhibit any antibacterial activity (data are not shown). bacteria were obtained from the stock cultures of the microbiology laboratory, faculty of technology, university of novi sad. all measurements were carried out in triplicate, and presented as mean sme or sd. regression analyses and significance of differences were carried out using a spss statistical software package (spss for windows, 8.0, 1997, spss inc., chicago, il, usa). kitaibelii (winter savory) were collected in the period may - june, 2005, in the region of mountain zlatibor, serbia, as labelled. the voucher specimen of the collected plant material was confirmed by biljana boin and deposited at the herbarium of the department of pharmacognosy, faculty of medicine, university of novi sad. all chemicals and reagents were of analytical reagent grade and were purchased from sigma chemical co. (st louis, mq, usa), aldrich chemical co. (steineheim, germany) and " zorka " (abac, serbia).. " vital ", oil and vegetable fat factory (vrbas, serbia). commercial preparation of liposomes (pro - lipo s, with 30% soybean phosphatidylcholine) was obtained from lucas - meyer, hamburg, germany. the dried aerial parts (moisture content 3.72%) of winter savory were ground in a grinder (dlfu bhler miag laboratory disc mill, germany) with a 2 mm in diameter mesh. this material (100 g) was macerated with 70 % methanol in water (2 2500 ml) at room temperature for 2 24 h. the obtained extract was concentrated under reduced pressure to remove methanol, and then extracted sequentially with petroleum ether (2 200 ml), chloroform (2 200 ml), ethyl acetate (2 200 ml) and n - butanol (2 200 ml). the solvent extractions were performed in separation funnels, shaking for 10 min. the petroleum ether, chloroform, ethyl acetate, n - butanol and remaining water extract were evaporated to dryness under reduced pressure at 40c with a water bath. the yields, average of triplicate analysis, of the extracts were : petroleum ether, m = 0.580.029 g ; chloroform, m=0.490.024 g ; ethyl acetate, m=0.550.028 g ; n - butanol, m=2.130.106 g and water, m=6.030.30 g. hydroxyl radicals were obtained during a fenton reaction by mixing 200 l 0.3 m 5,5-dimethyl-1- pyroline - n - oxide (dmpo), 200 l 10 mm h2o2, 200 l 10 mm fe and 10 l of methanol (blank). the influence of different types of extract on the formation and transformation of hydroxyl radicals was investigated by adding the petroleum ether, chloroform, ethyl acetate, n - butanol and water extracts in the fenton reaction system at the range of concentrations 0.10.5 mg / ml (probe). the esr spectra were recorded on a bruker 300e, after 5 min, with the following spectrometer settings : field modulation 100 khz, modulation amplitude 0.512 g, receiver gain 210, time constant 81.92 ms, conversion time 163.84 ms, centre field 3440.00 g, sweep width 100.00 g, x - band frequency 9.64 ghz, power 20 mw, temperature 23c. the antioxidant activity (aaoh) of the extracts was defined as : aaoh = 100 (ho - hx)/ho [% ] where ho and hx are the height of the second peak in the esr spectrum of the dmpo - oh spin adduct of the blank and probe, respectively. oxidation was investigated in a solution containing 1 ml of sunflower oil, 0.0018 g acva, 0.0274 g of -phenyl - n - tert - butylnitrone (pbn) and 380 l of various concentrations of methanolic solutions of the investigated extracts (final concentrations were in range 15 mg / ml). liposome suspension was prepared by adding 1 g of commercial liposomes to 10 ml water and incubated with stirring at 37c for 1h. oxidation was investigated in the solution containing 1 ml of liposome suspension, 0.0019 g aaph, 0.0274 g of pbn and 380 l of various concentrations of methanolic solutions of the investigated extracts (final concentrations were in the range 15 mg / ml). lipid oxidation in both systems was measured by following the formation of peroxyl radicals with esr spin trapping method. the esr spectra of the formed pbn - peroxyl radical spin adducts were recorded with the following spectrometer settings : field modulation 100 khz, modulation amplitude 1.021 g, receiver gain 510, time constant 20.48 ms, conversion time 327.68 ms, centre field 3440.00 g, sweep width 100.00 g, x - band frequency 9.64 ghz, power 20 mw, temperature 23c. the antioxidant activity value (aaloo) of the extract was defined as : aaloo=100(ho - hx)/ho [% ], where ho and hx are the height of the second peak in the esr spectrum of the pbn - ool spin adduct obtained in the absence and in presence of the investigated extracts, respectively. splitting constants were calculated from computer - generated second derivatives of the spectra, after optimizing signal - to - noise ratios, and were verified by computer simulations. for these investigations the disc diffusion method and microbroth dilution methods were applied. from the primary isolation medium 23 colonies of investigated bacteria were taken by flamed loop, suspended in mueller - hinton or sabouraud broth, and they were incubated at 37c. the suspension for inoculation the number of cells in 1 ml of suspension for inoculation measured by the mcfarland nephelometer was 110 cfu / ml. a volume of 1 ml of this suspension was homogenized with 9 ml of melted (45c) mueller - hinton or sabouraud dextrose agar and poured into petri dishes. for screening, sterile 6 mm discs (himedia, mumbai, india) were impregnated with 10 l of 100 mg / ml of satureja montana l. subsp. after incubation for 2448 hours in the thermostat, inhibition zone diameters, zi, (including disc) were measured and expressed in mm. the presence of the inhibition zone indicates the activity of tested extracts against bacteria or yeasts. the minimum inhibitory concentration (mic) was reported as the lowest concentration of the extracts capable of inhibiting the growth of the bacterium tested. the mic was determined by the broth macrodilution test using an inoculum of 110 cfu / ml. final concentrations of the investigated extracts were 6.25, 12.5, 25, 50, 75 and 100 mg / ml. pseudomonas aeruginosa (atcc 27853, gram negative), escherichia coli (atcc 25922, gram negative), staphylococcus aureus (atcc 25923, gram positive), sarcina lutea (atcc 9341, gram positive), bacillus cereus (atcc 10707, gram positive), saccharomyces cerevisiae (112, hefebank weihenstephan) and candida pseudotropicalis (clinical isolate) microorganism strains were employed for the determination of antimicrobial activity. penicillin (10 iu / disc) and amoxicillin (25 g / disc) were used as reference standards as obtained from bioanalyse co., ltd., ankara, turkey. in parallel with the antibacterial investigation of satureja montana l. subsp. kitaibelii extracts, pure solvent was tested too, and it did not exhibit any antibacterial activity (data are not shown). bacteria were obtained from the stock cultures of the microbiology laboratory, faculty of technology, university of novi sad. all measurements were carried out in triplicate, and presented as mean sme or sd. regression analyses and significance of differences were carried out using a spss statistical software package (spss for windows, 8.0, 1997, spss inc.,	the antioxidant activity of different satureja montana l. subsp. kitaibelii extracts was tested by measuring their ability to scavenge reactive hydroxyl radical during the fenton reaction, using esr spectroscopy. also, the influence of these extracts on lipid peroxyl radicals obtained during lipid peroxidation of : (i) sunflower oil (37 c, 3h) induced by 4,4-azobis(4-cyanovaleric acid) (acva) and (ii) liposomes induced by 2,2-azobis(2- amidino - propane)dihydrochloride (aaph) was studied. n - butanol extract had the best antioxidant activity (100% at 0.5 mg / ml in fenton reaction system ; 89.21% at 5 mg / ml in system i ; 83.38% at 5 mg / ml in system ii). the antioxidant activities of the extracts significantly correlated with total phenolic content. the antimicrobial activity of satureja montana l. subsp. kitaibelii extracts was investigated. petroleum ether, chloroform and ethyl acetate extracts expressed a wide range of inhibiting activity against both grampositive and gram - negative bacteria.
arteriovenous malformations (avms) present with hemorrhage, seizures, headache or focal neurological deficits. cysts associated with avms are uncommon, and are usually a sequelae of stereotactic radiosurgery or after a hemorrhage. untreated and unruptured avms with large cysts are uncommon, with only five cases reported in literature, and one in the pediatric population. we describe a case of an untreated and unruptured avm in the right parietal lobe, which was associated with a large cyst. an 8-year - old boy presented with history of generalized tonic clonic seizures for the last 2 years. there was no history of sudden onset headache or neurological deficits, suggestive of bleed. on examination, there were no focal neurological deficits. an mri was performed, which showed a nidus, seen as flow voids, in the medial parietal lobe on the right side with a large cystic area abutting it posteriorly. this was hyperintense on t2-weighted images and inverted completely on flair images [figure 1a and b ]. a digital subtraction angiogram was subsequently performed, which showed the nidus was receiving arterial feeders from the pericallosal branches of the right anterior cerebral artery and draining into the superior sagittal sinus via cortical veins [figure 1c and d ]. (a and b) sagittal t2-weighted (a) and axial flair (b) mr images showing the avm nidus anteriorly and medially with the large cystic component posteriorly located in the right medial parietal lobe. (c and d) digital subtraction angiogram images in ap and lateral planes showing the nidus receiving arterial feeders from the pericallosal branches of the right anterior cerebral artery. (e and f) post - operative angiograms in ap and lateral planes showing complete excision with non - visualization of the nidus on right ica injection various treatment options were discussed, and the patient was offered surgical excision of the avm as the first choice. the avm nidus and the draining vein was seen on the surface, and below that, there was a large cyst containing about 30 ml of clear fluid. histological examination of the nidus showed abnormal arterial and venous channels, and the cyst wall had numerous thin - walled vascular channels containing hemosiderin in the fibrous tissue. a post - operative angiogram was done on post - operative day two, which showed complete excision of the nidus [figure 1e and f ]. his post - operative stay in the hospital was uneventful, and was discharged on the third post - operative day. at 6-month follow - up, patient is neurologically normal, and is seizure - free on a single anti - epileptic drug. avms associated with cyst without undergoing radiosurgery are extremely rare. in previously reported literature of 5 cases, there were 2 females and 3 males, with age between 14 and 38 years. our patient is the youngest case of this type of association, and is only the second patient in pediatric age group reported in the literature [table 1 ]. three of the patients presented with seizures, 1 with headache and 1 with a focal neurological deficit in the form of a superior quadrantanopia. in 4 of the 5 patients, the avms were located in the temporal lobe and 1 was located in the parietal lobe. cysts accompanying avms have been attributed to cavity formation secondary to liquefaction of the hematoma, which forms due to the bleeding in the avm. the cases reported in literature had no history to suggest any previous episode of hemorrhage, and neither had received stereotactic radio surgery. in our case as well, there was no history suggestive of bleed or previous radiation treatment. multiple hemorrhages in a long period and exudation of fluid from part of the avm have been suggested as the etiology for cyst formation. reported the histology of the cyst wall and showed numerous thin - walled vascular channels and deposits in fibrous tissue, which are also seen in the membrane of the giant cysts associated with cavernous angiomas. hence, they suggest that a capsule forms around the minor hemorrhage from the avm resulting in the cyst and repeated subclinical hemorrhages from the neovascular channels of the cyst wall result in the growth of the cyst. itakura., have propounded the theory of gradual exudation of fluid into the surrounding parenchyma resulting in cyst formation. in the present patient, the fluid in the cyst was clear, without any xanthochromia or hemorrhage, and the characteristic of the fluid was similar to csf on imaging. the histology too showed no evidence of hemorrhage or slit like cavities in the cyst wall. hence, we are in concurrence with this theory of gradual exudation of fluid causing the cyst associated with the avm. these can occur secondary to microhemorrhages or exudation of fluid from the part of the avm.	cysts associated with arteriovenous malformations (avms) are either secondary to hemorrhage or after radiosurgery. untreated and unruptured avms with large cysts are rare. we here describe a child with medial parietal avm associated with cyst, without any history of hemorrhage or radiosurgery. surgical excision led to cure for the patient.
the drinking water distribution network is a source of disquiet regarding the contamination of water during delivery and regrowth of microorganisms that survive after treatment. it is often the scene of many physicochemical and biological reactions resulting from interactions between disinfectants, pipe walls, and the free and fixed biomass. the presence of natural organic matter provides a food source for bacteria that can colonize the inner walls of distribution pipes, forming biofilms that protect and support the growth of microorganisms, some of which are associated to hostile effect on human health and others through their interactions with disinfectants and pipe walls are sometimes the cause of the deterioration of the organoleptic properties of the water supply [2, 3 ]. in recent years, world health organization recognizes a. hydrophila as an opportunistic pathogen, implicated as a pathogenic agent in gastroenteritis, septicemia, cellulitis, colitis, meningitis, and respiratory infections [46 ]. to prevent bacterial regrowth, previous work has shown that the bacterium a. hydrophila is widespread in the environment, especially in water intended for human consumption [7, 8 ]. its concentration can sometimes reach 10 cfu / ml at the outlet of treatment plants for drinking water. this concentration may be higher in networks of drinking water distribution due to the growth of a. hydrophila on biofilms [7, 9 ]. the ingestion of water or contaminated food is the common way of progress in the case of aeromonas infection. numerous studies have been conducted in view of highlighting the inactivation of various waterborne pathogens by various disinfectants, including sodium hypochlorite, hydrogen peroxide, ozone, and chlorine dioxide. the mixture of naocl and h2o2 in water resulted in a redox reaction which gave the following equations. h2o2/h2o : 1,77 v and clo2/clo : 0,66 v (1)clo+2hoclo2+h2o+2e(2)h2o2 + 2h++2e2h2o(3)(1) and (2):clo+h2o2 + 2ho+2h+clo2+3h2o(4)clo+h2o2 + 2h2oclo2+3h2o(5)clo+h2o2clo2+h2o(6)na++clo+h2o2na++clo2+h2o(7)naclo+h2o2naclo2+h2o naclo2 is a very unstable compound that gives nacl + o2 (singlet oxygen). it resulted in (8)naclo+h2o2nacl+o21+h2o the reaction between these disinfectants produces singlet oxygen (o2), which is a powerful oxidant that rapidly kills bacterial cells. singlet oxygen short lifespan (100 nanoseconds in lipid media and 50 nanoseconds in the cytoplasm) can diffuse a short distance and react with certain amino acids leading to structural and functional alteration of the membrane causing lipoperoxidation. less data are available on the bacterial behavior or bacterial metabolism when both disinfectants are dissolved in water at the same time. less information are also available on the cell survival with respect to the both disinfectants concentrations. most studies carried out so far provided some information on the doses of disinfectants and adequate contact duration period to effectively control pathogens of public health importance that are commonly used to develop regulations and strategies treatment. chemical disinfectants cause lethal or nonlethal changes in proteins, lipids, membrane, and dna of microorganisms. in addition, the mechanisms of disinfection are also highly dependent on the type of microorganism, cell growth stage, and disinfectant. other studies have considered the impact of disinfectants on a. hydrophila adhered to the fragments of polythene immersed in water. it appears that naocl is more effective on a. hydrophila adhered to polythene than h2o2. in addition, a. hydrophila adhered to polythene under dynamic condition is more sensitive to each of the two disinfectants than that adhered under static condition. this study aims to evaluate in microcosm the synergistic effect of naocl and h2o2 on a. hydrophila cells from different cell growth phases and adhered to fragments of polythene immersed in water. the bacterium a. hydrophila was isolated from well water in yaound (cameroon) using membrane filtration technique, on ampicillin - dextrin agar medium [19, 20 ]. cell subculture was performed on standard agar medium (bio - rad laboratories, france). these cells are facultative anaerobic, nonsporulated, gram - negative bacilli, and ferment mannitol, produce indole, and are mobile. they do not possess urease, lysine decarboxylase (ldc), ornithine decarboxylase (odc), and arginine dihydrolase (adh). for the preparation of stocks of bacteria, colonies are inoculated into 100 ml of nutrient broth (oxford) for 24 hours at 37c. afterwards, cells were harvested by centrifugation at 8000 rpm for 10 min at 10c and washed twice with nacl (8.5 the stocks were then frozen stored. on the basis of previous studies regarding the different growth phases and biofilm formation, the growth of a. hydrophila in nonrenewed peptone liquid medium gives 4 growth phases : a lag growth phase from 0 to 2 hours, an exponential growth phase from 2 to 13 hours, a stationary growth phase from 13 to 22 hours, and a decline growth phase which begins as from the 22th hour. the mixture of two disinfectants was used : naocl, which belongs to the group of halogen derivatives, and h2o2 which belongs to the group of oxidants. naocl and h2o2 used are, respectively, colgate - palmolive (usa) and gilbert (france) brand. the ease use of these two disinfectants in drinking water treatment justified their choice for this study. the combination concentrations of each disinfectant used ranged from 0.1 to 0.3 and from 0.5 to 1.5, for naocl and h2o2, respectively. these concentrations were evaluated by simple method of dilution of crude solution obtained directly from the supplier. the choice of these combination concentrations is justified by their synergistic action. to count the surviving bacteria after disinfection treatment, sterile nacl solution (8.5 it differs from radical low dense polythene and linear low dense polythene by the molecular structure of its sparsely branched chains and its relatively high resistance to shocks, high temperatures, and ultraviolet rays [22, 23 ]. it is a plastic piping material obtained directly from the supplier and used in drinking water distribution. this polymerization is obtained from gaseous ethylene according to the following equation [24, 25 ] : (9) the polythene used in this study is commercialized by goodfellow sarl (france). the principle of this protocol consists in preparation of the mixtures of naocl (a (b assoc)) and h2o2 (b (a assoc)). for it, nine couples of disinfectant concentrations (a (b assoc), b (a assoc)) were studied simultaneously for the preparation of mixtures of disinfectants. the disinfectant concentrations used alone ranged from 0.5 to 1.5 and from 5 to 15 for naocl (a alone) and h2o2 (b alone), respectively. the contaminated substrates are getting in contact with these disinfectant concentrations for 25 to 30 min. the disinfecting effect was stopped by introducing substrates in 10 ml of sterile saline. antimicrobial activity was assessed after culture of surviving germs and appreciation of the reduction of the bacterial load. the effect of the association was estimated by calculating the fractional bactericidal concentration (fbc) according to maris : (10)fbc = a(b assoc)a(alone)+b(a assoc)b(alone), wherein a (b assoc) and b (a assoc) are the respective concentrations of naocl and h2o2 studied in the mixture. a (alone) and b (alone) are the respective concentrations of the two disinfectants studied alone. the synergy was then declared for a value of fbc less than or equal to 0.50. the study of this synergy was achieved at each stage of cell growth phase in stationary and dynamic regimes. on the basis of previous studies, parallelepiped shaped fragments of polythene with 13.28 cm of total surface area suspended with wire of 0.1 mm diameter were immersed in triplicate in the two sets a and b each in four flasks 250 ml duran a1, a1, and a1 and b1, b1, and b1, a2, a2, and a2 and b2, b2, and b2, a3, a3, and a3 and b3, b3, and b3, and a4, a4, and a4 and b4, b4, and b4 each containing 99 ml of nacl solution (8.5. meanwhile, the controls were made and coded a01, a02, a03, and a04 and b01, b02, b03, and b04. prior to the experiments, stocks frozen vial containing a. hydrophila cells were thawed at room temperature. then 100 l of the culture was transferred into test tubes containing 10 ml of nutrient broth (oxford) and incubated at 37c for 24 hours. cells from a specific cell growth phase were then harvested by centrifugation at 8000 rpm for 10 min at 10c and washed twice with sterile nacl solution (8.5 the pellets were then resuspended in 50 ml of sterilized nacl solution (8.5 after serial dilutions, the initial concentration of bacteria (data at t = 0) in each solution was adjusted to 6 10 cfu / ml by reading the optical density at 600 nm using a spectrophotometer (dr 2800) followed by culture on agar. 1 ml of the suspension was added to 99 ml of sterilized nacl solution (8.5 triplicate flasks were incubated under dynamic condition for 180, 360, 540, and 720 min at a stirring speed of 60 rev / min, using a stirrer (rotatest brand). in the same way another triplicate flasks were incubated under static condition for 180, 360, 540, and 720 min. all these incubations were done at laboratory temperature (25 1c). after each incubation duration, fragments of polythene were drained for 10 seconds in a sterile environment created by the bunsen burner flame and then introduced into test tubes containing 10 ml of diluted mixture of disinfectant of various concentrations. fragments removed from flasks a1, a2, a3, a4, b1, b2, b3, and b4 were introduced in mixture disinfectant solutions of 0.1 naocl and 0.5 h2o2. fragments removed from flasks a1, a2, a3, a4, b1, b2, b3, and b4 were introduced into mixture disinfectant solutions of 0.2 naocl and 1 h2o2. similarly, those removed from flasks a1, a2, a3, a4, b1, b2, b3, and b4 were introduced into mixture solutions of 0.3 naocl and 1.5 h2o2. fragments of polythene flasks from a01, a02, a03, and a04 and b01, b02, b03, and b04 were introduced into 10 ml of sterile nacl solution (8.5 g / l). the concentration of the disinfectant has not been evaluated after incubation. after 30 min of incubation at room temperature and under static condition, each fragment was then drained out under sterile condition. each fragment was then introduced into 10 ml of sterilized nacl solution (8.5 the unhooking of adherent cells was performed by vortex agitation at increasing speeds for 30 seconds in three consecutive series of 10 ml sterilized nacl solution (8.5 this technique allows for the unhooking of maximum adhered cells [28, 29 ]. the total volume of the suspension containing the unhooked bacterial cells was 30 ml. the isolation and enumeration of unhooked cells were made by culture on ampicillin dextrin agar, by using spread plat method, followed by incubation on petri dishes at 37c for 24 hours. the variation of the abundance of adhered a. hydrophila in each experimental condition was illustrated by semilogarithmic diagrams. correlation test was used to assess the degree of correlation between the abundance of adhered cells and other parameters considered. kruskal - wallis and mann - whitney tests were used to compare the mean abundance of cells adhered from one experimental condition to another. this isotherm was chosen because of the number and the relevance of the information it provides on the real adsorption mechanisms on one hand and its remarkable ability to match doses of adsorption on the other hand. the freundlich isotherm is described by the following equation [30, 31 ] : (11)cs = kfcl / n, where cs is the quantity of cells adsorbed in the presence of the mixture of disinfectant solutions, c is the concentration of cells adsorbed in the absence of mixture of disinfectant solutions, kf is the freundlich coefficient adsorption which is connected to the adsorption capacity, l / n is coefficient linearity, and n is the intensity of adsorption. here, cs is expressed as the number of adherent cells / mixture of disinfectant concentration and c is the number of adherent cells / cm of polythene. constructing linear regression log cs versus log c results in a line of slope l / n which intercepts the y - axis log kf. to ensure the synergistic action of the two disinfectants, only disinfectant concentrations giving fbc equal to 0.3 were used for the preparation of mixture of disinfectants. the densities of cells adhered ranged from 0.30 to 2.29 units (log (cfu / cm)) after the action of the mixture of naocl and h2o2 under static condition. the maximum abundance of cells adhered was recorded in the presence of the mixture of 0.1 naocl and 0.5 h2o2 and this is after 720 minutes with cells harvested from the lag growth phase. adhered cells were always partially decimated by the mixture of naocl and h2o2 (figure 1). with cells coming from the lag phase, the abundance of cells adhered under static condition to the control substrate varied throughout from 2.02 to 3.19 units (log (cfu / cm)) and was always superior to those of fragments tested for disinfection. maximum cell density was recorded after an adhesion test of 720 minutes. after the action of the mixture of naocl and h2o2, the densities of cells adhered ranged from 0.30 to 2.29 units (log (cfu / cm)). the effectiveness of the mixture of naocl and h2o2 decreased with the length of the adhesion duration test. the maximum cell abundance was recorded in the presence of the mixture of 0.1 naocl and 0.5 h2o2 after an adhesion test of 720 minutes. the lowest density of adhered cells was observed in the presence of the mixture of 0.3 naocl and 1.5 h2o2 with cells coming from the adhesion tests of 180 minutes (figure 1). the abundance of cells under static condition adhered to the control substrate during the exponential growth phase was lower than that tested for disinfection in the lag growth phase under the same condition. they generally fluctuated between 2.30 and 2.91 units (log (cfu / cm)). after disinfection test, it was noted that the effectiveness of the mixture of naocl and h2o2 decreased when the duration of adhesion test increased. abundance of cells adhered ranged between 0.70 to 1.81 units (log (cfu / cm)) (figure 1). the highest cell abundance was recorded in presence of the mixture of 0.1 naocl and 0.5 h2o2 after an adhesion test of 720 minutes. the lowest density of adhered cells was observed in the presence of the mixture of 0.3 naocl and 1.5 h2o2 with cells coming from the adhesion tests of 180 minutes (figure 1). the stationary growth phase shows the abundance of cells in static regime adhered to the control substrate which varies from 1.92 to 2.49 units (log (cfu / cm)). after disinfection test, abundance of cells adhered ranged between 0.90 and 1.89 units (log (cfu / cm)). as the duration of adhesion test increased, it was noted that the effectiveness of the mixture of naocl and h2o2 decreased. the highest density of cells adhered to the polythene was recorded in the presence of the mixture of 0.3 naocl and 1.5 h2o2 after 720 minutes incubation duration. the lowest density of adhered cells was observed in the presence of mixture of 0.3 naocl and 1.5 h2o2 after 180 minutes incubation duration (figure 1). the abundance of cells adhered in static regime to the control substrate during the decline growth phase varied from 1.95 to 2.48 units (log (cfu / cm)). adhered cells after the action of naocl relatively increased (figure 1). the maximum density of cells adhered to the polythene was recorded in the presence of the mixture of 0.3 naocl and 1.5 h2o2 after 720 minutes incubation duration. the minimum density of adhered cells was observed in the presence of mixture of 0.3 naocl and 1.5 h2o2 after 180 minutes incubation (figure 1). the abundances of cells adhered ranged from 0.85 to 2.27 units (log (cfu / cm)) after the action of the mixture of naocl and h2o2 under dynamic condition. the maximum abundance of cells adhered was recorded in the presence of mixture of 0.1 naocl and 0.5 h2o2 and this is after 720 minutes with cells harvested from the lag growth phase. the density of cells adhered under dynamic condition to the control substrate varied throughout from 2.35 to 3.25 units (log (cfu / cm)) from the lag phase and was always superior to those fragments tested for disinfection. the maximum cell abundance was recorded in the presence of the mixture of 0.1 naocl and 0.5 h2o2 after an adhesion test of 720 minutes. the lowest density of adhered cells was observed in the presence of the mixture of 0.3 naocl and 1.5 h2o2 with cells coming from the adhesion tests of 180 minutes (figure 2). after action of the mixture of naocl and h2o2, the densities of cells adhered ranged from 0.85 to 2.27 units (log (cfu / cm)). the effectiveness of the mixture of naocl and h2o2 decreased with the length of the adhesion test duration. abundance of cells adhered under dynamic condition to control substrate during the exponential growth phase was lower than that tested for disinfection in the lag growth phase under the same condition. they generally fluctuated between 2.47 and 3.19 units (log (cfu / cm)). after disinfection test, it was noted that the effectiveness of the mixture of naocl and h2o2 decreased when the duration of adhesion test increased. abundance of cells adhered ranged between 0.95 and 2.09 units (log (cfu / cm)) (figure 2). the maximum cell abundance was recorded in presence of mixture of 0.1 naocl and 0.5 h2o2 after an adhesion test of 720 minutes. the minimum density of adhered cells was observed in the presence of mixture of 0.3 naocl and 1.5 h2o2 with cells coming from the adhesion tests of 180 minutes (figure 2). the abundance of cells adhered in dynamic regime to the control substrate varied from 2.35 to 2.74 units (log (cfu / cm)) during the stationary growth phase. after disinfection test, abundance of cells adhered ranged between 1.30 and 2.13 units (log (cfu / cm)). as the duration of adhesion test increased, it was noted that the effectiveness of the mixture of naocl and h2o2 decreased. the maximum density of cells adhered to the polythene was recorded in the presence of the mixture of 0.3 naocl and 1.5 h2o2 after 720 minutes incubation duration, whereas the minimum density was observed in the presence of the mixture of 0.3 naocl and 1.5 h2o2 after 180 minutes incubation duration (figure 2). density of cells adhered in dynamic condition to the control substrate during the decline growth phase varied from 2.10 to 2.71 units (log (cfu / cm)). cells adhered after the action of naocl were relatively high (figure 2). the maximum density of cells adhered to the polythene was recorded in the presence of the mixture of 0.3 naocl and 1.5 h2o2 after 720 minutes incubation duration and the minimum in the presence of the mixture of 0.3 naocl and 1.5 h2o2 after 180 minutes incubation (figure 2). freundlich isotherms were constructed by considering only the combination concentrations, the number of cells adhered to the substrate, subjected to the test of disinfection, and obtained without exposure to the mixture of disinfectants for each stage of cell growth and each experimental condition. it can be noted that, no matter which growth stage cells are, the appearance of the isotherms differs from one incubation condition to another. the linearity coefficient l / n which is related to the adsorption intensity ranged from 0.01 to 0.21 and from 0.02 to 0.15, respectively, under static and dynamic incubation conditions. the adsorption coefficient kf which is related to the adsorption capacity ranged between 2 and 53 and between 2 and 54 cells adhered, respectively, under static and dynamic incubation conditions. the adsorption coefficient for the lag growth phase ranged between 4 and 53 and between 2 and 54 cells adhered, respectively, under static and dynamic conditions (table 2). the lowest adsorption coefficient after the mixture of disinfectant treatment was obtained with cell harvested from the lag growth phase (table 2). when considering each experimental condition, the adsorption coefficient of cells harvested from the lag phase was relatively higher after the mixture of disinfectant treatment than that of cell harvested from the other cells growth phases (table 2). it was also noted that for the whole cell growth phases and the whole incubation conditions, the adsorption coefficient values were relatively higher with the mixture of 0.1 naocl and 0.5 h2o2 concentration than those of the two other mixture of disinfectant concentrations (table 2). correlation coefficients between the abundances of cells adhered and incubation durations for each concentration of mixture of disinfectant and each experimental condition were assessed and are presented in table 3. it is noted that the increase in the incubation durations caused a significant decrease in the efficiency of 0.3 naocl and 0.3 h2o2 mixture of disinfectant concentration (p < 0.01). this could result in higher abundance of cells adhered as the duration of the cell adhesion process increased. r correlation coefficients between abundance of cells adhered and concentrations of the mixture disinfectants for each incubation duration and under each experimental condition were also assessed (table 4). under static as well as dynamic condition, it was noted that the effectiveness of the mixture of disinfectant concentrations on cells adhered to polythene increased leading to a significant decrease (p < 0.01) in the abundance of bacteria adhered after disinfection treatment. the degrees of relationship between the mixture of disinfectant concentrations and abundance of cells adhered harvested from each growth stage were also assessed (table 5). it resulted that an increase in the mixture of disinfectant concentration significantly increased (p < 0.01) the abundance of cells adhered to the substrate, with cell harvested from each cell growth phase. the h test of kruskal - wallis was performed in order to compare the mean abundance of cells adhered harvested from different cell growth stages and considering each mixture of disinfectants concentrations. it showed that there is an overall significant difference (p < 0.05) between the mean abundance of cells adhered to polythene for each mixture of disinfectant concentration at different cell growth stages. the pair two - by - two comparisons of the mean abundances were then performed using the u test of mann - whitney. it was noted that, at each cell growth stage, there was a significant difference (p < 0.05) amongst the mean abundance of cells adhered after the action of various mixture of disinfectant concentrations with cells coming from each cell growth phase. with the mixture of 0.1 naocl and 0.5 h2o2 and that of 0.3 naocl and 1.5 h2o2, a nonsignificant difference was observed only with cells harvested from the stationary cell growth phase (p 0.05) (table 6). the aim of this study was to determine the synergistic effect of naocl and h2o2 on a. hydrophila adhered to polythene immersed in water under static and dynamic conditions. by contrast, most previous studies have indicated only the effect of naocl on one hand and that of h2o2 on the other hand on the adhesion of a. hydrophila to polythene [18, 32, 33 ]. from the 9 pairs of concentration of disinfectants used for the preparation of mixture of disinfectants, three couples (0.1 naocl + 0.5 h2o2 ; 0.2 naocl + 1 h2o2 ; and 0.3 naocl + 1.5 h2o2) were used to evaluate the synergy as they presented an fbc equal to 0.3. a synergy is declared when a value of fbc is less than or equal to 0.50. the present study showed that the overall abundance of cells adhered to polythene after the action of the mixture of two disinfectants was lower than that obtained after the action of h2o2 alone. abundance of cells adhered to polythene ranged from 0.30 to 2.29 and 0.85 to 2.27 units (log (cfu / cm)) after the action of the mixture of naocl and h2o2 under static and dynamic conditions, respectively. previous studies showed that they sometimes reached 2.41 and 3.39 units (log (cfu / cm)) after the action of naocl and h2o2, respectively. these results suggest that the combination of naocl and h2o2 leads to a significant synergy in eliminating cells adhered to polythene. abundance of cells adhered to polythene after the action of the mixture of naocl and h2o2 was relatively higher than those obtained after the action of naocl alone. the maximum abundance of cells adhered to polythene was recorded under static condition in the presence of the mixture of 0.1 naocl and 0.5 h2o2 and this is after 720 minutes with cells obtained in the lag growth phase (figures 1 and 2). that obtained after the action of naocl was recorded during the lag phase under dynamic condition in the presence of 0.5 concentrations of naocl and this is after an adhesion test of 720 minutes. by cons, the abundance of cells adhered to polythene after the action of the mixture of naocl and h2o2 was considerably lower than those obtained after the action of h2o2. the maximum abundance of cells adhered after the action of h2o2 was recorded during the stationary growth phase under static condition in the presence of 5 h2o2 concentration after the same period of adhesion test. due to its highly oxidizing capacity - based production of free radicals that affect the biofilms matrix h2o2 in addition, h2o2 was chosen as it is highly effective disinfectant in inhibiting biofilms formation at a concentration of 0.05%. the reaction between naocl and h2o2 produces singlet oxygen (o2), which is a powerful oxidant that rapidly kills bacterial cells. in addition, oxygen singlet short lifespan (100 nanoseconds in lipid media and 50 nanoseconds in the cytoplasm) can diffuse a short distance and react with certain amino acids leading to structural and functional alteration of the membrane causing lipoperoxidation. naocl and h2o2 inhibit the brownian motion and control the growth of the microbial population. the adhesion of microorganisms to surfaces is the first step in biofilms formation, which is a form of microbial life in aquatic environments. the latter is the source of problems bioburden in various fields such as health, environment, food industry, and water purification [31, 39, 40 ]. adhesion is governed by physicochemical interactions of the van der waals and lewis acid - base types. fluctuating velocities of adhesion of cells observed during different stages of growth in stationary and dynamic regimes could be explained by changes in the physiology of bacterium at each stage of growth [41, 42 ]. there are three strategies against biofilms formation : (i) the disinfection time before the biofilms develop, (ii) the disinfection of biofilms using aggressive disinfectants, and (iii) inhibition fixing microbes choosing surface materials that do not promote adherence. by considering separately each condition, it was noted that the increase in incubation durations resulted in a significant decrease (p < 0.01) in the effectiveness of the mixture of 0.3 naocl and 1.5 h2o2 (table 3). indeed, a biofilm can be developed within in a few hours, allowing bacteria therein to become resistant to external agents causing any contamination [44, 45 ]. in static as well as dynamic condition, increasing the effectiveness of the mixture concentration of naocl and h2o2 on cells adhered to polythene resulted in a significant decrease in abundance of cells adhered after disinfection test (p < 0.01) (figures 1 and 2). the treatment of biofilms by combining antimicrobial agents has a synergistic effect on the removal of adherent bacterial cells. furthermore, this variation of the reaction of cells against the combination of disinfectants may be related to changes in the surface due to a change in their growth phase. it was also noted that for each incubation period and each cell growth phase, a rise in the concentration of disinfectant mixture increases significantly (p < 0.01) the abundance of cells adhered to the substrate (table 4). face with antimicrobial agent bacteria develops biofilm formation as a coping strategy [47, 48 ]. for each cell growth phase, a significant difference was observed between the mean densities of cells adhered after the action of the different concentrations of the mixture of disinfectants (p < 0.05). the effectiveness of any method of disinfection depends on biotic factors such as the physiological state and the intrinsic microbial resistance to lethal agents. the age of the culture also plays an important role since the adhesion of the bacterium is better during exponential growth phase than stationary growth phase. it is important to remember that bacteria in a biofilm have very different characteristics from their planktonic counterparts including the production of exopolymers, a significant increase in antimicrobial resistance and environmental stress [52, 53 ]. the matrix of exopolymers which presents itself as a mechanical barrier, reducing the penetration of environmental compounds through the biofilms, thus protects bacterial cells embedded in biofilm. this explains the fact that the increase in the concentration of the mixture of disinfectants for each stage of growth leads to a significant increase (p < 0.01) in abundance of cells adhered to the substrates. the adsorption coefficient (kf) was relatively higher in the static than in the dynamic regime no matter the cell growth phase or presence of a well - defined concentration of the mixture of disinfectant. cells adhered to polythene under dynamic condition were more sensitive than that obtained with the two combined disinfectants under static condition. this could be explained by the structure of adhered bacteria which depends on the hydrodynamic regime. although these enzymes often remain qualitatively unchanged with bacterial growth phase, they would quantitatively be modified from one cell growth stage to another. this study showed that the combination of naocl and h2o2 has a synergistic effect on cells adhered to polythene. abundance of cells adhered to polythene after the action of the mixture of naocl and h2o2 is relatively higher than that obtained after the action of naocl alone. by cons, it is significantly lower than that obtained after the action of h2o2 alone. under static as well as dynamic condition, an increase in the effectiveness of the concentrations of the mixture of naocl and h2o2 on cells adhered for each cell growth phase, the densities of cells adhered differed from a given concentration of a mixture of disinfectants to another. although the adsorption coefficient (kf) obtained from the freundlich isotherm is relatively higher in static state than in dynamic regime, cells adhered to polythene in the presence of the mixture of the two disinfectants under dynamic condition seem more sensitive than under static condition.	the synergistic effects of the combined treatments of naocl and h2o2 on the elimination of a. hydrophila adhered to polythene under static and dynamic conditions were evaluated. the concentrations 0.1, 0.2, and 0.3 naocl and 0.5, 1, and 1.5 h2o2 were used. the contact periods were 180, 360, 540, and 720 minutes. the abundance of cells adhered reached 2.47 and 2.27 units (log (cfu / cm)), respectively, under static and dynamic conditions after action of the mixture of disinfectants, whereas it reached 2.41 and 3.39 units (log (cfu / cm)) after action of naocl and h2o2 alone, respectively. increase in the incubation period resulted in a significant decrease in the abundance of cells adhered when the mixture of 0.3 naocl and 1.5 h2o2 was used (p < 0.01). for each cell growth phase, there was a significant difference amongst the mean densities of cells adhered after action of the mixture of disinfectants (p < 0.05). although the freundlich isotherm parameters relatively varied from one experimental condition to another, the kf value registered in the exponential growth phase was relatively higher in static state than in dynamic regime ; cells adhered under dynamic condition seem more sensitive to the synergistic action than those adhered under static condition.
great vessel injury is a rare but fatal complication that can occur during lumbar disc surgery. great vessel injury can result in massive retroperitoneal hemorrhage or present as a delayed pseudoaneurysm or arteriovenous fistula (avf)2,3,6,8,9,11). despite a very low incidence (0.016 - 0.17%), the reported mortality rate ranges from 15% to 100%, and this complication may elicit serious medicolegal problems2,4,5,7,9,10,12). in cases of great vessel injury, early detection and management is critical to ensure patient survival1,4,7). major vascular surgery is often required to prevent fatality ; however, procedure - related morbidity and mortality must also be considered4,5,10). we describe the case of a woman who sustained a common iliac artery and vein injury after l4 - 5 lumbar microdiscectomy. a 48-year - old woman referred to hospital for radiating pain in her lower back and right leg. magnetic resonance imaging of the lumbar spine showed a ruptured disc in l4 - 5. patient vital signs were stable during the operation and immediate postoperative period, and the radiating pain disappeared with no evidence of neurological deficit. although no symptoms such as abdominal cramps were noted, the postoperative hemoglobin level decreased from 13.5 g / dl initially to 8.6mg / dl postoperatively. 1). with the suspicion of postoperative hemorrhage, emergent abdominal ultrasonography and computed tomography (ct) were performed. imaging studies revealed a large retroperitoneal hematoma and a pseudoaneurysm near the right common iliac artery and right common iliac vein (fig. vital signs were monitored, with blood pressure at 110/70 mmhg and heart rate at 70 beats / min. while vital signs were stable, an endovascular intervention was planned. aortography demonstrated a traumatic avf between the right common iliac artery and the right common iliac vein with a 1cm - sized pseudoaneurysm (fig. seoul, korea) and a 94 cm - sized balloon at the right common iliac artery (fig. no retroperitoneal hemorrhage was identified on the 3-month follow - up ct scan (fig. the patient was given a 1-year supply of clopidogrel, and was stable and asymptomatic 3 years after the surgery. major vessel injury is a rare but well - known complication in lumbar disc surgery. despite advances in modern microscopic surgical techniques and laser - assisted surgery, since many cases remain undetected or are not reported, the true incidence of major vessel injury may be higher than what is reported in the literature. the common iliac artery and common iliac vein are located anterior to the l4-l5 lumbar and l1 sacral vertebral bodies, which is where great vessel injury most frequently occurs1,4). great vessel injury is the most frequent injury that occurs when operating on the l4 - 5 lumbar segments6,9). the injury is caused by the perforation of the anterior spinal ligament while attempting complete removal of disc material using a rongeur. the common iliac vessels and the disc are usually separated by the anterior spinal ligament alone at the lower lumbar spinal level. disc degeneration may weaken this ligament and alter the relationship between the ligament and the adjacent vascular structures2,3). the clinical course of vascular injuries can vary and can be categorized into acute, subacute, and chronic. acute blood loss may occur due to damage caused by a severe arterial laceration and lead to sudden hypovolemia10 - 12). diagnosis of avfs or pseudoaneurysms can be delayed because of unusual clinical symptoms and a lack of specific warning signs. vessel injuries may be detected quickly, especially in cases of profuse arterial bleeding and hypotension with tachycardia. however, as in the present case, bleeding may be minimal or go undetected in half of the injuries6,7). even when massive hemorrhage occurs, blood usually collects in the retroperitoneal space, and blood pooling in the disc space may not be recognized in the surgical field. moreover, the prone position in which the patients are operated may confer a degree of vascular compression during surgery, and as such, this may temporarily tamponade any vascular tears7,9). surgeons should suspect vessel injury if hemorrhage or fat tissue is observed during a lumbar discectomy even when vital signs are stable. after a vessel injury, the duration of detection and the treatment approach are associated with the mortality rate1,2,5,7,12). when the vessel injury is recognized intraoperatively and signs of life - threatening hypovolemic shock are observed, vigorous volume replacement and urgent repair of vascular laceration should be considered. in arterial injuries with critical bleeding, although the injury is not confirmed during surgery and the vital signs are stable, insignificant symptoms such as abdominal distension, pain, palpitation, unexplainable anemia, and blurring of the psoas muscle on radiographs are possible predictors of vessel laceration. abdominal ultrasonography and ct angiography are the preferred methods of diagnosis in confirming vessel injury and retroperitoneal hematoma11,12). when diagnosed, conventional angiography and interventional procedure should be performed. in the past, vascular injury following a lumbar discectomy was treated by direct surgical repair of the damaged vessels9,10). although the current outcomes are considered good, surgical repair of traumatic or postoperative vascular injury may result in considerable blood loss and complications4,10). endovascular techniques are an alternative method of managing the vascular injury following lumbar disc surgery. endovascular techniques using a stent graft to occlude avf or leakage show satisfactory bleeding control with few complications1,4,7,10,11). the advantages of these procedures are the absence of a lower abdominal incision, minimal blood loss, and reduced depth and length of anesthesia, which consequently allows for shorter hospitalization periods compared with conventional surgery. however, proper facilities and preparation of stent grafts and other materials are mandated for endovascular intervention7). delayed onset of symptoms or signs may occur in patients who undergo lumbar disc surgery because of an avf or pseudoaneurysm2,8,11). swelling of the legs, fatigue, shortness of breath, cardiac failure, classic machinery bruit in the abdomen, and delayed bleeding may lead to the diagnosis of avf6,8). most isolated vascular injuries of the great retroperitoneal veins remain clinically silent because of a tamponade effect of a perivascular hematoma1). therefore, the treatment of stable avfs or pseudoneurysms should not be delayed, and only arterial side sealing would be sufficient, as in the present case. vascular injury following lumbar disc surgery necessitates early recognition, diagnosis, and prompt surgical repair for prevention of fatal outcomes. an endovascular repair using a stent graft is a minimally invasive and efficient treatment modality with low morbidity.	great vessel injury is a rare but well - known complication of lumbar disc surgery, which may result in acute or fatal outcomes of delayed diagnosis. thus, early detection and proper management is vital. the authors report a case of retroperitoneal hemorrhage with arteriovenous fistula and pseudoaneurysm after lumbar microdiscectomy. the patient was successfully managed by endovascular intervention using a stent graft. endovascular repair is a minimally invasive and efficient treatment modality with considerably low morbidity and mortality.
estimation of prostate volume has been found to be important for choosing a surgical technique (ranging from minimally invasive to open surgery) as well as medical treatment. it is also important for prediction of the duration of surgery and blood loss, especially for surgeons with little experience. although prostate volume does not correlate with symptomatology, as patients with small prostate can have significant symptoms while those with large prostate mild symptoms, international prostate symptom score (ipss) remains relevant in all settings in determining whether it is mild, moderate or severe benign prostatic hyperplasia (bph). for the patients that may require medical therapy based on ipss, the size of the prostate determines those that will benefit from combination therapy (alpha adrenergic blocker and 5 alpha reductase inhibitor). prostate size estimation can be done clinically or with the aid of imaging techniques. over the years, supremacy of imaging techniques has been established over clinical estimation of prostate size by digital rectal examination. initially, estimation of prostate volume was done by transabdominal ultrasound but this has transitioned to the use of transrectal ultrasound. with respect to prostate volume estimation there is no significant difference between measured prostate volumes ; if the comfort of the patient is being considered, suprapubic ultrasound has an advantage over transrectal ultrasound (trus) since it is better tolerated. in most rural hospital centers in sub - saharan africa, contemporary imaging modalities are unaffordable and most times unavailable so relying on it to estimate prostate volume limits the surgeon. in such situations, relying on digital rectal estimation (dre) notable is the grading scale which categorized the prostate volume using a range from zero to 4 +. the grade is as follows : (1) normal gland (20 g) ; about the size of a chest nut _ _ _ _ _ _ _ 0. (2) enlarged prostate gland (about 25 g) ; about the size of a plum and occupies a bit of the rectum________2 +. (4) enlarged prostate gland (about 75 g) ; about the size of an orange and fills approximately three - fourth of the rectal diameter________3 +. (5) enlarged prostate gland (about 100 g) ; may attain the size of a grape fruit and fills so much of the rectal lumen that adequate examination is difficult_________4 +. this study is aimed at determining the reliability of dre in estimating prostatic volume using the sliding scale. establishing the reliability of digital rectal examination in estimating prostate volume would lend more credence to using it as a substitute in areas where ultrasound is unavailable. a total of 150 patients who presented with lower urinary tract symptoms at the urology outpatient clinics in jos university teaching hospital (juth) in 2008 were included in the study. all consented patients with psa greater than 4 ng / ml or abnormal digital rectal examination were subjected to transrectal biopsy specimen of the prostate for histological diagnosis. those with biopsy evidence of prostate cancer detected at any point during the study were excluded. the prostatic volume determination was categorized into not significantly enlarged (prostate volume 50 ml). for each patient, a dre was done by the same urologist (5 years experience) and the volume of the prostate was estimated and categorized into not significantly enlarged or significantly enlarged using the grading scale. not significantly enlarged : equivalent to grade 01 (grading scale) ; equivalent to size of a chest nut ; slightly protruding into rectum ; below 50 mlsignificantly enlarged : equivalent to grade 2 (grading scale) to grade 3 ; fills somewhat < of the rectum of the rectum to three - fourth of the rectum ; above 50 ml. not significantly enlarged : equivalent to grade 01 (grading scale) ; equivalent to size of a chest nut ; slightly protruding into rectum ; below 50 ml significantly enlarged : equivalent to grade 2 (grading scale) to grade 3 ; fills somewhat < of the rectum of the rectum to three - fourth of the rectum ; above 50 ml. subsequently, patients were then sent for transabdominal suprapubic ultrasound that is done within a week following dre. for the abdominal ultrasound, patients were requested to attend with a full bladder. in each patient, transabdominal ultrasound for all patients recruited for the study was performed by the same consultant radiologist (6 years experience) to remove interobserver difference, using a 3.5 mhz curvilinear scanner, with each patient scanned in the supine position. prostate volume (v cm) was then calculated using the formula v = \ 6 ; where is transverse diameter ; is anterior - posterior diameter ; is the longitudinal diameter ; \6 is 0.52. the data generated in the study was analyzed by stata / ic (stata corp lp. texas usa) 13.1 and microsoft excel sheet 2012 (microsoft campus, thames valley park reading, berkshire, rg6 1wg) ; level of significance was set at a two - tailed p < 0.01.. a value of between 0.4 and 0.75 implies a significant agreement between the two variables. the mean age was 65.6 9.84 years while the peak age group was 6069 years [figure 1.0 ]. age distribution of 100 patients with benign prostatic hyperplasia the mean prostate volume based on transabdominal ultrasound estimation was 72.79 44.38 ml and the range was 14.83223.82 ml. the median prostate volume was 62 ml of the 100 patients, three had no significant prostate enlargement on both digital rectal examination and suprapubic ultrasound. three patients had significant prostate enlargement on digital rectal examination only while 93 patients had significant prostate enlargement on both digital rectal examination and suprapubic ultrasound as shown in figure 1.1. bars depicting correlation between ultrasound and digital rectal examination in addition, following kappa 's reliability test for the above data, the kappa 's reliability test was 0.579832, the kappa 's standard error was 0.097768 and kappa 's t value was 5.93. the kappa 's reliability test fell into the good agreement range (0.4 - 0.75). this is further validated by the pearson 's correlation test ascertaining correlation between ultrasound and dre and generated a correlation coefficient of 0.59 (p = 0.00) as shown in figure 1.2. this implies a high positive correlation between ultrasound estimated prostate volume and that estimated by dre that is statistically significant (p < 0.01). in this study, there was significant agreement in the accuracy of dre in determining enlarged prostate compared to ultrasound. both varenhorst. and cheng. in separate studies proved that dre done by a urologist had a higher predictive value. the grading scale is one of such methods that tried to standardize clinical estimation of prostate size. though, dre is very important in initial evaluation of patients with lower urinary tract symptoms and suspected bph, it is a poor predictor of actual size of prostate, compared to trus, computed tomography scan, or magnetic resonance imaging. a study by streich. showed that dre despite the high diagnostic value, for a large part is subjective and needs to be objectified by means of ultrasound examination. estimation of prostate volume by dre appears bigger than evaluated by trus. in this study, three patients prostate volumes were overestimated by dre compared to ultrasound. cheng. have shown that the trained urologist is more accurate in estimating prostate volume with dre than a urology junior trainee, as the difference between their discrepancies is statistically significant. the difference between the discrepancies becomes insignificant if the trained urologist and the urology higher trainee are compared. although, evidences abound on the inferiority of determining prostate volume by dre compared to imaging studies. it is of note to emphasize its value in a resource poor setting where such imaging studies are unavailable. more so, dre estimated prostate volume has been proven by various studies including this study to correlate with prostate volume estimated by ultrasound if done by a urologist. as such relying on dre in a resource poor setting may not be completely out of place. it is important to emphasize the importance of detecting significantly enlarged prostate in medical management. significantly enlarged prostate (50 ml) respond effectively to combination therapy (alpha adrenergic blocker and 5-alpha reductase inhibitors). any guide to identify patients who will benefit from such therapy will aid appropriate management of bph. arguably, giving combination therapy blindly to those with not significantly enlarged prostate in the absence of any reliable guide will increase the cost of medical management, which will further increase the financial burden on the already impoverished populace. a combination of ipss to determine the severity of bph and dre estimated prostate volume to determine those with moderate or severe bph who will benefit from combination therapy will be a possible guide for cost effective treatment for bph in a rural setting. several studies have been done to assess the reliability of abdominal ultrasound in estimating prostate volume, and they demonstrated reliability. a local study conducted by ibinaye. at uch ibadan also demonstrated that the abdominal ultrasound correlated well with trus in the measurement of prostate volume. separate studies where prostate volume estimated by transabdominal ultrasound was compared with the actual prostate size following prostatectomy showed significant correlation. this is very important in an environment where esoteric laboratory facilities are not readily available, and the clinician has to depend mainly on his clinical acumen.	objectives : to determine the correlation between prostate volume estimated by digital rectal examination (dre) and that estimated by abdominal ultrasound in the same patients.patients and methods : men who presented to our urology outpatient clinic with lower urinary tract symptoms were recruited in this study. we estimated the prostate size by digital rectal examination using the sliding scale as a guide and subsequently measured the prostate volume by transabdominal ultrasound.results:a total of 100 patients completed this study. the mean age was 65.6 9.84 years. the kappa 's reliability test comparing the prostate size estimated by dre and the prostate size measured by transabdominal ultrasound was 0.579832, the kappa 's standard error was 0.097768 and kappa 's t value was 5.93. the kappa 's reliability test fell into good agreement range (0.40.75). this is further validated by the pearson 's correlation test ascertaining correlation between ultrasound and dre and generated a correlation coefficient of 0.59 (p = 0.00). this implies a high positive correlation between ultrasound estimated prostate volume and that estimated by dre that is statistically significant (p < 0.01).conclusion : estimation of prostate volume by digital rectal examination is reliable. this is very important in an environment where esoteric laboratory facilities are not readily available, and the clinician has to depend mainly on his clinical acumen.
the sensory strategy for postural control involves the visual, vestibular, and somatosensory systems. in particular, the vestibular system provides information regarding head position and movement with respect to gravity and inertial forces1. more specifically, the vestibulo - ocular and vestibulospinal reflexes are significantly associated with postural control, eye movement, and neck and trunk rotation2. a previous study reported that stroke patients and healthy elderly adults exhibit increased postural instability during head rotations in the standing position compared to that during static standing3. with regard to vestibular information related to cortical activation, areas of the superior temporal gyrus related to postural control are activated in subjects who primarily receive vestibular sensory input in the standing position4. increased brain activity has also been observed in the supplementary motor area during adjustment for postural perturbation5. however, it is unknown whether neck and trunk rotation speeds influence postural perturbation and cortical activation. thus, measuring changes in cortical activation and postural stability simultaneously would provide valuable information regarding vestibular function in activities of daily living. the purpose of this study was to determine if different neck and trunk rotation speeds influence standing postural stability or frontal and temporal cortical activity during rotation in healthy young adults. twelve healthy volunteers (mean age, 25.8 2.1 years) participated in this study. self - reported history of vestibular, balance, and mobility impairment was obtained from all subjects. written informed consent was taken from all volunteers prior to study participation, and the study protocol was approved by the ethics committee of university of saga, japan. the following three parameters were measured simultaneously for each subject : brain activity, center of pressure (cop), and head perturbation. evaluations were performed with a block design comprising an initial resting during standing condition (15 seconds), a task condition (30 seconds), and a second resting during standing condition (15 seconds). evaluation of cortical activity during postural stability requires an accurate definition of rotation stimulation. postural stability was evaluated using a force platform (gs-31 ; anima, inc., tokyo, japan) and head sensor (tsnd121 ; atr - promotions, kyoto, japan). a custom turn - table operated by one of the experimenters was placed on the platform to assess postural perturbation. subjects were asked to stand barefoot on the turn - table with their feet together ; the platform rotated at an angle of up to 180. subjects were randomly assigned to either the high- or low - rotation speed condition (180/s or 90/s peak angular velocity, respectively). to avoid excessive eye movement during testing, subjects were asked to keep their head as still as possible while focusing on a target placed at a distance of 5 m in front of them. subjects were permitted to familiarize themselves with the platform s movement prior to the measurement. to evaluate postural stability, we measured cop for both, area of body sway and total body length. a sensor was placed on the top of each subject s head to detect perturbations. the following two head movements were evaluated : head velocity was measured in the anterior posterior, left - right, and up - down directions, and angular velocity was measured in the roll, pitch, and yaw planes. cop positions and head perturbations cerebral cortex activity was evaluated using a functional near - infrared spectroscopy (fnirs) system (omm-3000 ; shimadzu corp., kyoto, japan) equipped with 16 light sources and 16 detectors. this system captured changes in oxygenated hemoglobin (oxyhb) level through 51 channels. we adopted an interoptode distance of 3.0 cm from the near - infrared light source to ensure propagation to the gray matter underlying the optodes. the light source at the center of the third row was set in a position that corresponded to cz, t3, t4, f3, and f4 of the 1020 international system. we defined each fnirs channel by the midpoint of the corresponding light source - detector pair. regarding anatomical information, the location of each optode on the plastic cap was marked using a 3d digitizer (fastrak ; polhemus, inc. the estimated locations of the fnirs channels on the cortex were transformed using the affine transformation matrix in the fusion software program (shimadzu corp). data were analyzed with nirs - spm using matlab 2014a software (the mathworks, inc., all statistical analyses were performed using spss version 21 software (ibm corp., armonk, ny, usa), with the significance level set at p 0.05). the aim of the present study was to determine whether different neck and trunk rotation speeds influence standing postural stability or frontal and temporal cortical activity during rotation. there was an increase in the subjects cop and head perturbation in the roll, pitch, and yaw planes during high - speed rotation. with regard to movement strategies, ankle and hip strategies are critical for fine motor coordination and dynamic motor coordination, respectively1. the high rotation speed in this study increased body perturbation in order to control postural stability ; this would affect both ankle and hip strategies. moreover, a previous study showed that the obliquus capitis inferior, rectus capitis posterior major, and splenius muscle responses are affected during body rotation when the head position is fixed2. these muscles have a high spindle density, and a high rotation speed could be one factor that allows for highly sensitive postural control. the fnirs results in this study showed that oxyhb levels did not increase significantly during high- or low - speed rotation compared to those in the resting condition. regarding the effect of rotation during standing, automatic postural responses suggest that the spinocerebellum and basal ganglia play complementary roles in adapting postural responses to changing conditions7. the cerebral cortex exerts more control over anticipatory postural adjustments than automatic postural responses7 ; therefore, neck and trunk rotation during standing may not significantly activate the cerebral cortex. thus, regardless of rotation speed, rehabilitative neck and trunk actions might be able to activate automatic postural responses. on the other hand, the vestibular system senses head position during tilting and acceleration1. one reason for the lack of significant differences between speed conditions could be that subjects were asked to keep their head as still as possible during platform rotation. first, although spinal reflexes are associated with postural stability, they were not evaluated here. second, in experimental conditions, the distance between a subject and a visual target is usually set at 2 m, but we chose 5 m due to space restrictions. in addition, the scalp and the skull could have interfered with fnirs signal measurement, and fnirs fiber movement during neck and trunk rotation could have introduced an artificial noise source. future studies should employ other neuroimaging methods to clarify the neural mechanisms of postural control.	[purpose ] the aim of the present study was to determine whether different neck and trunk rotation speeds influence standing postural stability or frontal and temporal cortical activity during rotation in healthy young adults. [subjects and methods ] twelve healthy volunteers participated in this study. a custom turn - table operated by one of the experimenters was placed on a platform to assess postural perturbation. subjects were asked to stand barefoot on the turn - table in an upright position with their feet together, and measurements were obtained during high- and low - speed rotations. postural stability was tested using a force platform and a head sensor. cerebral cortex activity was measured using functional near - infrared spectroscopy. brain activity, center of pressure, and head perturbation were measured simultaneously for each subject. [results ] significant differences were found in the center of pressure and the head angular velocity between high- and low - speed rotations. however, compared to baseline, oxygenated hemoglobin levels were not significantly different during high- or low - speed rotations. [conclusion ] automatic postural responses to neck and trunk rotation while standing did not significantly activate the cerebral cortex. therefore, the response to stimuli from the feet may be controlled by the spinal reflex rather than the cerebral cortex.
	pellagra is a nutritional disorder secondary to niacin deficiency. the classical triad is dermatitis, diarrhea, and dementia. we report the case of a young girl with hypermobility - type ehlers danlos syndrome who exhibited the classical pellagra symptoms, despite apparent adequate nutritional intake. her condition resolved after oral niacin supplements were administered. although this association has not previously been recognized, an inherited connective tissue disorder may be related to the appearance of pellagra.
human immunodeficiency (hiv) is a disease which results in decreased chemotaxis, defective granuloma formation and maintenance, impaired antigen processing and presentation, and generalized loss of cd4 + t cells. india alone accounts for over 2.5 million people living with hiv with an estimated prevalence of 0.91%. the risk of occupational transmission of the virus from a patient to a health - care provider has been estimated at 0.3% after a single percutaneous exposure to hiv - infected blood. we can improve the medication tolerance / effectiveness, treatment success rate, and quality of life by providing good oral care to hiv - positive individuals. with improved survival rates, in the near future more hiv - positive patients will seek dental care. previous reports have shown that approximately 90% of the hiv infections among health - care workers occur in developing countries where occupational safety is a neglected issue. in the last two decades all over the world unwillingness to treat patients with hiv by the dentists has been associated with inadequate knowledge of disease process, transmission, diagnosis, and treatment of hiv infected patients which in turn has led to fear regarding contagion or aids phobia. dental faculty should act as a role model for the dental students regarding the dental treatment of aids patients. in the studies conducted in various countries, although satisfactory knowledge level among the study participants was there, a stigma was reported regarding the treatment of hiv / aids patients. studies done in south africa, brazil, japan, and sudan demonstrated that dental students had insufficient knowledge regarding hiv, its mode of transmission, and management of hiv positive patients. lack of knowledge, fear of contracting the infection during the course of treating hiv infected patients, resistance of support staff, and perceived lack of clinical skills act as barriers to treating hiv positive individuals among dentists. the studies done in india by aggarwal. and fotedar. have demonstrated good knowledge score among the dental students. according to the world health organization (who), hiv - positive patients should be treated. despite all these recommendations, dentists are reluctant to treat hiv / aids patients because of lack of knowledge and ignorance about the disease. infectious diseases including hiv / aids and cross - infection control should form a part of the dental course curriculum. the studies done till date in kerala to assess the knowledge and attitude of dentists towards hiv are rare. the purpose of the present study was to assess the knowledge, attitude, and practice towards hiv patients among the dentists of trichur district, kerala. a cross - sectional survey was conducted among the registered dental practitioners of trichur district, kerala using a pretested 26-item questionnaire. out of the 26 questions, 5 questions were based on sociodemographic factors and the rest were knowledge, attitude, and practice questions. the correct knowledge response rate was estimated to be at 65.25 = (1.96) 65.25 34.75/(65.25 10/100) = 204.59 the data collection was done by direct interviews, telephoning, and mailing. the questionnaire consisted of 5 questions based on sociodemographic factors, 9 questions on knowledge, 9 questions on attitudes, and 5 questions on practice. participants who were legally registered dental practitioners of trichur district, kerala were included in the study. the study was conducted for a period of 6 months from january to june 2013. the statistical analysis was done using the ibm statistical package for the social sciences software version 20, and the statistical test used here was the chi - square test. in the present study, out of 206 study participants, 67% and 70.4% agreed that if they treated patients with hiv or aids, they would be placed at an increased personal risk and that it would be difficult to deal with staff fears about patients with hiv / aids, respectively. in the present study, 89.6% disagreed that they do not have an ethical responsibility to treat patients with hiv. in the present study, a total of 46.4% of dentists agreed that, if they treat patients with hiv / aids, other patients may discontinue treatment in their dental office. only 4.4% correctly answered the question what is the risk of contracting hiv infection from an hiv - contaminated needlestick injury. in the current study, 60.7% of the dentists agreed that it is possible to be infected with hiv by mother 's breast milk. in the present study, 69.9% of the study participants knew that white lesions on the lateral parts of the tongue with fissured or hairy surface is hiv manifestation. only 60.5% knew that positive anti - hiv findings indicated that a patient suffered with hiv. in the present study, out of 38 study participants who agreed that they did not have an ethical responsibility to treat hiv infected patients, 89.5% were unwilling to treat hiv infected patients. a statistical significance was found to exist between q12 (i am willing to treat hiv infected patients) and q6 (i do nt have an ethical responsibility to treat hiv infected patients) (p = 0.0001). in the present study, 10% of the study participants agreed that they used hard containers for disposing sharps. table 1 shows that, out of 81 study participants who were unwilling to treat hiv infected patients, 39.5% were in the age group of 5059 years. a statistical significance was found to exist between q12 (i am willing to treat hiv infected patients) and age groups (p = 0.0001). age group versus q12 (i am willing to treat hiv infected patients) table 2 shows that, as attitude towards hiv patients increases, knowledge and practice towards hiv also increases. as knowledge towards hiv patients increases, practice also increases. correlations between attitude and knowledge, attitude, and practice and knowledge and practice table 3 shows that there is a significant difference between males and females regarding attitude and knowledge with females showing an increase in both attitude and knowledge towards hiv (p = 0.048 for attitude and p < 0.001 for knowledge, t = students t - test). knowledge, attitude, and practice versus gender table 4 reveals that as age increases knowledge, attitude, and practice towards hiv is significantly reduced (p < 0.001, vhs ; f f test). knowledge, attitude, and practice versus age group table 5 shows that there is a significant difference in the attitude and knowledge among dentists in rural, urban, and semirural areas (p = 0.001 ; vhs). in the current study, 70.4% of the dentists agreed that it would be difficult to deal with staff fears about patients with hiv. this is comparable to the study done by bodadhe. in india where 61.4% agreed to the same. in a study conducted by mc carthy. in canada, this difference can be attributed to the differences between canadian and indian population. in the present study, 67% of the dentists agreed that they would be placed at greater personal risk if they treat hiv patients. where 62% agreed to the same. in the present study, 46.4% of dentists agreed that, if they treat patients with hiv / aids, other patients may discontinue treatment in their dental office. this is consistent with the study done in uae by haroun., where 59% of the students responded that the university should treat hiv infected personnel or students. only 4.4% of the study participants correctly answered the question of the risk of contracting hiv infection from an hiv - contaminated needlestick injury., where only 10.9% correctly answered this question. in the current study, 60.7% of the dentists knew that it is possible to be infected with hiv by mother 's breast milk. this is similar to the study done by li. among chinese students, where majority of the students answered correctly about the routes of transmission and also in contrast to the study done by rehan. in pakistan where 47.3% students answered incorrectly regarding the mode of transmission this may be due to the difference in study population and the difference in training given at the under graduate level and at the continuing education level. in the present study, only 67% of the study participants knew that aids can not be diagnosed using urine and only 78.6% knew that aids can be diagnosed using blood. this is lower than the study conducted by park. in korea where 98% and 87% of the dentists correctly answered these questions, respectively. in the present study, 10% agreed that they used hard containers for disposing sharps, whereas in the study conducted by borax. a statistical significance was found to exist between willingness to treat hiv patients and age groups. respondents younger than 30 years were least likely to refuse to treat h1v infected patients. this may be because of the fact that they had received more formal training related to hiv than older dentists. there is a significant difference between males and female dentists in attitude and knowledge, with females showing an increase in positive attitude and knowledge towards hiv than males ; however, in studies by aggarval. and hashemipour., there was no significant gender difference. there was a positive correlation between knowledge and attitude, which is similar to the study done by mc carthy. as age increases, knowledge, attitude, and practice towards aids decreases probably because the junior dentists had received more formal training related to hiv than older dentists had. in the present study, dentists practicing in urban areas showed significant increase in positive attitude and practice, which is similar to the study done by bodadhe. one of the main limitation of the study was that the results of the study are based on subjective assessment. as the participants were selected according to convenient sampling from a single district, the results can not be generalized to the general population of india. one of the main concerns of the study was regarding the effect of age and sex of the dentists on the knowledge and attitude level towards hiv patients. further studies can be done to assess the effect of continuing dental education programs / health education related to hiv on the attitudes, knowledge, and practice among dentists. staff fears and increased personal risk are found to be the most frequently reported concerns in treating hiv patients among dentists of trichur district, kerala. another alarming finding is that only very few dentists reported that they knew how to treat an hiv patient safely. moreover most of the dentists were not treating all patients as if they would treat an hiv positive patient.. the dentists should make decisions with regard to the type of dental treatment provided. dentists should not refuse to treat a patient solely based on their hiv positive status. continuing dental education has to be given to reduce the dentists refusal to treat hiv infected patients.	aims and objectives : discrimination by some health care workers, including dentists, against human immunodeficiency virus (hiv) infected persons has been noted. the main aim of the present study was to assess the knowledge, attitude, and practice towards hiv patients among the dentists of trichur district, kerala.materials and methods : a cross - sectional survey was conducted among 206 dentists practicing in trichur district of kerala. data was collected using a pretested, self - administered 26-item questionnaire and was statistically analyzed using spss software version 20.results:out of 206 participants, 39.3% were unwilling to treat hiv patients. a statistical significance was found between willingness to treat hiv infected patients and age groups (p = 0.0001) as well as between the willingness to treat hiv infected patients and ethical responsibility (p = 0.0001).conclusion : staff fears and increased personal risk are found to be the most frequently reported concerns in treating hiv patients among dentists of trichur district, kerala. senior dentists showed more reluctance to treat hiv positive individuals.
pseudotyping envelopes of viral vectors are heterologous glycoproteins with the key role of mediating vector entry into target cells. thus, their nature, function, and density on the vector surface may deeply influence the transduction ability of the vectors. a powerful strategy to increase the expression of heterologous proteins in eukaryotic cells is codon optimization (co), which is an artificial process through which dna sequences are modified by the introduction of silent mutations, generating synonymous codons. by degeneracy of the genetic code, all amino acids (aa) except met and trp are encoded by more than one codon ; i.e., synonymous codons. genetic code redundancy makes dna triplets tolerant for point mutations, which do not result in corresponding aa mutations (silent mutations). codon optimization is exploited to overcome species - specific codon usage bias and ultimately improve heterologous protein production. the frequency of codon distribution within the genome (codon usage bias) is variable and differs depending on species. it follows that trnas corresponding to synonymous codons are not equally abundant in different cell types and species. therefore, for a certain aa, there are synonymous codons more often used, influencing the timing and efficiency of protein translation.2, 3, 4 the codon adaptation index (cai) technique measures synonymous codon usage bias in a given species. the cai uses a range (between 0 and 1, where 1 is the maximum translational efficiency) of high - rate expression genes (i.e., ribosomal proteins and elongation factors) to assess the relative contribution of each codon in a specific organism, allowing comparison with the nucleotide sequence of interest. thus, it is possible to increase the expression of a certain gene in a specific organism / cell type by simply changing rare codons with more frequent ones, resulting in modification of the cai. codon optimization has been extensively used to increase the production of either recombinant proteins or viral vectors.6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 rd114-tr is a chimeric mutant deriving from the feline endogenous retrovirus rd114 envelope, in which the tr domain of the gamma retroviral vector (-rv) moloney leukemia virus (mlv) amphotropic 4070-a envelope, fused at the c - terminal end of rd114, increases envelope incorporation into lentiviral vector (lv) particles. rd114-tr is first translated in a non - functional precursor (pr) that is then processed by the membrane - associated endoprotease furin in the surface (su) and transmembrane (tm) active subunits. rd114-tr processing occurs either in furin - rich compartments of the trans - golgi network, where the pr accumulates during its way to the plasma membrane or in the recycling endosomes close to the plasma membrane. the cleavage and post - translational glycosylation of rd114-tr are crucial for trafficking to the plasma membrane and for incorporation into nascent virion coats. the tm subunit mediates plasma membrane anchoring of the su subunit. upon recognition and engagement of functional subunits to specific receptors, fusion between viral and cell membranes mediates the entry of the vector into target cells. rd114-tr - pseudotyped retroviral vectors are suitable for both ex vivo and in vivo gene therapy applications because they can be concentrated by centrifugation and are resistant to human serum complement inactivation.20, 21, 22, 23 to improve and simplify the expression of the rd114-tr envelope during development of the rd - molpack packaging technology for stable and constitutive manufacturing of lvs,21, 23 we codon - optimized the entire rd114-tr open reading frame (orf). this idea stemmed from our previous observation that rd114-tr expression is achieved only when the -globin intron (bgi) is inserted between the promoter and the rd114-tr cdna of the expression cassette of many different expression plasmids tested. to explain this constraint, we hypothesized that bgi may attenuate the negative effect of interfering sequences existing in the rd114-tr cdna. to eliminate these sequences and to simplify the vector design, we decided to codon - optimize the entire rd114-tr orf. in fact, the elimination of the interfering sequences would have avoided using the bgi, therefore reducing the size of the vector. unexpectedly, we found that, despite the high level of transcription / translation and cytosol export, rd114-trco is functionally dead. our data strengthen the conclusion, also supported by other studies, that codon optimization may not always lead to functional improvement of the gene of interest. we initially analyzed the expression of the rd114-trwt envelope in rd3-molpack - gfp producer cells and in their derived lvs to confirm previous studies describing proper processing and trafficking to the plasma membrane of the wild - type (wt) envelope. rd3-molpack - gfp cells contain 12 copies of the integrated self inactivating (sin)-rd114-trwt - in - rev - responsive element (rre) transfer vector (tv) (figure 1a, scheme 2), and the originated rd114-trwt pseudo - typed lvs are proficient in cell transduction, as reported previously. we used two specific antibodies (abs), each recognizing either the pr and su (anti - su) subunits or the pr and tm (anti - tm) subunits, respectively (figure 1b). to visualize the expression of rd114-trwt at the rd3-molpack - gfp plasma membrane, we carried out pull - down (pd) of biotinylated and de - glycosylated total cell extracts. because, in sds - page, glycosylated pr and su molecules co - migrate, we first pulled down membrane proteins, which were first biotinylated in vivo and then deglycosylated by peptide n - glycosidase f (pngasef) treatment in vitro. pngasef cleaves the link between asparagine and n - acetylglucosamine residues (complex oligosaccharides) that are added in the endoplasmic reticulum (er) and the golgi stack. we here confirmed the results previously reported by sandrin., showing that both tm and su subunits are correctly localized at the plasma membrane, whereas the pr does not reach and/or accumulate on it (figure 2a, lane 8). the very low level of pr detected in the pngasef - treated sample likely derives from contamination of the endoplasmic reticulum or other membranes (figure 2a, anti - su, top right panel, lane 8). to further characterize rd114-tr glycosylation and trafficking to the plasma membrane, we treated in vitro producer cellular and derived vector extracts not only with pngasef but also with endoglycosidase h (endoh) enzyme. the latter is active on n - linked high - mannose oligosaccharides (simplex oligosaccharides), added in the er compartment, but not on high - glucose residues attached later during glycosylation in the golgi apparatus. it follows that glycoproteins carrying complex oligosaccharides become resistant to the attack of endoh (endoh - resistant proteins). of note, we observed that, in both cells and derived lvs, pr and tm subunits are endoh - sensitive (figure 2b, lanes 3 and 6, anti - tm). on the contrary, the su subunit is endoh - resistant because it carries complex oligosaccharides (figure 2b, lanes 3 and 6, anti - su). the tm contains one putative n - linked glycosylation site (nxs and nxt, where x is any aa), whereas su contains 11 sites (figure s1). it is possible that this unique n - linked site in tm is glycosylated with simplex and not complex oligosaccharides and that the tm subunit is transported to the plasma membrane linked to the su. furthermore, the average titer of rd3-molpack - gfp lvs tested in this study is 1.6 10 4.7 10 sem transducing units (tu)/ml (n = 5), in line with our previous collective data.21, 23 overall, these findings demonstrate that expression of rd114-trwt in rd3-molpack - gfp producer cells and stemmed lvs is correctly achieved. in an attempt to enhance the transduction efficiency of rd3-molpack - derived lvs by increasing the expression and stability of rd114-tr glycoprotein, we codon - optimized its complete cdna. after recoding, the cai of the rd114-tr orf shifted from 0.64 to 0.98, and the average gc content increased from 48% (wt) to 61% (co), resulting in 73% identity between the wt and co sequences (figures s2 and s3). to test the function of rd114-trco, the new orf, cloned into the pires - puro3 expression vector, was transiently co - transfected in pk-7 cells together with the sin - gfp tv to produce rd114-trco - expressing lvs. we analyzed the expression of rd114-tr proteins by western blot, treating cell and virion extracts with or without pngasef and endoh (figure 3). surprisingly, the pattern of rd114-trco subunits greatly differed from that of the wt counterparts. in fact, both cell and lv protein extracts showed very high levels of prco and very low levels or even absence of processed suco and tmco subunits (figures 3a and 3b). in contrast, the expression profile of rd114-trwt in cell and vector extracts was identical to that of rd3-molpack - gfp producer cells and the lvs shown in figure 2. in agreement with these data, the viral titer of rd114-trwt pseudotyped lvs calculated on cem a3.01 cells was 3.9 10 7.1 10 sem tu / ml (n = 3), whereas that of rd114-trco - pseudotyped lvs was consistently undetectable. to better understand the difference between prwt and prco processing, we tested whether codon optimization might have somehow compromised furin - mediated cleavage of rd114-trco. to this purpose, we treated cell extracts derived from pk-7 cells transfected with either the rd114-trwt or rd114-trco plasmid with recombinant furin overnight at 16c. untreated and treated extracts were then analyzed by western blot using the anti - tm ab (figure 4a). we observed that, after furin treatment in vitro, the level of tmco subunit clearly increases (figure 4a, lane 4), even though it is difficult to appreciate the corresponding decrement of prco because of its high level of expression. on the contrary, the amount of prwt is clearly decreased, although it is difficult to appreciate the corresponding increase of tmwt because the wild - type protein is already abundantly cleaved before cell protein extraction. overall, these results support the idea that codon optimization does not compromise furin - mediated cleavage of the envelope, at least in vitro. based on this notion, we then tried to understand why the prco is not correctly processed in vivo. one possible explanation was that a large amount of prco could trigger the phenomenon known as excess substrate inhibition. to exclude this possibility, we transfected hek293 t cells with a scalar amount of rd114-trco plasmid and tested the corresponding cell extracts in a western blot to find the lowest possible dose of prco substrate not inhibiting endogenous furin action (figure 4b). we observed that, even at the lowest amount of plasmid generating detectable prco, the tmco subunit was not visible, indicating that in vivo prco is not processed (figure 4b, lane 3). we next evaluated whether partial recoding of the orf could restore the function of the rd114-trco envelope. to this aim, we generated two cdnas recoded only in the 5 or 3 half of the cdna sequence. we transiently transfected either the rd114-tr5co or rd114-tr3co chimera, cloned into the pires - puro3 plasmid, into pk-7 cells together with the sin - egfp tv. we then tested cellular and lv extracts in a western blot and lv titer in cem a3.01 cells. immunoblot analysis demonstrated that, for both chimeric rd114-tr glycoproteins, prco processing was impaired (figure s4). furthermore, although we transfected equal amounts of rd114-tr, rd114-tr5co, and rd114-tr3co plasmid dna, the expression of rd114-tr3co was lower than that of rd114-tr5co and rd114-trwt (figures s4a and s4c, lanes 3 and 4). the tm3co and tm5co subunits were not detectable in the respective lvs, whereas, after pngasef treatment, su3co and su5co were barely visible and visible, respectively. we explain the difference between anti - tm and anti - su staining with an intrinsic difference in the specific affinity of the two abs. in agreement, to see whether rd114-trco differs from rd114-trwt in its subcellular localization, we carried out confocal microscope imaging in cos-7 cells transfected with pires - rd114-tr plasmids. forty - eight hours after transfection, rd114-tr expression was visualized together with that of calnexin and vamp8/endobrevin, which are er and early and late endosomal markers, respectively. as shown previously by sandrin., rd114-trwt is expressed in the cytosol and perinuclear region and is co - localized mostly with calnexin and very poorly with endobrevin / vamp8. a similar staining pattern and subcellular localization was observed for rd114-trco in either the cos-7 (figure 5) or pk-7 cell experimental setting (figure s5), indicating that er and early and late endosome trafficking of rd114-tr is not affected by codon optimization. because many groups have demonstrated that silent mutations affect correct pre - mrna splicing by introducing cryptic splice sites or altering splicing control elements (i.e., exonic splicing enhancers and silencers),3, 25, 26 we and two service provider companies analyzed rd114-trco mrna both in silico and in vitro for the presence of potential cryptic splicing sites. the first in silico service - provided analysis identified one consensus (cryptic) splice donor site that was nullified by codon optimization, whereas the second service - provided analysis recognized no cryptic sites (figures s2, s3, s6, and s7). we also examined the rd114-trwt and rd114-trco orfs in silico using the netgene2 server, which calculates the probability of cryptic splicing sites in pre - mrna sequences. we did not pinpoint any differences between wild - type and codon - optimized orfs. to further confirm these results, we assessed rd114-trwt and rd114-trco mrna transcripts derived from pk-7 cells transiently transfected with the sin - rd114-trwt / co - in - rre constructs (figure 1a, schemes 2 and 3) by northern blot (figure 6a). two sequence - specific probes targeting rd114-trwt and rd114-trco, respectively, recognized qualitatively comparable rd114-tr mrna transcript patterns (figure 6a). similar results were obtained by using a probe directed against the packaging signal (), which is a sequence common to both constructs. the overall steady - state level of rd114-trco rna detected by the probe was only slightly reduced compared with the wild - type counterpart, but no extra spliced bands were observed. these results indicate that the two lentiviral vector plasmids were equally transfected and correctly expressed from the 5 long terminal repeat (ltr)-cytomegalovirus (cmv) vector promoter. these findings indicate that no cryptic splicing sites are present either in the orf or in the vector backbone (figure 6a). to assess whether mrna metabolism differs between rd114-trwt and rd114-trco, we studied mrna nuclear - cytoplasm export in the pk-7 cell setting using the pires - puro3-based expression vectors, which generate only one mrna transcript. northern blot analysis of total, nuclear (nucl), and cytoplasmic (cyt) mrnas and quantification by typhoon phosphorimager of the band intensity normalized by cellular equivalents loaded revealed that the unique codon - optimized mrna is exported 1.4-fold more (wt cyt / nucl band intensity = 1.1 and co cyt / nucl band intensity = 1.6 ; 1.6/1.1 = 1.4) than wild - type mrna (figure 6b). qrt - pcr analysis, using the expression of nuclear u6 and cytosolic / total gapdh genes as an internal normalizer, revealed that rd114-trco mrna is exported 3.6-fold more than rd114-trwt mrna (figure 6c). overall, these data establish that recoding affects nuclear export but not transcription and splicing processes. we then investigated whether codon optimization could influence mrna secondary structure and, thereafter, protein translation, as reported recently by several groups.3, 4, 27 thus, we examined rd114-trwt and rd114-trco mrna sequences by mfold software (figures 7a and 7b). this computational analysis predicts the most thermodynamically stable rna configurations (up to 50) based on the free energy value (g) of the molecules, where a lower g indicates a higher stability. we retrieved 33 different configurations for rd114-trwt and 37 for rd114-trco (figure 7e). as expected, wild - type structures are very different from codon - optimized ones ; the average g for rd114-trwt mrna is 462.25 (where g = 468.70 is the most stable configuration), whereas the average g for rd114-trco mrna is 679.39 (where g = 687.60 is the most stable configuration). this finding indicates that recoded mrna molecules are more stable than wild - type counterparts (p < 0.0001) (figure 7e). because 5 end and 3 end substructures are fundamentally important for translational dynamics and protein folding, we scanned the 5 and 3 ends of all wild - type and recoded mrnas to identify any possible structural conserved domains. over the 33 conformations of rd114-trwt mrna, we identified a conserved domain at both the 5 end (nucleotides [nt ] 1320) and 3 end (nt 1,3081,677) (figure 7c). these 5 end and 3 end domains are also conserved in the corresponding region of the rd114-trco mrna structure. above the 37 structures calculated by mfold for rd114-trco mrna, we identified nt 1330 at the 5 end and nt 1,3901,677 at the 3 end (figure 7d). to evaluate the similarity between identified domains, we studied rd114-trwt and rd114-trco mrna 5 end and 3 end substructures with simtree software. this software compares each node complexity (branch - loop) of two structures that eventually grades in a similarity score (between 01, where 1 is the maximum). the score is normalized by the number of nucleotides of the substructures from two rna structures showing the lowest g in mfold (table s1). at the 5 end of the rd114-trwt mrna substructure, 26 complexities were identified, which corresponded to only ten complexities in rd114-trco (normalized symmetric similarity [nss ] = 0.5213) (figure 7a). at the 3 end mrna substructures, 42 complexities were found in rd114-trwt and only 18 in rd114-trco (nss = 0.6178) (figure 7b). these results point out that codon optimization of the rd114-tr gene introduced significant alterations at both the 5 end and 3 end of the rd114-trco mrna secondary structure. codon optimization and de - optimization have been used extensively for a lot of different biotechnological practices, primarily in heterologous systems to increase recombinant protein yield and as an adaptive response to environmental conditions and natural host selection in bacteria, yeasts and viruses.29, 30, 31, 32, 33, 34 in some eukaryotes (i.e., c. elegans and drosophila melanogaster), it has been exploited to control intracellular trna to modulate translational efficiency.35, 36, 37, 38 in autologous hosts, such as the mammalian chinese hamster ovary (cho) or hek293 cell lines, which are the most widespread systems for manufacturing pharmaceuticals, codon optimization is a valuable strategy to prevent transcriptional silencing, mrna destabilization, or inefficient translation other than being a powerful tool to increase immunogenicity in dna vaccinology applications.9, 10, 16, 17, 39 finally, hiv - derived lv production has also benefited from codon optimization by enhancing production of structural and functional viral proteins (i.e., gag and pol);9, 11, 12 neutralizing cis - repressive sequences present in gag / pol genes, thereby making the expression of these genes rev - independent;13, 14 and eliminating homology between packaging gag - pol genes and the cis - regulatory packaging () sequence contained in the packaging construct and tv, respectively, therefore reducing the risk of generating a replication - competent lentivirus (rcl). based on these premises, we analyzed dna codon optimization of the rd114-tr gene with the aim of improving envelope translation in rd3-molpack producer cells. in fact, codon optimization would have neutralized the interfering sequences contained in the rd114-tr orf, thereby sparing the use of the bgi in vector design and, at the same time, increasing the production and density of rd114-tr on the cellular plasma membrane and, consequently, on virion coats. we asked for two recoding analyses by two independent companies, which provided similar results (figure s8). therefore, we believe that the quality of the analysis could not have affected the final output. the consistently negative results obtained with the chimeric rd114-tr3co and 5co support this idea. in this study, we show that rd114-trwt expressed either stably (rd3-molpack-24 cells) or transiently (pk-7 cells) naturally traffics from the er through the golgi network to reach the plasma membrane. prwt is processed in suwt and tmwt subunits, which are eventually embedded into nascent lvs. in contrast, rd114-trco reaches the plasma membrane mainly as unprocessed prco, and maturation into suco and tmco functional subunits is drastically reduced or even absent. as a consequence, a high level of prco is erroneously incorporated into budded viral particles, which become defective vectors. because n - linked glycosylation is crucial for maturation of different envelope (env) proteins, such as the hepatitis c virus glycoprotein e2 and human t cell leukemia virus type i (htlv - i) envelopes,39, 40, 41 we studied the glycosylation status of rd114-trwt and rd114-trco. here we confirm the conclusions reached by sandrin., showing that prwt, suwt, and tmwt are deglycosylated by pngasef. furthermore, we also expanded glycosylation studies showing that, in contrast to suwt, tmwt is endoh - sensitive. this result suggests that tmwt either reaches the plasma membrane anchored to su or loses complex oligosaccharides when on the membrane, or, alternatively, does not need complex oligosaccharides for its function. the analysis of prco glycosylation demonstrates sensitivity to both pngasef and endoh enzymes, suggesting defective glycosylation of the recoded protein. further studies will clarify whether a correlation between the observed defective glycosylation and maturation of rd114-trco does exist. although prco is cleaved in vitro by recombinant furin both under reducing and non - reducing conditions (data not shown), it is not cleaved in vivo. to explain this result, we reasoned that cleavage in vivo could be prevented by an excess of substrate : prco is, in fact, much more abundant compared with prwt. alternatively, the deficit of prco processing could be secondary to a deficit of retrograde transport of prco from the cell membrane to the endosomes, where the active form of furin is accumulated. we ruled out the first hypothesis because furin is not active in vivo, even with a very low amount of prco substrate, whereas the second hypothesis requires further analysis to be formally accepted. synonymous mutations have been considered ineffective for a long time, and, for this reason, they are also named silent mutations. however, their nature has been recently re - evaluated because evidence has shown that these mutations have a great effect on pre - mrna splicing and mrna secondary structure formation, therefore affecting protein translational efficiency and folding.4, 43 even a single synonymous codon substitution can have a significant effect on protein folding and function. protein dysfunction can be caused either by disruption (or introduction) of splicing enhancers, by altering mrna stability at the global and local level, or by altering the kinetic of protein production, the ribosomal pausing sites, and co - translational folding.27, 44 our results exclude that codon optimization has introduced aberrant pre - mrna splicing sites. rather, they establish that rd114-trco mrna is exported more efficiently into the cytosol than rd114-trwt mrna and support the theory that some alterations occurred at the mrna secondary structure, thereby influencing protein translation. we focused our study on the mrna 5 end and 3 end because previous findings from others demonstrated that these two domains crucially influence translation dynamics, such as translation initiation and rna global and local stability.45, 46, 47 the in silico mfold and sintree software analyses highlighted that the secondary structures of rd114-trwt and rd114-trco mrna are significantly different. especially some conserved domains at the 5 end and the 3 end of rd114-trwt mrna are lost in the rd114-trco isoform. interestingly, the generation of chimeric rd114-tr5co and rd114-tr3co led to even worse functional impairment. these findings suggest that rd114-trco inactivity is not due to single mutations clustered at the 5 end or 3 end but, more likely, due to conformational modifications distributed along the mrna molecule that affect global mrna stability and, thereby, protein folding and processing. demonstrated that modifications in the cytoplasmic tail of rd114 and rd114-tr alter pr subunit transport from the cell membrane to the trans - golgi network. in particular, transport of the envelopes associated with core protein (i.e., gag) to the endosomal compartment, where active furin accumulates, is important because it affects cleavage efficiency. we observed, by confocal microscope imaging, that both rd114-trwt and rd114-trco are localized mostly in the er compartment when assessed either in the presence (pk-7 setting) or in the absence (cos-7) of gag protein. to this extent, secondary structure modifications identified in rd114-trco mrna might result in alteration of protein folding, which, in turn, is responsible for protein dysfunction altogether, this study suggests that rd114-tr is not suitable for codon optimization and that this strategy can not be applied to improve its performance. cosset (inserm), encodes the chimeric rd114-tr envelope that derives from fusion of the extracellular and transmembrane domains of the feline endogenous retrovirus rd114 envelope and the cytoplasmic tail (tr) of the amphotropic (a)-mlv 4070 envelope (figure 1a, scheme 1). the pires - rd114-trwt - internal ribosome entry site (ires)-puro - woodchuck hepatitis post - transcriptional regulatory element (wpre) plasmid was obtained by excising the cmv - rd114-trwt cassette from the phcmv - rd114-tr plasmid and cloning it into the pires - puro 3 plasmid (clontech laboratories, a takara bio company) (figure 1a, scheme 7). the generation of the sin - rd114-trwt and sin - rd114-trco vectors (figure 1a, schemes 2 and 3) as well as the constructs encoding the hiv gag, pol, and rev genes (figure 1a, schemes 4 and 5, respectively) have been described previously. the sin - gfp tv encoding the egfp gene was kindly provided by l. naldini (tiget, osr) (figure 1a, scheme 6). the rd114-tr orf was codon - optimized, synthesized, and cloned in either the pmk - rq or pms - rq plasmid by geneart. we further cloned the rd114-trco orf into either the pires - puro3 or sin - lv plasmid. four different molecules were generated : pires - cmv - rd114-tr - flco, in which full - length (fl) cdna was codon - optimized and cloned into the ecorv and nsii sites of the pires - puro3 plasmid by excising the orf from the pmk - rq - rd114-tr - flco plasmid ; pires - cmv - rd114-tr-5co, obtained by recoding only the 5-half sequence (789 bp) of the rd114-trwt orf (rd114-tr5co was modified by adding eco47iii and nsii restriction sites at the 5 end and 3 end of the gene sequence, respectively, and the orf was then cloned into the eco47iii and nsii sites of pires - puro3 plasmid) ; pires - cmv - rd114-tr-3co, obtained by recoding only the 3-half sequence (789 bp) and cloning into the eco47iii and sphi restriction sites of the pires - puro3 plasmid by excising the orf from pmk - rq - rd114-tr-3co (figure 1a, scheme 7) ; and sin - rd114-trco fl (figure 1a, scheme 3), generated by inserting rd114-trco into a sin - lv through a three - step cloning strategy. first, the rd114-trwt - ires - puro - wpre fragment was excised from the sin - rd114-trwt - in - rre vector (figure 1a, scheme 2) and cloned into the ecori site of a pgem - t plasmid, generating the pgem - rd114-trwt - ires - puro - wpre plasmid. then, rd114-trwt - ires - puro was excised from pgem - rd114-trwt - ires - puro - wpre (obtaining the pgem - wpre intermediate) using bamhi, and the rd114-trco - ires - puro orf was excised from the pires - cmv - rd114-tr - fl - co using ecorv / xbai enzymes. rd114-trco - ires - puro was then cloned into the pgem - wpre intermediate through blunt ligation, obtaining the pgem - rd114-trco - ires - puro - wpre plasmid. finally, the rd114-trco - ires - wpre orf was cut out from pgem - rd114-trco - ires - wpre and cloned into the ecori site of sin - rd114-trwt - in - rre, generating the sin - rd114-trco - in - rre vector. t cells and their derivative pk-7 clone, which constitutively expresses the hiv gag - pol - rev genes, were propagated in iscove s modified dulbecco s medium (imdm) (biowhittaker, lonza group) supplemented with 10% australian fetal calf serum (fcs) (biowhittaker) and a combination of 1% penicillin - streptomycin and glutamine (psg) (lonza). the cem a3.01 t cell line was grown in rpmi 1640 medium (biowhittaker) supplemented with 10% fcs and 1% psg. cos-7 cells were grown in dmem (biowhittaker) supplemented with 10% fcs and 1% psg. briefly, 35 10 cells were plated on 100-mm tissue culture dishes (becton dickinson). after 24 hr of culture, the egfp tv, rev, packaging, and envelope constructs were co - transfected at a 4:1:0.88:0.48 ratio using either profection mammalian transfection system calcium phosphate (promega) or fugene 6 transfection reagent (roche diagnostics) according to the manufacturer s instructions. transfection efficiency was calculated 48 hr later by analyzing the percentage of egfp - positive cells by fluorescence - activated cell sorting (facs) analysis. the cem a3.01 cell line was transduced by spinoculation at 1,024 g for 2 hr at 37c in the presence of polybrene (8 g / ml) (sigma - aldrich). physical titer was evaluated by measuring the level of p24gag released in the culture supernatant with the alliance hiv-1 p24 antigen elisa kit (perkinelmer) according to the manufacturer s instructions. pk-7 cells were transfected with the pires - rd114-tr or sin - rd114-tr tv plasmid encoding either rd114-trwt or rd114-trco. forty - eight hours after transfection, total, nuclear, and cytoplasmic rnas were extracted by trizol reagent (life technologies) following the manufacturer s instructions and analyzed by northern blot assay. five micrograms rna / sample was run on 0.8% agarose - formaldehyde gel, transferred onto a hybond - n membrane by capillary transfer, and finally probed with 1 10 dpm / ml of a p - labeled 550-bp rd114-trwt or rd114-trco probe in perfecthyb plus hybridization buffer (sigma - aldrich). membranes were extensively washed and then exposed to x - ray films at 80c or to a typhoon phosphorimager 9000 (ge healthcare) for direct quantification of the radioactive signal. after stripping, membranes were re - hybridized with an internal control probe encompassing the packaging sequence () to detect full - length mrnas. total, nuclear, and cytoplasmic rnas, obtained as described above, were retrotranscribed with the superscript first - strand synthesis system kit for rt - pcr (invitrogen). the cdna (1.25 ng) was quantified by qpcr sybr green technology with the following specific primers : rd114-trwt (for 5 aac ggg tca gtc ttc ctc tg ; rev 5 atc aat ggc agg aat ggg ga), rd114-trco (for 5 ccg tgc agt tca ttc ctc tg ; rev 5 ctc agc ttg gtg tac tgg gt), u6 (for 5 ctc gct tcg gca gca ca ; rev aac gct tca cga att tgc gt 5), and gapdh (for 5 tgc acc aca act gct tag c ; rev 5 ggc atg gac tgt ggt cat gag). normalization was calculated using gapdh for total and cytosolic mrna and u6 for nuclear mrna. cellular extracts and viral proteins derived from isolated cell - free virus - like particles (vlps) or lvs were prepared as described previously.21, 49 briefly, lv supernatants were concentrated by centrifugation at 15,000 g for 90 min at 4c. then, the liquid phase was gently removed, and pelleted virions were directly lysed by adding 5 l of pbs/0.5% np-40 (calbiochem, merck - millipore, # 492016). proteins were size - fractionated on 8%, 12%, or 4%15% gradient sds - page (mini - protean tgx gels, # 456 - 1084, bio - rad). then, proteins were electroblotted on either hybond enhanced chemiluminescence (ecl) nitrocellulose membranes (ge healthcare) or transblot turbo transfer pack membranes (bio - rad, # 170 - 4159). membranes were blocked in 5% low - fat dry milk tris - buffered saline (tbs), 1% tween 20 (tbs - t) and then incubated with the appropriate primary antibody diluted in 5% bsa and tbs - t. the anti - tm rd114-tr rabbit serum, kindly provided by f.- the anti - su rd114-tr mab, generated by areta international, was diluted 1:50. the anti - extracellular signal - related kinase-1 (erk) rabbit ab was diluted 1:1,000 (cell signaling technology, # 16). the anti - calnexin rabbit ab was diluted 1:2,000 (santa cruz biotechnology, g1910). the extravidin horseradish peroxidase (hrp) ab was diluted 1:2,000 (sigma - aldrich, # e2886). the anti - hiv human serum, obtained from an aids patient, was kindly donated by g. poli (osr) and diluted 1:1,000. the secondary hrp - linked abs anti - human (# na933v) and anti - rabbit (na934v) (ge healthcare) were diluted 1:5,000. the anti - mouse (# a2066) ab (sigma - aldrich) was diluted 1:10,000. ecl western blotting detection reagent (ge healthcare, rpmn2106) was used for the chemiluminescence reaction. pk-7 and cos-7 cells were transfected with pires - rd114-tr plasmids encoding either rd114-trwt or rd114-trco, seeded on poly - l - lysine - coated glass slides (thermo fisher scientific). forty - eight hours after transfection, cells were fixed with pbs and 3% paraformaldehyde (pfa)/0.1 mm cacl2/0.1 mm mgcl2, permeabilized with pbs and 0.1% triton x-100, and then stained with the following abs : rabbit anti - vamp8 (endobrevin synaptic systems, catalog no. 1047 302) at 1:200 dilution, rabbit anti - calnexin (santa cruz, h-70, catalog no. the secondary abs were alexa fluor goat anti - rabbit a488 (invitrogen, catalog no. a11034) and alexa fluor goat anti - mouse a568 (invitrogen, catalog no. slides were mounted with fluorescence mounting medium (dako), and images were captured with a laser - scanning confocal microscope (leica tcs sp5) with an hcx pl apo blue 63 (na 1.4) objective in oil immersion. images were acquired using the leica application suite (las) advanced fluorescence (af) software (leica microsystems) and processed by the public domain imagej image processing and analysis software (http://rsb.info.nih.gov/ij/) (image processing and analysis in java). protein extracts from either cells or virions were treated with pngasef and endoh enzymes according to the manufacturer s instructions (new england biolabs, # p0704s and # p0702s, respectively). briefly, proteins were first denatured for 10 min at 99c and then digested for 1 hr at 37c with 250500 u of pngasef or endoh enzyme. 4 loading buffer containing -mercaptoethanol was added to samples that were then boiled for 5 min at 99c and finally loaded onto 4%15% sds - page precast gels (mini - protean tgx gels, # 456 - 1084, bio - rad) for western blot analysis. in vitro furin digestion was carried out by treating 35 g cellular extracts with 4 u of recombinant furin (neb, # p8077s) for 16 hr at 16c following the manufacturer s instructions. rd3-molpack - sin - gfp producer cells were plated in 60-mm culture dishes at 1 10 cells / cm density. forty - eight hours after cell seeding, rd3-molpack - sin - gfp cells reached about 90% confluency. cellular monolayers were gently washed in pbs supplemented with 1 mm mgcl2 and 0.1 mm cacl2 to keep epithelial junctions tight and impermeable to molecules. cells were then incubated on ice for 30 min with 0.5 mg / ml ez - link sulfo - nhs - lc - biotin (thermo scientific) in pbs/1 mm mgcl2/0.1 mm cacl2 and gently shaken. after biotinylation, cell monolayers were washed for 5 min with pbs/100 mm glycine/1 mm mgcl2/0.1 mm cacl2 for quenching the biotin excess. cells were finally lysed as described previously,21, 49 and protein extracts were quantified by protein assay (bio - rad, # 500 - 0006). one milligram of proteins was incubated with 5060 l of biotin binder magnetic beads (dynabeads myone streptavidin t1, # 65602, invitrogen) for 1 hr at room temperature by gentle rocking. beads were washed four to five times with 1 ml of pbs/0.1% bsa, and then protein / bead complexes were processed with pngasef. after addition of 4 loading dye and boiling for 5 min at 99c, proteins were separated by sds - page on 4%15% precast gels (mini - protean tgx gels, # 456 - 1084, bio - rad) and analyzed by western blot assay. matched p24gag equivalents of vector particles were incubated with 5 l of anti - su ab (0.9 mg / ml) for 3 hr at 4c under rocking conditions. the pd was performed by washing three times 100 l of dynabeads (sheep anti - mouse immunoglobulin g [igg ] dynabeads, invitrogen, # 422.01) with pbs/0.5% bsa/2 mm edta and then rocking them for 30 min at room temperature in the presence of lv particles and the anti - su ab. after virion pd, the dynabeads were washed several times, 4 loading dye was added, and proteins were separated by sds - page on 4%15% precast gels for western blot analysis. prediction of rna splice sites was generated by the software made available by the netgene2 server (http://www.cbs.dtu.dk/services/netgene2/) and prediction of mrna structure by the software mfold (http://mfold.rit.albany.edu/?q=mfold/rna-folding-form) using the following parameters : linear rna sequence ; 37c folding temperature ; 1 m nacl ionic condition : number of calculated folding ; differences between the calculated foldings = default parameters ; maximum extension of the calculated loops = 30 ; maximum asymmetry between the calculated loops = 30 ; and no limit in base pairing distance. statistical analysis was performed using jmp statistical software and by running the wilcoxon - mann - whitney ranked - sum non - parametric test. contributed to the conception, acquisition, analysis, and interpretation of data and drafting the article. contributed to the final approval of the version to be published. c. bovolenta contributed to the conception, design, analysis, and interpretation of the data, drafting and approving the final version to be published	lentiviral vectors (lvs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. their optimization is therefore very important for the field of vectorology and gene therapy. a key molecule for lv function is the envelope because it guides cell entry. the most commonly used in transiently produced lvs is the vesicular stomatitis virus glycoprotein (vsv - g) envelope, whose continuous expression is, however, toxic for stable lv producer cells. in contrast, the feline endogenous retroviral rd114-tr envelope is suitable for stable lv manufacturing, being well tolerated by producer cells under constitutive expression. we have previously reported successful, transient and stable production of lvs pseudotyped with rd114-tr for good transduction of t lymphocytes and cd34 + cells. to further improve rd114-tr - pseudotyped lv cell entry by increasing envelope expression, we codon - optimized the rd114-tr open reading frame (orf). here we show that, despite the rd114-trco precursor being produced at a higher level than the wild - type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into lvs, making them inactive.
staphylococcus aureus may cause community - acquired and nosocomial bacterial meningitis and is associated with significant mortality. it is usually associated with neurosurgical interventions (including csf shunts), staphylococcal bacteriemia or a parameningeal focus. methicillin - resistant s. aureus (mrsa) is an important cause of hospital - acquired central nervous system infections [17 ]. over the last decade, since the available treatment options are limited, treatment of infections, particularly meningitis, is problematic. however, glycopeptides achieve relatively low cerebrospinal fluid (csf) concentrations and treatment failure is not rare. there are a few case reports in which teicoplanin, fucidic acid and daptomycin have been used successfully. to date, experience with linezolid for the treatment of meningitis is anecdotal and limited to case reports or series. the aim of this study was to compare the efficacy of vancomycin and linezolid in a mrsa rabbit meningitis model. s. aureus atcc 43300 (vancomycin mic : 1 mg / l, linezolid mic : 0.025 mg / l measured in duplicate using the etest ; ab biodisk, solna, sweden) was used as the infecting bacteria. this solution was further diluted 1/300 to achieve a concentration of 10 colony - forming units (cfu)/ml. drugs used were vancomycin (lilly, indianapolis, usa) and linezolid (pfizer pharmaceuticals group, new york, usa). new zealand white rabbits weighing 22.5 kg were anaesthetized by intramuscular injections of ketamine (35 mg / kg) and xylazine (5 mg / kg) before each intraventricular intervention including induction of meningitis and csf sampling [911 ]. then 0.5 ml of the bacterial solution of mrsa was injected directly into the cisterna magna of each rabbit using a 22 g spinal needle (hayat ticaret, istanbul, turkey). csf white cell count more than 1000/mm (counted by thoma slide) and a bacterial count greater than 10 cfu / ml were accepted as the indications of meningitis. the linezolid-10 group received 10 mg / kg linezolid every 12 h (q12h) similar with normal human dosage (at 16 h and 28 h after the induction of meningitis). the linezolid-20 group received 20 mg / kg linezolid every 12 h (q12h) (at 16 h and 28 h after the induction of meningitis). the vancomycin group received 20 mg / kg vancomycin every 12 h (q12h) (at 16 h and 28 h after the induction of meningitis). vancomycin and linezolid were administered through a peripheral ear vein. at the end of the study period (24 h after the end of incubation period or start of treatment, 40 h after bacterial inoculation), animals were humanely killed by intravenous infusion of high dose nembutal. bacterial concentrations in csf were measured at the end of the 16 h (end of incubation period and before the first dosage of vancomycin or linezolid) and the 40 h of the study (end of treatment) by plating undiluted and serial 10-fold and 100-fold dilutions of csf (10 l) on 5% sheep blood agar and incubated at 37c for 24 h. bacterial response was evaluated in 3 categories full response, sterilization of csf ; partial response, any decrease in bacterial count ; and bacteriological failure, an increased bacterial count. data were evaluated by spss 11.0 package program using mann - whitney u test, kruskal wallis test and fisher s test. a p - value less than 0.05 was considered significant. the study was approved by the local ethics committee on animal studies (approval no : 2005 - 23). s. aureus atcc 43300 (vancomycin mic : 1 mg / l, linezolid mic : 0.025 mg / l measured in duplicate using the etest ; ab biodisk, solna, sweden) was used as the infecting bacteria. this solution was further diluted 1/300 to achieve a concentration of 10 colony - forming units (cfu)/ml. drugs used were vancomycin (lilly, indianapolis, usa) and linezolid (pfizer pharmaceuticals group, new york, usa). new zealand white rabbits weighing 22.5 kg were anaesthetized by intramuscular injections of ketamine (35 mg / kg) and xylazine (5 mg / kg) before each intraventricular intervention including induction of meningitis and csf sampling [911 ]. then 0.5 ml of the bacterial solution of mrsa was injected directly into the cisterna magna of each rabbit using a 22 g spinal needle (hayat ticaret, istanbul, turkey). csf white cell count more than 1000/mm (counted by thoma slide) and a bacterial count greater than 10 cfu / ml were accepted as the indications of meningitis. the linezolid-10 group received 10 mg / kg linezolid every 12 h (q12h) similar with normal human dosage (at 16 h and 28 h after the induction of meningitis). the linezolid-20 group received 20 mg / kg linezolid every 12 h (q12h) (at 16 h and 28 h after the induction of meningitis). the vancomycin group received 20 mg / kg vancomycin every 12 h (q12h) (at 16 h and 28 h after the induction of meningitis). vancomycin and linezolid were administered through a peripheral ear vein. at the end of the study period (24 h after the end of incubation period or start of treatment, 40 h after bacterial inoculation), animals were humanely killed by intravenous infusion of high dose nembutal. bacterial concentrations in csf were measured at the end of the 16 h (end of incubation period and before the first dosage of vancomycin or linezolid) and the 40 h of the study (end of treatment) by plating undiluted and serial 10-fold and 100-fold dilutions of csf (10 l) on 5% sheep blood agar and incubated at 37c for 24 h. bacterial response was evaluated in 3 categories full response, sterilization of csf ; partial response, any decrease in bacterial count ; and bacteriological failure, an increased bacterial count. data were evaluated by spss 11.0 package program using mann - whitney u test, kruskal wallis test and fisher s test. a p - value less than 0.05 was considered significant. the study was approved by the local ethics committee on animal studies (approval no : 2005 - 23). at the beginning of the study, 60 rabbits were inoculated with bacteria, of which 47 were alive and had developed meningitis at the end of 16 h incubation time. mean bacterial concentrations of these 4 groups were similar as log10 cfu / ml at 16 h (table 1, p>0.05). although at 40 h the differences in mean csf bacterial counts were significant between vancomycin and control groups (p=0.037) and between linezolid 10 or 20 and control groups (p=0.01), the mean cfu / ml in both vancomycin and linezolid-10 or -20 groups was similar (p=0.71 and 0.54) (table 1). at the end of treatment the decrease in bacterial counts in the vancomycin group was approximately 2 logs higher than the linezolid-20 group (p>0.05) and approximately 4 logs higher than in the linezolid-10 group (p : 0.037) (vancomycin group : 2.8604.495 vs. linezolid-20 : 0.7244.360, vs. linezolid-10 : 1.393.37). during the study mortality was relatively higher in the linezolid-20 group (2/11 in vancomycin, 6/11 in linezolid-20, 3/14 in linezolid-10 and 5/11 in control group, p>0.05), but this difference was not statistically significant. at the end of the study, rates of full (2/9 in vancomycin group, 0/11 in linezolid-10 and 2/5 in linezolid-20 group, p>0.05) and partial success rates were similar (5/9 in the vancomycin group, 1/5 in the linezolid-20 and 3/14 in the linezolid-10 group, p > 0.05). full or partial bacteriological response was higher in vancomycin vs. linezolid-10 (p : 0.01) but not vancomycin vs. linezolid-20 or linezolid-10 vs. linezolid-20 groups (7/9 in vancomycin group, 3/5 in linezolid-20 and 3/11 in linezolid-10 group). despite developments in intensive care facilities and antibiotic agents, meningitis is still associated with significant mortality and morbidity. s. aureus is an important pathogen, causing both community - acquired and hospital - acquired meningitis. the main option for the treatment of mrsa meningitis is vancomycin, but teicoplanin, linezolid, fucidic acid and daptomycin may be used as salvage therapy. this decision is based primarily on published series, but not controlled human studies. despite available treatment modalities, prognosis of mrsa meningitis recent guidelines for treatment of meningitis from the european federation of neurological sciences suggest linezolid as the main therapy option for methicillin - resistant staphylococcal meningitis. recently, sipahi. reported microbiological eradication of mrsa in 7 of 8 mrsa meningitis cases. however, to our knowledge, there is no human or animal study comparing vancomycin and linezolid in the treatment of mrsa meningitis. this study was performed to compare antibacterial activity of vancomycin (20 mg / kg) and linezolid (10 or 20 mg / kg) against mrsa in a rabbit meningitis model. the vancomycin dosage of the study was adopted from previous meningitis studies in rabbit models. there was only 1 meningitis study related to linezolid in a rabbit model using 20 mg / kg. hence, we used 2 different dosages of linezolid 10 mg / kg q 12 h (the usual dosage in humans) and 20 mg / kg q 12 h. our findings showed that linezolid was not statistically inferior to vancomycin in the treatment of mrsa meningitis at 20 mg / kg dosage, but was inferior at 10 mg / kg. however, the rate of full bacteriological response was very low in each treatment group. the low full bacteriological success with vancomycin is probably due to the low csf penetration of the drug and is in concordance with previous published data. sipahi compared vancomycin (20 mg / kg - same dosage used in this study) and teicoplanin (6 mg / kg) in a mrsa (the same strain used in this study) rabbit meningitis model. although they found teicoplanin as effective as vancomycin, the rates of full (2 in 11 rabbits) or partial (2 in 11 rabbits) bacteriological response rates were very low, in concordance with the present study. they analyzed trough antibiotic levels and reported that only 4 rabbits in the teicoplanin group and 2 rabbits in the vancomycin group had antibiotic levels higher than the lowest drug detection limit of the bioassay (> 1 mg / l). also analyzed the vancomycin levels in the rabbit csf after the same vancomycin dosage used in our study. / l, respectively, while peak serum concentration was 36.88.0 mg / l and trough serum concentration was 4.21.0 both studies confirm that penetration of vancomycin in the csf is very poor. in the present study, at the end of treatment bacterial counts were significantly lower linezolid group in comparison to untreated controls. however, although not statistically significant, the mean decrease in bacterial counts in the vancomycin group was approximately 2 log cfu / ml more than in the linezolid-20 group and approximately 4 log cfu / ml more than linezolid-10 group (p<0.05). in the linezolid-10 group there was partial bacterial response in only 3 of 14 rabbits at 24 h. our data are in concordance with the study of cottagnoud. in which they compared linezolid with ceftriaxone in penicillin - susceptible streptococcus pneumoniae and with ceftriaxone+vancomycin in penicillin - resistant s. pneumoniae meningitis of rabbits, and reported that linezolid had lower anti - bacterial activity (more than 2 log 10 cfu difference at 8 h) than both comparators. the relatively low full bacteriological response rate with linezolid in both studies is probably due to the fact that it is mainly a bacteriostatic agent [1517 ]. although mortality was somewhat higher in the linezolid-20 group, the difference was not statistically significant. linezolid shows good penetration into the cns, achieving levels in csf 30% to 70% of those present of serum in humans. the only study that analyzed penetration of linezolid into rabbit csf was by cottognaud, who reported that linezolid showed 384% csf penetration with a dosage 20 mg / kg via high performance liquid chromatography technique. linezolid csf levels peaked at 9.5 mg / l and troughed to 1.8 mg / l. in the present study, a total of 13 rabbits (21.6% of total) died during the incubation period. this rate is somewhat higher than the 13.3% mortality in a previous study performed with the same bacteria and inoculum, possibly due to inadequate surgical intervention. in previous studies, main limitation of our study is the lack of pharmacokinetic data, which were not analyzed since the main aim was to compare antibacterial efficacy of the drugs, the presence already existing data related to the distribution of vancomycin and linezolid in rabbits (which are summarized above), and economic reasons. are rather time dependent and linezolid s efficacy is slow, it would have been of major interest to have examined antibiotic efficacy over a longer period of time and to have tested different dosages, but this was not possible because of economic reasons. in addition, previous studies that analyzed antibacterial efficacy in a rabbit meningitis model lasted only 24 h or less [911 ]. we did not evaluate efficacy of vancomycin+linezolid combination, but previous reports of this combination was reported as indifferent or slightly antagonistic. finally, the fact that animal experiments, in particular with rabbits, do not permit large cohorts, limits the power of statistical analysis. according to the us food and drug administration, linezolid is not yet indicated for the treatment of meningitis. experience with linezolid is limited to single case reports or small series for the treatment of meningitis caused by gram - positive pathogens [2,3,57 ]. our results suggest that linezolid is not statistically inferior to vancomycin in the treatment of mrsa meningitis in an experimental rabbit model in 20 mg / kg q12 h dosage, but it is inferior in 10 mg / kg q12 h dosage. additional data should be gathered to confirm these findings in advance of clinical trials to assess efficacy in humans.	summarybackgroundthe aim of this study was to compare the antibacterial efficacy of vancomycin and linezolid in a rabbit model of methicillin - resistant staphylococcus aureus (mrsa) meningitis.material/methodsmeningitis was induced by intracisternal inoculation of atcc 43300 strain. after 16 h incubation time and development of meningitis, the vancomycin group received vancomycin 20 mg / kg every 12 h. the linezolid-10 and linezolid-20 groups received linezolid in 10 and 20 mg / kg dosages every 12 h, respectively. the control group did not receive any antibiotics. cerebrospinal fluid bacterial counts were measured at the end of 16-h incubation time and at the end of 24-h treatment.resultsbacterial counts were similar in all groups at 16 h. at the end of treatment the decrease in bacterial counts in the vancomycin group was approximately 2 logs higher than the linezolid-20 group (p>0.05) and approximately 4 logs higher than in the linezolid-10 group (p : 0.037) (vancomycin group : 2.8604.495 versus linezolid-20 : 0.7244.360, versus linezolid-10 : 1.393.37). full or partial bacteriological response was higher in vancomycin versus linezolid-10 (p : 0.01), but not vancomycin versus linezolid-20 or linezolid-10 versus - linezolid-20 groups.conclusionsour results suggest that linezolid is not statistically inferior to vancomycin in the treatment of mrsa meningitis in an experimental rabbit model in 20 mg / kg q12 h dosage ; however, it is inferior in 10 mg / kg q12 h dosage. additional data should gathered to confirm these findings in advance of clinical trials to assess efficacy in humans.
in december 2011, a 51-year - old woman was admitted to our hospital due to massive hemoptysis. she had suffered from recurrent hemoptysis for five years and had undergone bronchial artery embolization many times. the patient s vital signs were stable. on chest examination, decreased breathing sound and crackle were audible in the right lower lung field. a chest x - ray showed patchy consolidation in the right lower lung (fig. 1a). a computed tomography scan of the chest showed a small nodule, ground glass appearance, and consolidation around the nodule in the lateral basal segment of the right lower lobe (fig we performed an operation for the differential diagnosis between the possibility of a malignancy or life - threatening, massive hemoptysis. under general anesthesia with a double lumen endotracheal tube, we resected the right lower lobe through a posterolateral thoracotomy at the sixth intercostal space. in the operation field, we could palpate a round, hard mass 2.03.0 cm in the lateral basal segment of the right lower lobe. the resected specimen consisted of the right lower lobe, weighed 177.5 g, and was sized 11.511.23.0 cm. on multiple serial sections, a cavity lesion measuring approximately 2.02.5 cm was identified (fig. histopathological examination confirmed it to be a vegetable foreign body, and clumps of actinomyces, indicating actinomycosis, were present within the abscess cavity (fig. actinomycosis is a chronic, suppurative pulmonary infection usually caused by actinomyces israelii, which is present in the oropharynx of humans. pulmonary infection with species of actinomyces is uncommon, and usually results from aspiration of oropharyngeal secretions in those with chronic dental infections, extension from a cervicofacial infection, or hematogenous spread from a distant source. bronchial involvement, a rare form of thoracic actinomycosis, has been reported to be associated with foreign bodies. the first report of endobronchial pulmonary actinomycosis induced by a foreign body was the case of a spanish patient in 1991, when a chicken bone was aspirated. according to chouabe., eleven cases of pulmonary actinomycosis secondary to endobronchial foreign body aspiration have been described. most cases have occurred in middle - aged males with known risk factors such as a chronic debilitated state and poor dentition. the main symptoms include cough, fever, expectoration of yellow pus, chest pain, and weight loss. chest computed tomography shows a thickened bronchial wall, dense pulmonary alveolar opacity, atelectasis, pleural effusion, bronchiectasis, lymphadenopathy, or a radiopaque foreign body. obstructive endoluminal masses have been found on bronchoscopy, but the actual foreign bodies are not easily detected in some cases. in the present case, usually the foreign body is likely to have become a host for subsequent actinomyces contamination once the patient had developed poor dentition. mucosal breaches secondary to foreign body impaction must occur for actinomyces species to colonize and cause a marked granulomatous inflammatory reaction. we could find only one case of pulmonary actinomycosis induced by a vegetable foreign body. our case represents a very rare pathogenesis of pulmonary actinomycosis, which is an important disease in the differential diagnosis of a pulmonary mass.	a 51-year - old woman visited our hospital with massive hemoptysis. she had suffered from recurrent hemoptysis for five years and had undergone bronchial artery embolization many times. the patient had a history of pulmonary tuberculosis and bronchiectasis. chest radiography showed consolidation around the nodule in the lateral basal segment of the right lower lobe. we successfully performed a right lower lobectomy. the histological study of the resected specimen showed a vegetable foreign body and clumps of actinomyces, indicating actinomycosis, which was suggested to be the cause of the hemoptysis. this was a very rare case of hemoptysis caused by a vegetable foreign body and actinomycosis.
commercialisation of biotech or genetically modified (gm) crops was started in 1996. since this date a 94-fold increase (from 1.7 million hectares in 1996 to 160 million hectares in 2011) was observed, which makes biotech crops the fastest adopted crop technology in the history of modern agriculture. in 2011, 29 countries were planting biotech crops of which 19 developing countries and 10 industrial ones. between the developing countries, brazil is the leader [1, 2 ] with 30.3 million hectares and an increase of 20% was seen compared to 2010. the other main countries are argentina (23.7 million ha), india (10.6 million ha cotton), china (3.9 million ha), and south africa (2.3 million ha). the united states of america are still the lead producer of biotech crops amongst the industrial countries with 69 million hectares of biotech crops and an increase of 5% in the last year. since 1996, sixty countries worldwide have granted regulatory approvals for biotech crops for import for food and feed use and for release into the environment, corresponding to a total of 1045 approvals for 196 gm events in 25 crops. usa still tops the list of number of approved gm events followed by japan, canada, mexico, south korea, australia, the philippines, new zealand, the european union, and taiwan. in many countries legislations concerning gmo commercialisation have been adopted and although they differ from country to country, some issues are common. the assessment of new gmo, for example, is in any state done on a case - by - case basis. further, a distinction is always made between the purpose of the gmo namely if it is intended for contained use or for release into the environment. in addition, the countries make also a division between growing of gmo or using it in the food and feed chain (e.g., as raw or processed material). concerning the labelling threshold for food and feed, however, there is no consistency as this is only in place in some but not all countries and it may vary from 0.9% in the eu to 5% in japan. in some countries like russia, on the other hand, food products such as oil, syrups, and starch, do not require labelling. the various regulations worldwide governing the authorisation of gmo lead to the fact that some gmo approved in an exporting country may enter involuntarily via feed and food importation in another country where the status of approval of the gmo is not the same (i.e., unauthorised gmo or ugm). indeed, until now, the gmo in food and feed currently commercialised have been developed by american and european biotech companies. these developers have a major interest in requesting authorisation for the introduction of their gm event on the world market as their products are meant for exportation. however, in 2015 more than half of the gmo will be developed by research institutions [2, 6 ]. this trend has already started worldwide. in asia, for example, the international rice research institute (irri) in the philippines has successfully bred different gm varieties which are moving forward in the regulatory pipelines for the philippines and bangladesh. in brazil, a genetically modified phaseolus bean, resistant to the golden mosaic virus was developed by the brazilian public agricultural research corporation (embrapa) and has been approved for commercial release by the national technical commission on biosafety (ctnbio). further, projects concerning the improvement of the level of micronutrients in banana are being led by the queensland university of technology (australia) in partnership with the national agricultural research organisation of uganda (see http://www.grandchallenges.org/). as these gmo are mainly intended for local consumption or limited export no worldwide application for authorisation will be filed and therefore these gmo will be considered as unauthorised as soon as they appear outside these markets. as such, the various sources of gmo producers will increase the risk of the presence of ugm in food and feed samples. furthermore, escapes from field trial releases are another source of ugm that can represent a significant risk to health and the environment. in recent years, a number of such cases of accidental releases have been observed. for example, soybean and sorghum fields planted on former gm maize test plots have been found to be contaminated with a gm maize developed by the us biopharmaceutical company prodigene to produce an experimental pig vaccine [8, 9 ]. zapiola and coworkers also showed evidence for transgene flow of glyphosate resistant creeping bent - grass (agrostis stolonifera l. ; a golf grass) from the field trials not only during the time the plants were setting seeds but also after the fields were taken out of production. also the finding of traces of genetically modified liberty link 601 rice in us exports shows the difficulties of full containment of field trials. it is expected that the number of gmo planting countries will increase in the coming years and that consequently the number of regulatory approvals will follow the same trend. in addition, new crops and new traits will be commercialised. in this paper, we will briefly summarise the currently used detection methods in food and feed samples. subsequently, we will elaborate on the envisaged evolution of the gmo crops and the challenge for the detection methods this will create. it is clear that the increase in the number and diversity of gmo and the growing possibility of the presence of ugm will necessitate a reevaluation of the one - by - one approach presently used in gmo detection / identification as this will become practically impossible and too costly. consequently, there is an urgent need for efficient strategies for the detection of all these gmo both to comply with the unique regulation of each country but also to limit the potential health and environmental risks associated with ugm. most of the current plant gm events are created by inserting a transgenic dna construct into the host genome. the resulting organism will exhibit new properties due to the fact that the foreign dna sequence encodes a new protein expressed in this plant. from these characteristics, basically, two types of methods were developed to detect gmo, namely, protein- and dna - based methods ([1215 ] and references therein). protein - based methods have some drawbacks as previously discussed [13, 16 ]. in addition, this type of methods has another limitation based on the fact that they target the product resulting from the genetic modification and not directly the genetic modification that is at the origin of the gmo which is the fundamental advantage of dna - based methods (see below). consequently, the methodology only allows identifying the presence of the transgenic protein but does not permit to differentiate between different events containing the same transgenic construct. they do thus not allow an unequivocal identification of the gmo, which can be a problem to ensure traceability. furthermore, some proteins are instable and most of them are nearly impossible to be reliably detected in processed products. several review publications are available concerning dna - based methods [12, 1722 ]. in addition, the compendium established by the joint research centre (jrc) focuses as well on dna - based reference methods for gmo analysis. at present, the most commonly used dna - based methods are based on the amplification of a specific dna fragment (i.e., element and construct - specific methods) or the unique junction between the transgenic insert and the host genome (i.e., event - specific methods) using the polymerase chain reaction (pcr) technology (http://users.ugent.be/~avierstr/principles/pcr.html). the method relies on subsequent cycles of repeated heating and cooling of the dna strands and enzymatic replication of the dna. the basic technique of detection using pcr is the use of a heat stable enzyme (taq polymerase) allowing the exponential amplification of the dna followed by the migration of the amplified dna fragment(s) by agarose gel electrophoresis. this technique allows estimating the size of the dna fragment(s). at the present time, a more advanced quantitative real - time pcr technology exists and enables to determine the content of a gmo in a sample. the amount of product synthesised during the pcr is measured in real time by the detection of a fluorescent signal. the most common chemistries used to produce the fluorescent signal are the use of a specific fluorescent probe (taqman) or using dsdna binding (intercalating) fluorescent dyes (e.g., sybr green i). by recording the amount of fluorescence emission at each cycle, it is possible to monitor the reaction during its exponential phase and subsequently to correlate the fluorescent signal with the initial amount of target template. beside these two main real - time pcr chemistries, others have been applied for the detection of gmo [2426 ] but will not be further discussed. in sybr green chemistry, the fluorescent dye sybr green i, which binds to the minor groove of dsdna, is used. this dye will bind to every dsdna, including nonspecific pcr products and primer dimers. a melting curve analysis can be performed at the end of the pcr, that is, measuring the dna dissociation in function of the temperature. each dsdna has a specific melting temperature as a direct property of its nucleotide content. based on this melting temperature, it is possible to distinguish the nonspecific fragments from the specific pcr products. this analysis allows us to detect not only the specific products but also to get an idea on the presence of closely related targets. in addition, the sybr green chemistry is cost saving as there is no need for the use of a fluorescent - labelled probe, which is the case with the taqman chemistry. indeed, the taqman chemistry uses three specific oligonucleotides, that is, two primers and a probe [2931 ]. as long as the two dyes are in each other 's vicinity, the quencher prevents the emission of fluorescence of the reporter. during the elongation phase of the real - time pcr, the taq polymerase cleaves the annealed probe (5 3 exonuclease activity). the fluorescence increases proportionally to the dna quantity present in the reaction with each amplification cycle. however, at the same time it reduces the flexibility of the system as it will not allow detecting mutated sequences (e.g., caused by genetic variation of species varieties). in addition, taqman amplicons need to be longer as both primers and probe need to be designed in a conserved region. this is an additional drawback when working with processed samples in which the dna might be degraded. taqman real - time pcr is also a technology that allows multiplexing by using different fluorescent dyes for different targets to be simultaneously detected in one sample [3234 ]. however, seeing the limitation of the number of fluorescent dyes that can be detected by the current real - time pcr instruments and the fact that multiplex pcr becomes more prone to false positives if more than five to 10 real - time pcr systems are to be combined, only a maximum of 5 - 6 targets is possible. one should further guarantee that the detection and quantification of the different targets in a single tube is not impaired, that is, the sensitivity and pcr efficiency should be equal as when the reactions are run in simplex. in addition, attention should be paid to the design of the different primers so that they can not interact with each other, that is, primer dimer formation. apart from real - time pcr, new alternative and advanced technologies have been proposed including the use of high - throughput systems or platforms for the detection of multiple targets, for example, microarrays, mipc, pcr combined with capillary gel electrophoresis (fingerprinting), and next generation sequencing ([12, 18, 3538 ] and references therein). however, at the present stage they are often more expensive, difficult to standardise and validate, and require extensive, specialised work and equipment. they are still at the proof of concept stage and therefore not applicable in routine testing at the moment. consequently, these methods will not be discussed as this paper focuses on upcoming challenges for gmo detection in food and feed by enforcement laboratories. the genetically modified plants that have been developed and commercialised so far, are mainly transformed using a transgenic insert. this cassette consists of a regulatory promoter region, a coding sequence (trait), and a terminator (scheme in figure 1). the promoter and terminator elements used in the first gmo were mostly the cauliflower mosaic virus (camv) 35 s promoter (p35s ;) and the agrobacterium tumefaciens nopaline synthase terminator (tnos ;). the traits were also limited to genes conferring herbicide tolerance (ht) and insect resistance (ir) and were introduced into few commodity crops such as maize, soybean, and oilseed rape. the main ht sequences are the bacterial phosphinotricin - n - acetyltransferases from streptomyces viridochromogenes (pat) and from streptomyces hygroscopicus (bar) and the 5-enolpyruvylshikimate-3-phosphate synthase (epsps) from the agrobacterium tumefaciens strain cp4 or from plant origin (in casu petunia) [42, 43 ]. for the ir trait, artificial versions of the bacillus thuringiensis (bt) -endotoxin encoding genes (e.g., the cryiab / ac) have been utilised. at the present time, this first - generation of traits and regulatory elements remains prominent in commercial crops and their derived food and feed products. indeed, in 2011, the main gm crop was still soybean with 47% of the global biotech crop area followed by maize (32%), cotton (15%), and oilseed rape (5%). throughout the 16 years of biotech crop commercialisation insect resistant crops account for 15% of the total area of planted transgenic crops. in more recent years, new regulatory sequences have been introduced [44, 45 ] such as the cauliflower mosaic virus 35 s terminator (t35s), the figworth mosaic virus promoter (pfmv ;), the agrobacterium tumefaciens nopaline synthase promoter (pnos), the rice actin promoter (pact ;), and the maize ubiquitine promoter (pubizm ;). furthermore, new genes from the bt -endotoxin family are also being used now (cry3bb, cry3a, cry1f,). moreover, more species like rice, cotton, sugarbeet, and potato are currently used for transformation. in the coming years, an even larger panel of genes encoding various traits and new plant species are being genetically modified and brought onto the feed and food market. hawaiian researchers have developed a papaya (carica papaya) resistant to the papaya ringspot virus which was commercialised in 1998 and is being exported to canada since 2003. they further collaborated with other countries such as brazil, jamaica, and thailand to develop resistant transgenic papaya suitable for these countries [50, 51 ]. also eggplant (solanum melongena l.) for instance, has been modified to confer resistance to the colorado potato beetle (leptinotarsa decemlineata say ;) by the expression of bt genes. cassava (manihot esculenta crantz), on the other hand has been modified to reduce the amylase content in the starch which is important for industrial purposes such as paper and textile. in addition to these single trait gmo, a new trend of gene stacking has been observed. this implies the introduction of different transgenic inserts into the same plant through conventional crossbreeding, co-, or retransformation to combine different traits [55, 56 ]. this trend is confirmed by the considerable boost in 2011 of planting of stacked events with an increase of 31% compared to the year before. in 2011, 12 countries have been growing events with two or more traits. the total area of stacked events accounts for more than 26% of the 160 million hectares of biotech crops planted [1, 2, 6 ]. in this context, rice (oryza sativa), a predominant food source in asian countries, has been genetically modified by the insertion of two genes (encoding phytoene synthase and carotene desaturase) to improve the production and accumulation of -carotene in the grains [57, 58 ]. it is the first plant gmo with eight combined events for multiple modes of insect resistance and herbicide tolerance. the smartstax is a combination of yieldgard vt triple (monsanto), herculex xtra (dow), roundup ready 2 (monsanto), and liberty link (dow). it is currently available for maize, but cotton, soybean, and speciality crop variations are to be released. in the above described gmo, the introduced genetic elements are mainly coming from a noncrossable organism (so - called donor organism). these elements are further combined with plant - specific markers such as the rice actin promoter and the maize ubiquitin promoter to construct the transgenic insert. however, other types of modifications in the genome can be introduced to alter the plant 's characteristics. several of these techniques have already been adopted by commercial plant breeders (especially in the usa). one of these is the introduction of a dna sequence which is completely derived from the recipient species itself in a process that is called cisgenesis. this technique together with oligonucleotide directed mutagenesis (odm) and agro - infiltration are the most used new breeding techniques. zinc finger nuclease (zfn) technology, rna - dependent dna methylation (rddm), grafting on gm rootstocks, and reverse breeding are less used. it should however be noted that discussions are still ongoing whether the organisms created by these plant breeding methods will fall under the current gmo regulation or not. due to the broad range of gmo, authorised and unauthorised, possibly present on the worldwide and local market, it rapidly becomes unrealistic to use a one - by - one based identification strategy. this step is necessary and an important task of the enforcement laboratories to unequivocally identify each gmo, that is, by targeting the dna fragment on the junction between the plant genome and the transgene (event - specific method ;). already now, strategies like simplex or multiplex real - time pcr (limited to 5 - 6 targets when using the real - time pcr technology) for the identification of known junction sequences (i.e., gm authorised events) are not rational as it is extremely expensive and time consuming. in the near future moreover, no event - specific methods are available for the ugm, especially the unexpected ones coming from field trials, and so they are undetectable with such kind of methodology. therefore, most of the enforcement laboratories have elaborated a screening strategy, in which a minimum set of pcr tests (targeting specific genetic elements) should allow making conclusions on the absence / presence of as many gm events as possible. only in the positive cases, a second step will specifically identify which individual event(s) is (are) present in the sample. the most common recombinant elements in the current gm crops are p35s and tnos [44, 45, 62 ], both transcription regulating sequences. consequently, in order to assess the presence of gm material in a product, a screening pcr for those generic recombinant markers is often performed. several methods have already been published for the detection of the p35s and tnos markers in a broad range of matrices using classical pcr as well as more advanced real - time pcr with a taqman probe or the sybr green dye [28, 6367 ]. this basic screening was very efficient when only few gmo were present on the food and feed market. however, as the number of gm events is growing exponentially, limiting the screening to those common targets (with high - coverage power) has the disadvantage that a too large number of gm needs to be confirmed using the event - specific detection methods. therefore, additional targets (gm - specific elements) such as hr genes (pat, bar, epsps) and ir genes (cry gene family) can be added to reduce the number of putative gm events to be identified in the sample (due to a higher discriminative power). several detection methods have been published targeting such types of elements [6870 ]. the majority of the pcr methods described above were designed by different groups and consequently are using different methodologies (classical pcr, real - time pcr using the taqman, or sybr green chemistry) as well as different pcr programmes and protocols (e.g.,). this makes the simultaneous use in a single run in a 96-well plate format impossible by enforcement laboratories. to reduce the number of analyses and facilitate high - throughput analysis however, these require multichannel detection devices and often include costly detection probes for at least some of the targets [3234 ]. furthermore, the multiplex strategy misses the flexibility of a modular system, that is, each time a new method needs to be added, the complete system needs to be validated. in addition to this multiplex approach, a new type of modular screening strategy can be used, that is, the so - called combinatory or matrix - approach. in this screening approach, a limited set of simplex real - time pcr methods targeting various types of elements (endogenous sequences, generic markers, and gm - specific elements) is selected in such a way that multiple gmo can be detected within a single analytical run. such a matrix - based approach was developed by the wiv - isp [71, 72 ] and is used in routine gmo detection. a careful selection of the markers was performed allowing developing an approach wherein not only most gmo are detected but also discriminated [28, 68, 72, 73 ]. special attention was given to the size of the amplicon, that is, the methods were developed to amplify a short amplicon (around 100 bp) that is particularly important to detect transgenes in processed food where the dna might be degraded. combined with an informatic decision support tool named cosyps (for combinatory sybr green qpcr screening), such gmo screening represents an example of a very useful approach in managing the experimental analysis of samples for regulatory or enforcement purposes and is further elaborated below. the cosyps for gmo detection was first designed to be able to combine the results obtained with 11 sybr green real - time pcr methods which were in - house developed to be performed under similar conditions and hence to be run in a single 96-well plate (table 1). hereto, two generic recombinant markers (p35s, tnos) and the 4 major gm elements (cryiab / ac, epsps, pat, and bar) have been designed. many of the elements used in transgenic constructs are derived from donor organisms such as bacteria and viruses. one such element is the 35 s promoter of the cauliflower mosaic virus (camv) which has brassica plants as its natural host. to discriminate between the p35s present in a gm event and the one due to its possible natural presence this marker targets the reverse transcriptase gene of camv and will thus allow the detection of the virus. these gm markers are combined with the rbcl plant kingdom marker and a lectin (lec), an alcohol dehydrogenase (adh), and a cruciferine (cru) species marker for the detection of materials derived from soybean, maize, and oilseed rape, respectively. as this approach is modular, three additional markers were developed to answer the actual need in gmo detection. two are targeting the promoters of the figworth mosaic virus (pfmv) and the a. tumefaciens nopaline synthase (pnos), one targets the -endotoxin encoding gene cry3bb from b. thuringiensis. to keep covering the newly developed gmo, three more species - specific methods have been developed, that is, targeting rice (pld), cotton (sad1), and sugarbeet (glu3). based on the decision values (lod expressed as ct value, tm) of the different screening methods, the cosyps allows to decide which gm events may be present in an analytical sample. the present version of the cosyps allows covering the presence of all currently eu authorised gmo [28, 68, 72, 73 ]. an example of the efficient use of this decision support system is shown in figure 2 for the detection of maize event nk603. as this decision support system is a modular tool, screening methods targeting new sequences present in new gm events can be added at any moment. this is necessary to add discriminative power to the cosyps system and to keep covering the increasing number of gmo. additionally, the use of the various markers in combination with the cosyps is a powerful tool in the detection of ugm. indeed, in principle, the elements that are positive in the screening real - time pcr should be covered by the eu authorised events or the gm events included in the low level presence (llp) legislation. if this is not the case, one might suspect the presence of an ugm in the sample and further confirmation of the event would be needed. this might require first the use of alternative and advanced tools such as anchor - pcr fingerprinting followed by confirmatory sequencing of the suspected amplified fragment(s) or dna walking to identify the junction between the transgene and the plant genome (for which later on a real - time pcr event - specific method can be developed). alternatively, the next generation sequencing technology can be used to screen whole genomes for foreign dna and their respective junctions. the roadmap for these ad hoc scenarios should be drawn based upon the evaluation of the perceived risk for health, environment, and economy. not only these will add to the complexity of detecting authorised gmo by the enforcement laboratories in food and feed samples, but also the occurrence of ugm which will be steadily increasing in the coming years. most likely there will also be a growing number of gmo with traits of industrial or pharmaceutical relevance but not intended for food or feed use. these could also enter the agricultural supply chains and cause ethical and religious concern or even pose a significant risk to human and animal health. therefore, there is an evident need for a continuous development of appropriate detection methods and strategies. as long as the development of gmo is following the same way and genetic modifications are being created by the introduction of a transgenic cassette originating from a foreign organism, the current detection / identification approaches can be continued to be used, even for stacked events. however, gene stacking poses a new challenge for gmo detection laboratories as there is no way (except when the analysis is done on individual seeds and plants) to discriminate between the presence of the gmo separately in a sample or combined as a stacked event [16, 55, 82 ]. in response to the increasing diversity of gmo on the market, new screening markers for the new species - specific sequences (e.g., eggplant and melon), and new genetic elements (e.g., coding for pathogen resistance) will need to be designed and developed. the challenge will be more in collecting the massive amount of data sequences (especially for ugm) to develop standardised real - time pcr methods and then combining the obtained results in an appropriate decision system, like cosyps, for an efficient manner of gmo testing. eventually, also the event - specific methods should be developed, to unambiguously identify the gmo in a second step. the developed sybr green screening methods will offer the possibility for the detection of potential allelic variation in gm markers, as is, for example, the case for the cry1ab gene in bt11, bt176, and mon810 maize, as compared to the natural (non - gm) donor bt ssp. the haplotypes of the gene in the three gm maize events have been differently optimised for expression in plants / maize. the snp can however be detected by using a technique originating from human genetic diagnostics, that is, high resolution dna melting analysis (hrm) to screen for mutations in dna pools. this requires only a simple real - time pcr step (using an intercalating saturating dye such as syto9 or lcgreen plus+ and the primers developed for the screening markers) followed by dna melting curve analysis. these are identified as differential melting temperature curves in comparison to homoduplexes (e.g.,). as such, hrm is a cost - effective, high - throughput mutation screening method with high potential for gmo analysis, including ugm analysis. however, the screening methodology will no longer fully be suited for the detection of new types of genetic modifications that are under development through new plant breeding techniques. in the case of cisgenesis, for example, detection of the inserted elements alone can no longer be used as evidence of the genetic modification. however, the order of the different elements and the insertion loci into the plant genome still may offer an opportunity for the detection of these modifications. provided this information is available, these event - specific sequences can be exploited to develop new real - time pcr - based methods if needed. also for other types of genetic modifications introduced by these new plant breeding techniques (e.g., zfn technology, odm) a minimum amount of information about the dna sequence of the modification and the neighbouring sequence needs to be known to be able to detect them. without prior knowledge, detection of these small modifications would be unlikely to be used in routine laboratories as more complex technologies are needed (e.g., full - genome sequencing). for the others, the detection of the genetic modification is currently not possible (e.g., rddm, grafting, reverse breeding, and agroinfiltration). in addition, crops resulting from most of the techniques can not be distinguished from conventionally bred crops or from crops produced by natural genetic variation, and identification is therefore not possible. these new advances in molecular biotechnology pose challenges to regulators as to whether they fall within the scope of their regulatory authority. in 2011, the european commission, with the cooperation of different member states, published an overview of these techniques. at the moment it is, however, not clear yet if these new breeding techniques lead to organisms that will be classified under the currently used gmo definition and thus the gmo legislation. if they would be classified as gmo, they would require control and traceability. as the current methodologies will be insufficient, new approaches, probably involving a combination of different analytical methods, need to be developed. if they are not classified as gmo, the currently used detection methods would remain appropriate for now. nevertheless, to anticipate the increasing number of gm events, the development of additional screening markers, potentially in multiplex set - ups (which might involve technologies beyond the real - time pcr platforms), should be continued to arrive in the near future to a robust and cost - efficient solution to the gmo challenge.	biotech crops are the fastest adopted crop technology in the history of modern agriculture. the commercialisation of gmo is in many countries strictly regulated laying down the need for traceability and labelling. to comply with these legislations, detection methods are needed. to date, gm events have been developed by the introduction of a transgenic insert (i.e., promoter, coding sequence, terminator) into the plant genome and real - time pcr is the detection method of choice. however, new types of genetic elements will be used to construct new gmo and new crops will be transformed. additionally, the presence of unauthorised gmo in food and feed samples might increase in the near future. to enable enforcement laboratories to continue detecting all gm events and to obtain an idea of the possible presence of unauthorised gmo in a food and feed sample, an intensive screening will become necessary. a pragmatic, cost - effective, and time - saving approach is presented here together with an overview of the evolution of the gmo and the upcoming needs.
the increase in frequency of arrhythmias and in symptoms during pregnancy may be a result of the associated hemodynamic, hormonal, autonomic, and emotional changes. we report a patient who developed bradycardia followed by atypical wenckebach block, after subarachnoid block for cesarean section. a 150 cm, 58 kg, 28-year - old second gravida, with history of previous lscs, presented with full term pregnancy with twins with transverse lie for lscs. electrocardiography (ecg), non - invasive blood pressure, and pulse oximetry monitoring was started. subarachnoid block was given through the l3 - 4 intervertebral space with 10 mg 0.5% hyperbaric bupivacaine using 25-g whitacre pencil point spinal needle. five minutes after the spinal injection, when the level of analgesia was t5, ecg showed sinus bradycardia of 48/min and a prolonged pr interval followed by atypical wenckebach atrio - ventricular (av) block [figures 1 and 2 ]. bradycardia following spinal blockade atypical wenckabach block one healthy male and one female infant, with normal neurologic status, were delivered. approximately two hours after the initiation of the spinal anaesthetic, ecg again showed progressive prolongation of the p - r interval in an atypical wenckebach type heart block [figures 3 and 4 ]. bradycardia two hours following spinal blockade atypical wenckabach block seen postoperatively echocardiograph showed normal sized cardiac chambers with no regional wall motion abnormality, global ejection fraction of 63%, no diastolic left ventricular dysfunction, and normal right ventricle systolic function. valves were structurally normal and no other intra - cardiac defect, intra - cardiac mass or pericardial effusion was seen. direct physiological effects of hormones, changes in autonomic tone, hemodynamic alterations, and hypokalemia of pregnancy can potentially result in arrhythmias during pregnancy, labour, and delivery. intraoperative arrhythmias could be due to parasympathetic over - activity following chemical sympathectomy resulting from subarachnoid block due to a reduction in venous return, and subsequent to a vasovagal attack. surgical maneuvers, like externalizing the uterus, also can result in increased vagal tone and cause arrhythmia. decreased threshold for cardiotoxicity with bupivacaine during pregnancy due to the increased unbound bupivacaine present in the hypoproteinemia of pregnancy or to the selective cardiac depressant effects of progesterone combined with bupivacaine can also precipitate arrhythmia. in patients undergoing cesarean section under subarachnoid block, shen. found the incidence of second degree av block to be 3.5%, and that of severe bradycardia (heart rate < 50 beatsbullmin1) to be 6.7%. mobitz i second - degree av block (wenckebach block) consists of progressive lengthening of conduction time in any cardiac tissue (most often the av node or junction) with ultimate dropping of a beat. it usually occurs due to excess vagal tone and the site of the block is within the av node. mobitz ii second - degree av block, is generally caused by conduction block in the his bundle or lower in the conduction system. when mobitz type ii block is associated with a wide qrs complex, it is due to disease of the his - purkinje system. these patients have a greater mortality and definite measures such as pacemaker insertion are needed to stop progression of the av block. second - degree av block whether mobitz type ii or atypical wenckebach (pseudo mobitz type ii) is usually asymptomatic. however, some patients may complain of presyncope or syncope. the latter is observed in more advanced conduction disturbances such as mobitz ii av block. a history of intake of medications that alter av node function (digitalis, beta - blockers, calcium channel blockers) should be taken. levels of electrolytes should be assessed in the case of second - degree av block whenever increased potassium levels are suspected. the patient was classified as atypical wenckebach block, as she failed to meet one or more criteria for typical wenckebach periodicity. typical wenckebach periodicity (wenckebach and winterberg) is considered to be present when : first pr interval of a cycle is the shortest ; there is progressive lengthening of the pr interval with the increment between the first and second conducted beats being the largest ; and there is progressive decrease in the rr intervals. a significant electrocardiographic difference between an atypical wenckebach block (pseudo - mobitz ii arrangement) in the av node and his - purkinje system is the length of the pr interval in the opening beat of the wenckebach cycle. in av nodal block it usually remains significantly prolonged (026 to 038 s, with an average of 032 s). mobitz type ii second - degree av block and a wide qrs complex indicate diffuse conduction system disease and is an indication for pacing, even in the absence of symptoms as it may degenerate into third - degree heart block. shen. found that when patients had narrow qrs complexes with second degree av block and it occurred transiently during spinal anesthesia, the prognosis was benign. our patient had atypical wenckebach block with narrow qrs complexes but it was transient and hence benign. the bradycardia was due to parasympathetic over activity, which initiated the second degree heart block. our patient did not have any hypotension, as reported by others, possibly because the patient was volume loaded. however, the arrhythmia recurred postoperatively when all surgical stimulation had been removed. second - degree av block associated with symptomatic bradycardia is an indication for pacing. in asymptomatic patients with second - degree av block, type i with a narrow qrs complex, cardiac pacing may be required if the level of block is infranodal as found on electrophysiological studies, because the progression to complete heart block is common. sympathetic block is slower to regress than the sensory block, and considerable sympathetic blockade can persist, when the patient is in the postoperative recovery. atropine is contraindicated though it improves conduction in the av node minimally it increases the sinus rate markedly. this can enhance the degree of block due to an increase in atrial rate. to conclude, we successfully managed a patient who developed bradycardia followed by atypical wenckebach block, after subarachnoid block for cesarean section.	arrhythmias in pregnancy are common and may cause concern for the well - being of both mother and fetus. generally, no previous history of heart disease is elicited and majority of the arrhythmias are benign. bradycardia is commonly seen following subarachnoid block for cesarean section. however, the incidence of subsequent heart block is low. this case report highlights the occurrence of perioperative arrhythmias following sympathetic blockade in pregnant patients and their early detection by vigilant monitoring.
lynch syndrome (ls), formerly referred to as hereditary nonpolyposis colorectal cancer (hnpcc) accounts for 2 4 % of all colorectal cancers (crcs) 1. affected family members have a germline mutation in one of the dna mismatch repair genes mlh1, pms2, msh2, or msh6, and a lifetime risk for development of crc of 25 centralized organizations of surveillance for lynch syndrome families have been established in finland, netherlands, germany, and canada, and follow - up results have been published since 1995, with essential information on the efficiency of prevention of crc incidence and mortality 3 4 5 6. colonoscopy has been shown to decrease both by 63 % 7 8. in spite of colonoscopic surveillance, interval cancers (defined as crc found within 2 years after a negative colonoscopy or leaving a colon clear of polyps) these 29 cases, with a median age of 52 years, and a median time since previous colonoscopy of 17 months to the diagnosis of interval cancer, were mlh1 and msh2 mutation carriers. of these, 39 % had previous crc, and those who had never undergone colonic resection developed proximal lesions in 84 % of cases ; 77 % were stage t1 3, n0m0. in 9 %, an incomplete previous colonoscopy was reported, and the tumor was located in the unexamined colon. in six cases, the authors concluded that mlh1 and msh2 mutation carriers, previous crc, incomplete colonoscopy, and incomplete polypectomy were risk factors for diagnosis of interval cancer in ls 9. several factors may predict failure to prevent interval cancer in ls : more lesions in the right colon ; more flat (nonpolypoid) and lateral growing polyps ; small adenomas may already harbor high grade dysplasia or a high percentage of villous components and become advanced adenomas. overall, there is an acceleration of the adenoma carcinoma sequence, synchronous lesions have a high prevalence and, importantly, patients with ls are younger and less tolerant to colonoscopy (need more sedation) ; and repeated colonoscopies are needed for lifelong surveillance (patient experience is important for compliance) 12. thus, a very meticulous and precise procedure is required with removal of all of the polyps. nonpolypoid lesions, defined as a lesion with height less than half the diameter, is a characteristic of ls 13. studied 59 ls patients in comparison with 590 controls, and found that 43.3 % and 58.1 % of adenomas and serrated adenomas were nonpolypoid, in comparison with 16.9 % and 20.4 % in controls, respectively (p < 0.001) 13. regular colonoscopy should be performed repeatedly and at short intervals when ls is suspected according to amsterdam ii criteria, or when a mutation in one of the dna mismatch repair genes (mmr) is found. the procedure should be performed in an endoscopic unit experienced with ls, to minimize the potential for missing polyps and thus exposing patients to interval cancer. four endoscopic methods have been tested in order to improve the diagnostic yield of colonoscopy and the polyp / adenoma detection rate : high resolution white light endoscopy (hr), chromoendoscopy, narrow band imaging (nbi) and autofluorescence endoscopy (afe) (table 1). four prospective trials have compared chromoendoscopy to hr and nbi in back - to - back studies 14 15 16 17. lecomte. examined 33 patients with ls and found an additional 45 lesions (11 adenomas) with chromoendoscopy performed after hr colonoscopy 14. similar findings have been described by hurlstone and colleagues who found an additional 52 lesions (32 adenomas) in 25 patients 15. huneburg. performed colonoscopy in 109 patients in two investigational arms : chromoendoscopy was found to be better than hr colonoscopy in 47 patients, with 1.5 and 0.5 lesions per patient, respectively (p < 0.01), and also better than nbi, with 1.8 and 0.7 lesions per patient, respectively (p = 0.032). comparing nbi with hr colonoscopy in 62 patients with ls according to amsterdam ii criteria, nbi added 21 polyps (6 flat adenomas) (p < 0.001) 17. chromo, chromoendoscopy ; hrc, high resolution colonoscopy ; wle, white light endoscopy ; nbi, narrow band imaging ; hdwl, high definition white light endoscopy ; afe, autofluorescence endoscopy. the first method listed is the better method. the method of back - to - back endoscopy has a methodological bias, since additional lesions can be found in the second procedure. stoffel. performed conventional colonoscopy in 54 patients with ls (46 with mmr gene mutation) 18. then patients were randomized into one of two arms : chromoendoscopy or intensive inspection. chromoendoscopy detected more polyps than intensive inspection (p = 0.04), but adenoma detection was not significantly different (p = 0.27). however, the second colonoscopy (chromoendoscopy or intensive) doubled the diagnostic yield for adenoma. the only procedure that demonstrated a higher diagnostic yield for adenoma than hd colonoscopy, without the methodological bias of back - to - back colonoscopy, was autofluorescence endoscopy (afe) 19, but this observation has not yet been confirmed in other studies. seventy - five ls or familial crc patients were examined with either white light colonoscopy (wle) followed by afe, or with afe followed by wle, by two blinded endoscopists. all lesions were removed on the 2nd endoscopy (or the 3 rd if missed). white light endoscopy identified 65 adenomas in 28 /41 patients with adenomas, less than afe which identified 87 adenomas in 37/41 patients, an increase of 32 %. sensitivities of afe and wle were 92 % and 68 %, respectively (p = 0.001). the adenomas additionally detected with afe were smaller than those detected with wle, with means of 3 mm versus 4.9 mm, respectively (p < 0.01). quality indicators for colonoscopy are constantly published and are all directed towards complete examination and removal of all polyps. a correlation is suspected between the incidence of interval cancer and the quality of colonoscopy in screening average - risk as well as high risk populations 20. validated quality indicators are even more important to follow in ls, since the adenoma fifty - four patients with a known pathogenic mutation in mlh1 or msh2 underwent colonoscopy every 1 2 years, with a mean follow - up period of 9.3 years. the polyp dwell time was 35.2 22.3 months, and shorter for the right than the left colon, 28.7 vs. 43.6 months, respectively. thus, a withdrawal time of more than 6 minutes, an excellent preparation, complete examination (photographic evidence showing the ileocecal valve and appendix orifice), u - turn in the rectum, adequate adenoma detection rate (adr) and polyp detection rate (pdr), and complete polypectomy by experienced endoscopists are cornerstones for surveillance endoscopy in ls. guidelines recommend colonoscopy every 1 2 years, starting at age 20 25 years, or 10 years younger than the age of first diagnosis in the family (whichever is first), and yearly after the age of 40 years 4 22 23 24 25 26. in four us cancer genetics clinics, 181 patients with ls (according to amsterdam criteria or with a proven mutation) were screened. only 132 (73 %) had appropriate surveillance according to the guidelines 27. personal history (or 2.81), first degree relative with crc (or 2.61), and genetic evaluation (or 4.62) were associated with appropriate surveillance. stuckless and co - investigators looked at the impact of colonoscopic screening in male and female ls carriers with the msh2 mutation 28. they compared 54 male and 98 female ls mutation carriers who had been surveyed (screened carriers) with 94 male and 76 female carriers (unscreened carriers). in men, the median age to develop crc was 58 years versus 47 years in screened and unscreened patients, respectively (p < 0.0001), and the median survival was 66 years versus 62 years, respectively (p = 0.034). in women, the median age to develop crc was 79 years versus 57 years in screened and unscreened patients, respectively (p < 0.0001), and the median survival was 80 years versus 63 years, respectively (p = 0.001). twenty percent of men and 7 % of women developed crc within 2 years of previous colonoscopy. crc development may be further reduced by decreasing the screening interval to 1 year and improving the quality of colonoscopy. the risk factors for adenoma or cancer in ls patients, on top of genetic propensity, are male gender, mlh1 and msh2 mutations, cigarette smoking, not participating in colonoscopic surveillance, previous crc, incomplete colonoscopy, and residual adenomatous tissue after polypectomy 7 8 29 30 31 32. in ls patients and individuals fulfilling the amsterdam ii criteria, surveillance colonoscopy should be performed using modern high resolution technology by experienced endoscopists every 1 2 years, starting at age 20 25 years, or 10 years younger than the age of first diagnosis in the family (whichever is first), and annually after the age of 40 years. colonoscopy in ls patients should include meticulous inspection and precise removal of all polyps, with special attention to the right colon and alertness to flat lesions. at the moment, none of the new endoscopic techniques have shown convincing superiority over high resolution white light colonoscopy in ls patients.	lynch syndrome (ls) accounts for 2 4 % of all colorectal cancers. affected family members have a germline mutation in one of the dna mismatch repair genes mlh1, pms2, msh2, or msh6, and a lifetime risk for development of colorectal cancer of 25 75 %. current guidelines recommend annual to biannual surveillance colonoscopy in mutation carriers. several factors may predict failure to prevent interval cancer in ls : more lesions in the right colon ; more flat (non polypoid) and lateral growing polyps ; small adenomas may already harbor high grade dysplasia or a high percentage of villous component and become advanced adenomas ; there is a short duration of the adenoma carcinoma sequence ; synchronous lesions have high prevalence ; patients are younger and less tolerant to colonoscopy (need more sedation) ; and repeated colonoscopies are needed for lifelong surveillance (patient experience is important for compliance). in order to prevent cancer in ls patients, surveillance colonoscopy should be performed in an endoscopic unit experienced with ls, every 1 2 years, starting at age 20 25 years, or 10 years younger than the age of first diagnosis in the family (whichever is first), and yearly after the age of 40 years. colonoscopy in ls patients should be a very meticulous and precise procedure (i. e. taking sufficient withdrawal time, documentation of such warranted), with removal of all of the polyps, special attention to the right colon and alertness to flat lesions. following quality indicators such as successful cleansing of the colon and removal of every polyp will probably improve prevention of interval cancers. at this moment, none of the new endoscopic techniques have shown convincing superiority over conventional high resolution white light colonoscopy.
von voss - cherstvoy syndrome is a part of a group of syndromes with radial and hematologic abnormalities, and until now approximately ten cases have been reported in the literature. this syndrome is characterized by a triad of radial ray defects, occipital encephalocele, and urogenital abnormalities. we report a neonate from indian ethnicity who was diagnosed with von voss - cherstvoy syndrome. the neonate had radial ray defect, occipital encephalocele, tetralogy of fallot, and bilateral agenesis of kidney, ureter, and bladder. the neonate was suspected to have von voss - cherstvoy syndrome on the basis of clinical features, which was further confirmed by fibroblast analysis showing somatic mosaicism for del(13q). von voss - cherstvoy syndrome is a very rare syndrome that can be suspected on the basis of typical clinical features and confirmed by fibroblast analysis showing somatic mosaicism for del(13q). von voss - cherstvoy syndrome is a very rare syndrome that is characterized by a triad of radial ray defects, occipital encephalocele, and urogenital abnormalities.1 the inheritance of this syndrome is thought to be autosomal recessive in the online mendelian inheritance in man library as few case reports showed consanguinity and the majority of the case reports were sporadic in nature. the inheritance pattern still remains undetermined, and this syndrome has equal sex predilection.2 we report a case of von voss - cherstvoy syndrome that was suspected on the basis of clinical features and confirmed by fibroblast analysis showing somatic mosaicism for del(13q). this adds a second case of this chromosome anomaly described in this syndrome, and the chromosome anomaly provides further evidence of at least some cases of von voss - cherstvoy syndrome. a preterm male child, indian in origin with a birth weight of 2.4 kg and with an apgar score of 7, 8, and 8 at 1 minute, 5 minutes, and 10 minutes, respectively, was referred to our hospital at the age of 2 days in view of fracture of femur and malformation. the mother had one antenatal scan done, which was suggestive of severe oligohydramnios with an amniotic fluid index of 2.1. the infant was born by a cesarean section with fetal transverse lie with difficult extraction. the fracture of femur was thought secondary to energetic traction during the time of birth. there was no history of any drug intake or any substance abuse by the mother. the detailed examination of the infant showed wide - open bulging anterior fontanel, all cranial sutures wide opened, small occipital meningocele (figure 1), low - set ears, downward slanted eyes, short neck, wide - spaced nipples, cleft palate, anteverted nostrils, bilateral microtia, bilateral absence of thumbs, shortening of both upper limbs with aplasia of both radial deviation and medial deviation of the hands, and single umbilical artery (figure 2). examination of the cardiovascular system showed grade 2/6 pansystolic murmur in the left parasternal border. the baby was evaluated with echocardiography that was suggestive of tetralogy of fallot, and ultrasound of abdomen and kidney was suggestive of bilateral agenesis of kidney, ureter, and bladder. the ultrasound of the head was suggestive of hydrocephalus with ventricle sizes of 20 mm each and left arachnoid cyst, with agenesis of the corpus callosum. the radiograph of the infant was suggestive of bilateral absence of radius bone, with hypoplastic low - volume lungs (figure 3). the fracture of femur was managed with immobilization of femur as advised by the pediatric orthopedician. the infant expired on the third day after birth as the parents were not willing for aggressive management of the neonate. the karyotype analysis of the lymphocyte showed 46xy pattern. based on the physical features, the diagnosis of von voss - cherstvoy syndrome was kept, and the fibroblast analysis of the skin taken from the left lower limb showed somatic mosaicism for del(13q) that confirmed our diagnosis of von voss - cherstvoy syndrome. written informed consent was obtained from the patient s legal guardian including publication of images. the ethics committee of civil hospital, palwal does not require ethics approval to be sought for case reports. von voss - cherstvoy syndrome (mendelian inheritance in man [mim ] 223340) is also known by various other names that include dk phocomelia or phocomelia, thrombocytopenia, encephalocele, and urogenital malformations syndrome. the first case was reported by von voss in 1979, and the syndrome was named after him. they reported a child with phocomelia, meningoencephalocele, and hypoplastic thrombocytopenia, among other findings.1 the second case was reported by cherstvoy,3 and they coined the term dk phocomelia syndrome, which is a misnomer as the limb anomalies are usually radial bone abnormalities. till date, there are very few case reports in the medical literature that describe this syndrome. the clinical findings and fibroblast analysis of our patient confirmed that our patient had von voss - cherstvoy syndrome. the analysis of our patient strengthens the findings of bamforth and lin.4 this syndrome is included in the family of syndromes with radial ray and hematologic abnormalities. in all case reports, only the upper limbs are affected with severity ranging from radial agenesis and phocomelia to virtual amelia. this syndrome involves majority of the systems, including the central nervous system, cardiovascular system, respiratory system, hematological system, urogenital system, skeletal system, and gastrointestinal system. the close differential diagnoses of von voss - cherstvoy syndrome include fanconi syndrome (mim 227650),5 thrombocytopenia absent radius syndrome (mim 274000),6 and roberts syndrome (mim 268300).7 the common clinical features of thrombocytopenia and limb defects suggest the possibility of involvement of the homeobox family of genes, as these genes are expressed in both cell lineages. the inheritance pattern has been unknown as all the cases have been sporadic, with no definite inheritance pattern. the somatic mosaicism for del(13)(q12) in the mesoderm lineage has been held responsible for this rare syndrome.4 the children with this syndrome do not die universally immediately after birth because the mortality will depend on the severity of the associated malformation in the case. in the index case, the neonate had bilateral renal agenesis and other system involvement that led to neonatal mortality. the long - term outcome of these patients is elusive because of very few cases reported till now and still there are no conclusive data over the long - term outcome. there have been reports of delayed development and seizures in these patients, but one case report showed transient thrombocytopenia and normal psychomotor development.8 a comparison between our case and the patients reported in the literature is shown in table 2. a recently published case report described the neurocognitive profile of a young adolescent with von voss - cherstvoy syndrome. the 12-year - old male with von voss - cherstvoy syndrome underwent comprehensive neuropsychological evaluation. the evaluation showed mild impairment in intellectual functioning, with more significant impairment in adaptive skills and academic achievement. there was a distinct pattern of strengths and weaknesses that showed functional compromise of posterior brain regions with relatively well - preserved functioning of more anterior regions. the impairments were more evident in perceptual reasoning, visual perception, and visuomotor integration, whereas normal or near - normal functioning was evident in memory, receptive language, social cognition, attention, and most aspects of executive functioning.12 in another recently published case report, the authors suggested that the various syndromes that have encephalocele and radial defects in common such as vacterl association (vertebral anomalies, anal atresia, cardiac defects, tracheoesophageal fistula and/or esophageal atresia, renal and radial anomalies, and limb defects), oculo - auriculo - vertebral spectrum, von voss - cherstvoy syndrome, and edwards syndrome (trisomy 18) should be considered as one entity and proposed the name encephalocele radial, cardiac, gastrointestinal, anal / renal anomalies or froster - iskenius and meinecke syndrome.13 the therapeutic option in our index case could have been going for renal dialysis to take care of the renal system as the neonate had renal agenesis. however, the parents were not willing for aggressive management and hence renal dialysis was not done. a novel aspect in the index case was that we were able to show somatic mosaicism for del(13)(q12) in the fibroblast analysis. the findings of our index case add to the hypothesis that somatic mosaicism for del(13)(q12) is responsible for von voss - cherstvoy syndrome. von voss - cherstvoy syndrome is a very rare syndrome and involves the central nervous system, cardiovascular system, hematological system, urogenital system, and respiratory system, with various manifestations. the diagnosis requires high index of suspicion, and confirmation must be done by identification of somatic mosaicism for del(13q) using the fibroblast analysis.	introductionvon voss - cherstvoy syndrome is a part of a group of syndromes with radial and hematologic abnormalities, and until now approximately ten cases have been reported in the literature. this syndrome is characterized by a triad of radial ray defects, occipital encephalocele, and urogenital abnormalities.case presentationwe report a neonate from indian ethnicity who was diagnosed with von voss - cherstvoy syndrome. the neonate had radial ray defect, occipital encephalocele, tetralogy of fallot, and bilateral agenesis of kidney, ureter, and bladder. the neonate was suspected to have von voss - cherstvoy syndrome on the basis of clinical features, which was further confirmed by fibroblast analysis showing somatic mosaicism for del(13q).conclusionvon voss - cherstvoy syndrome is a very rare syndrome that can be suspected on the basis of typical clinical features and confirmed by fibroblast analysis showing somatic mosaicism for del(13q). this adds a second case of this chromosome anomaly described in this syndrome.
tuberculosis (tb) is the second leading cause of death from infectious diseases worldwide. in the year 2012, an estimated 8.6 million people developed tb, and 1.3 million died from the disease. pelvic and peritoneal tb may resemble advanced ovarian cancer due to the presence of ascites, complex adnexal mass, peritoneal deposits, and raised ca-125 level. symptoms such as weight loss, reduced appetite, and dull abdominal pain are also common to these two conditions. quite often, the diagnosis of tb is made after a laparotomy for suspected ovarian cancer. hence, it is important to review these two entities in detail to arrive at a diagnosis prior to performing a major surgical procedure. we have earlier reported four women with pelvic tb who were diagnosed after laparotomy or laparoscopy for suspected ovarian cancer. from july 2012 to june 2015, we treated ten women in peri- and post - menopausal age group where this diagnostic dilemma arose of whom seven underwent laparotomy for suspected ovarian cancer. we report four perimenopausal and six postmenopausal women who presented with features mimicking ovarian malignancy but were finally diagnosed to have pelvic - peritoneal tb. all were parous with no history of infertility, hiv negative, had a normal chest x - ray, and raised ca-125 level. all presented with abdominal pain and distension of varying duration of 1 month to 3 years. two women had a history of loss of appetite and weight also, and only one woman gave a history of low - grade fever for a month. radiological imaging in these women showed complex adnexal masses ranging from 3 to 15 cm ; eight among them had ascites. ascitic fluid analysis was done in four which showed a lymphocytic predominant pattern with adenosine deaminase (ada) levels ranging from 18 to 70 (normal 30 iu / l) and is negative for malignant cells. fnac was used as a diagnostic modality in two of our cases. the median serum ca-125 level in epithelial ovarian cancer was reported to be significantly higher than among women with peritoneal tb (p 0.01). among 48 women with peritoneal tb and 370 with epithelial ovarian cancer, only one (2.1%) with peritoneal tb had a serum ca-125 level > 2000 iu / ml. however, 109 women (29.5%) with epithelial ovarian cancer had a serum ca-125 level > 2000 iu / ml. at the ca-125 ranges of 400 to 599 and 600 to 799, the proportions of those with peritoneal tb were 24% and 21.9%, respectively. at a serum ca-125 level > 1000 iu / ml, the proportion of women with peritoneal tb was much lower (2.1%). in seven of our cases, ca-125 was < 600 iu / ml (146571). in one woman, the ca-125 was 1643 iu / ml and peritoneal tb was not suspected even at laparotomy. a laparoscopic evaluation with biopsies is another option when the diagnostic dilemma persists, and we do not want to miss diagnosing ovarian cancer. whenever peritoneal tb is suspected intraoperatively, frozen section histology should be utilized so that unnecessary surgery is prevented. a diagnostic tool which may differentiate between the two conditions in the future is the xpert mtb / rif assay. it is a real - time pcr assay that can be used by operators with minimal technical expertise, enabling diagnosis of tb and assessment of rifampicin resistance and has been endorsed by the who recently. xpert mtb / rif may be used as a replacement test for usual practice (conventional microscopy, culture, and histopathology) for testing of specific nonrespiratory specimens (lymph nodes and other tissues) from patients presumed to have extrapulmonary tb. it is a diagnostic challenge to differentiate pelvic - peritoneal tb from ovarian cancer which has entirely different management and prognosis and peritoneal and pelvic tb is a differential diagnosis to be remembered while evaluating women with bilateral complex adnexal masses, ascites, and moderately raised ca-125 level. ascitic fluid showing lymphocytic predominance, raised ada, and no malignant cells are pointers to obtain a histopathological diagnosis by ultrasound - guided needle biopsy or laparoscopic biopsy or frozen section at laparotomy.	pelvic and peritoneal tuberculosis may resemble advanced ovarian cancer due to the presence of ascites, complex adnexal masses, peritoneal deposits and raised ca-125 level, especially in peri- and postmenopausal women. other common features among women with these two conditions are abdominal pain and distension, weight loss and reduced appetite. as the treatment of pelvic - peritoneal tuberculosis is completely different from that of ovarian cancer, it is important to reach a correct diagnosis. sometimes women with pelvic - peritoneal tuberculosis may be subjected to a laparotomy for suspected ovarian cancer which is likely to increase their morbidity. in the present article, we report ten women in the peri- and post - menopausal age group where this diagnostic dilemma arose of whom seven were diagnosed only after a laparotomy had been performed for suspected ovarian cancer due to adnexal masses with ascites and raised ca-125 level. ascitic fluid showing lymphocytic predominance, raised ada level and absence of malignant cells are pointers to consider the possibility of pelvic- peritoneal tuberculosis, especially in endemic countries like india. in such situations, an effort should be made to obtain a cytological or histopathological diagnosis of either condition by ultrasound guided needle biopsy or laparoscopically obtained biopsy rather that proceeding with laparotomy for suspected ovarian cancer.
the hyperimmunoglobulin e (hyper - ige) syndromes were initially described as a single entity job syndrome in 1966 in two girls who presented with recurrent cold staphylococcal abscesses, severe eczema, recurrent bronchopulmonary infections, and elevated serum ige. generally, the hyper - ige syndromes were thought of as primary immunodeficiency diseases and could be grouped into three general categories : autosomal dominant (ad), autosomal recessive (ar), and sporadically occurring. autosomal dominant hyperimmunologlobulin e syndrome is characterized by a mutation in the stat3 gene and is associated with the classical picture of immunodeficiency as well as non - immunological features including characteristic facial features, retained primary teeth, hyperextensibility, recurrent pathological fractures, and scoliosis. autosomal recessive hyperimmunoglobulin e syndrome is a distinct disease entity characterized by more severe viral infections and neurological manifestations and without the skeletal or dental abnormalities seen in their autosomal dominant counterparts. in general, non - immunological manifestations of hyperimmunoglobulin e syndrome do not appear until well into childhood, but are almost always present after the age of 8. a three and a half year old boy was referred to pediatric infectious disease clinic with a history of asthma, environmental allergies, multiple lung infections, long bone fractures, and hand - foot - mouth disease, for evaluation of recurrent skin abscesses and bullae. by the time of evaluation in pediatric infectious disease clinic a, referral to genetics had already been initiated to evaluate the child for osteogenesis imperfecta because of the history of recurrent long bone fractures. according to the patient s mother, the abscesses and bullae usually occurred on the face and were generally of rapid onset. most often, they began as a pimple on the cheek in the morning and progress forming large mildly erythematous bullae by the afternoon. the mother first noticed very bad lesions on the patient s face as a small infant, initially attributing these lesions to bad baby acne ; however, the complexion was much worse than her other children, as well as other children who she knew. additionally, the patient also experienced multiple, severe blisters over the leg when a cast was removed due to a distal tibia fracture. additionally, approximately one and a half months prior to evaluation the patient was admitted to an outside hospital for recurring bullae on the medial aspect of the left foot. these bullae were surgically debrided and the fluid expressed was cultured, revealing budding yeast. the patient had a similar infection when he was three years old. at that time the patient was the product of a 37 week 2 day uncomplicated pregnancy, but was hospitalized in the nicu for ten days, requiring mechanical ventilation secondary to pulmonary edema. a review of the patients past medical history revealed a combination left tibia and fibula fractures at age 21 months, a combination left radius and ulna fracture at 29 months, and a fracture of the left tibia at 41 months. laboratory studies obtained from the outside hospitalization revealed a white blood cell count of 11.4 cell / cm, hemoglobin of 12.0 g / dl, and platelets of 385,000 cell / cm. the differential demonstrated 52% lymphocytes, 5% monocytes, 11% eosinophils, 31% neutrophils, and 1% basophils. the erythrocyte sedimentation rate (esr) and c - reactive protein (crp) were both normal. a biopsy of the debrided tissue on his left foot revealed dermal changes consistent with a hypersensitivity reaction to a fungal infection. the subtyping of igg were within normal limits, igg1 of 894 mg / dl, igg2 of 164 mg / dl, igg3 of 17 mg / dl, and igg4 of 133 mg / dl, with a normal antibody response to the diptheria toxoid and tetanus toxoid and t and b cell subtypes were normal. partial immunizations had been given to date and the patient history revealed allergies to sulfa drugs, eggs, and some wheat products by skin test. his family history was significant for a paternal grandmother with cvid and asthma, asthma and allergies on the paternal side, and the patient s mother and maternal grandmother had systemic lupus. on physical exam, in pediatric infectious disease clinic at the university of michigan, the height was in the 89 percentile, the weight is in the 96 percentile, and he appeared as a playful young boy in no apparent distress. there were multiple small, non - erythematous, hard papules over the cheeks and bridge of his nose. no blue tint of hue was noted in the sclera and the pupils were equal round and reactive to light. the abdomen was soft, non - tender, non - distended with no hepatosplenomegaly. the medial aspect of the left foot had new granulation tissue with a lesion approximately 3 cm by 4 cm from a previous bulla. muscle mass, strength and tone were within normal limits and the joints were not hyper - extensible. laboratory studies obtained from pediatric infectious disease clinic revealed an immunoglobulin panel with an iga level of 214 mg / dl (normal 15 - 160 mg / dl), igg level of 1260 mg / dl (normal 405 - 1160 mg / dl), igm level of 117 mg / dl (normal 40 - 190 mg / dl), and an ige level of > 10,000 ku / l (normal 0 - 150 the subtyping of igg were within normal limits, igg1 of 947 mg / dl, igg2 of 92 mg / dl, igg3 of 18.3 mg / dl, and igg4 of 42 mg / dl, with a normal antibody response to the rubella. a ch50 level was normal at 101 units (normal 52 - 128 units). the esr and crp were normal and a gram stain and culture from the left foot were negative. this patient was referred to the pediatric infectious diseases and immunology service at the university of michigan, with suspected osteogenesis imperfecta (oi), for evaluation of the immune system due to a history of frequent infections. oi is a rare genetic disorder (85 - 90% autosomal dominant) caused by a mutation in one of the type i collagen genes. type i collagen is an important component of the bone matrix and collagen homeostasis is important for both bone and connective tissue integrity. clinical findings in conjunction with the multiple fractures include loose joints and muscle weakness blue sclera, triangular face, brittle teeth, hearing loss, short stature, barrel - shaped rib cage, respiratory problems, underdeveloped lungs, spinal curvature, coxa vera, and extremely poor skeletal mineralization, none of which were present in this patient. de novo mutations in col1a1 or col1a2 account for a majority of the cases of oi which can be confirmed by molecular genetic testing or collagen biopsy analysis. while frequent fractures and a variety of collagen related problems are characteristic of oi, recurrent infections and susceptibility to staphylococcus aureus and candida species are not. however, infections with both organisms as well as frequent fractures can be found in patients with hyperimmunoglobulin e syndrome. attempts have been made to establish diagnostic criteria for hyper - ige, many of which utilize non - immunologic factors as guides. an nih scoring system consists of 20 different characteristics and assigning a varying number of points to each fulfilled criteria has been proposed. using this system, scores of greater than 40 out of a theoretical maximum of 109 made the diagnosis more likely and scores less than 20 made the diagnosis less likely. relying on non - immunologic factors to aid in diagnosing hyperimmunoglobulin e syndrome presents a unique challenge to early detection in childhood, because many skeletal features such as scoliosis and characteristic facial features occur with disease progression rather than with initial symptoms. there have been many reports of this disorder being undiagnosed or misdiagnosed as osteogenesis imperfecta, ehlers - danlos syndrome, medication non - compliance, and non - accidental trauma, particularly in the young patient. the nih scoring system that has been utilized in many studies has an age correction factor to address this very issue, early diagnosis is difficult. the patient, presented in the case report, would have a score of 37 on presentation to the pediatric infectious diseases clinic (table 1). because of the unusual cutaneous manifestations of yeast serum ige levels were obtained [> 10,000 ku / l (normal 0 - 150 ku / l) ] increasing the nih score to 47 increasing the likelihood of a diagnosis of hyper - ige syndrome. genetic testing was performed by confirming a mutation in stat 3 and a diagnosis of autosomal dominant hyper - ige syndrome. these patients have less skeletal and dental involvement but are more susceptible to viral infections such as severe molluscum contagiosum. in a recent review of 100 patients, the most frequent manifestations, characteristic face (82.8%), failure to shed deciduous teeth (69%), and lung cyst formation (68%) were not present in our patient, likely because he was too young to do so. however, frequent pathologic fractures was present in our patient, but was seen in only 34% of the cohort studied (table 2). the nih scoring system still represents a means of prioritizing the diagnostic evaluation of a patient with frequent fractures.	we present a case of hyperimmunoglobulin e (hyper - ige) syndrome in a three year old boy. there are many pitfalls in diagnosing this disease in the very young population, mainly due to the ambiguity of some diagnostic criteria in this population. recognizing this syndrome early in life can potentially be very beneficial to the patients involved and the medical system as a whole. early diagnosis can lead to fewer diagnostic tests, fewer referrals, and more focused exams, thus potentially reducing medical cost while also reducing the number of serious infections later in life, including those which are potentially fatal. additionally, a wellknown association between lymphoma and hyper - ige syndrome has been established ; while no recommendations are currently in place for screening, early diagnosis could help medical providers have a higher threshold for diagnosis of this disease.
a systematic way of guidelines was put forth for human beings to lead a secured social life so that people will not be harmed by one 's lifestyle. any deviated behavior from the normal will be punished or may be kept behind bars depending upon the crime committed ; sometimes, the crime conducted may also go unnoticed too. studies on such abnormal behavior of human beings are carried out to know the reason for the criminal mind but until date many have ended up with very few definitive results. in this context, the association of behavior of a person and sex chromosomal anomalies has become an exciting challenge for the researchers. sex chromosomal abnormality and its association with psychopath are not only of academic interest but also aim at providing justice to the individual who have been determined as convicts. before the 19 century, discussion of crime was conducted entirely on moral and philosophical terms. it was only in 1876, an italian anthropologist cesar lombroso published his theories of criminal behavior with scientific approach to understand the criminal behavior of a person. he also stated physiological theories of criminality focusing on person 's physical form as a mark of criminality. in 1949, sheldon advanced lombroso theory and suggested that some crime might be attributed to a chromosomal abnormality. eventually evidence - based studies established that xyy men are more in offender population when compared with general population. syndrome where men with this condition were thought to be overly aggressive and more likely to become criminals. similarly, in one of the study it was reported that approximately 1% of all individuals institutionalized with mental retardation had an xxy karyotype and approximately half of all mental retardation seen in males originates from a defective gene on the x - chromosome. while the fragile - x site has been implicated in most x - linked mental retardation disorders, other regions of the x - chromosome thus, genes involved in human cognition reside on the x - chromosome. as the syndrome is associated with extra x chromosome, these patients will show positive sex chromatin material in epithelial cells similar to normal females which are termed as barr bodies (bb). the presence of an extra x - chromosome in males is suggestive of klinefelters syndrome (ks), which affects 167/100,000 men. individuals with ks generally presents with severe personality disorders, neuroses and psychoses which are usually in the form of paranoid states and schizophrenic reactions. a study was conducted on ks males as a genetic model for psychotic disorders on ks males and compared with an equal number of normal males followed by an examination of brain mri of these individuals. ten out of eleven ks males had psychiatric disturbance, four of whom had auditory hallucinations compared with normal, where none of the normal individuals showed any abnormality. bartholomew studied psychopath, sex chromosome abnormalities and criminal law and stated that multiple x syndromes in men especially in ks showed inadequate behavior when compared with large control hospital group and also showed aggressiveness in behavior which may include criminality. to examine the crime characteristics of men, we focused on determination of extra x - chromosome in male jail inmates (xxy syndrome) and normal healthy male individuals. thus, the present study was concentrated on the determination of extra x - chromosome in jail inmates using epithelial cells from buccal smears (bs) and neutrophils in peripheral blood smears (pbs), to detect bb and correlate the same with criminal behavior among jail inmates. this study was performed on male jail inmates and comparison was carried out with equal number of the normal healthy males or controls without any criminal background. a total of 100 male subjects (fifty jail inmates from central jail and fifty controls) of above 18 years of age were considered for the study. clearance was obtained from the ethical committee of the institute to conduct a study on jail inmates as well from controls, and written consent was taken from the higher authorities of central jail. a detailed written consent explaining the need for the study was obtained from the jail inmates and control group. to avoid error, the crime history of the jail inmates 46 had committed murder, two were convicted for sexual abuse and remaining two had been convicted of robbery. the identification of the extra x - chromosome was carried out in the study by detecting the presence of bbs in pbs and bs. pbs was prepared by obtaining blood by pricking the finger (capillary blood) with all aspetic precautions. pbs and bs were stained using leishman 's stain and cresyl violet stain, respectively. a total of 100 cells were counted in each smear at 100 magnification for the detection of the bbs. in pbs, neutrophils were analyzed for bb as they are present on the nuclear lobes of the neutrophils as drumstick shaped structure [figure 1 ]. in the case of bss, bbs were found to be attached to the nuclear membrane of epithelial cells [figure 2 ]. statistical analysis was performed using t - test and mean number of bb was calculated using chi - square test and results were tabulated. photomicrograph showing barr bodies attached to nuclear lobe of neutrophil in peripheral blood smears (leishman 's stain, 1000) the presence of the barr bodies attached to the nuclear membrane of the epithelial cells of buccal smears (cresyl violet stain, 1000) a total of 100 male subjects (fifty jail inmates from central jail and fifty controls) of above 18 years of age were considered for the study. clearance was obtained from the ethical committee of the institute to conduct a study on jail inmates as well from controls, and written consent was taken from the higher authorities of central jail. a detailed written consent explaining the need for the study was obtained from the jail inmates and control group. to avoid error, the crime history of the jail inmates 46 had committed murder, two were convicted for sexual abuse and remaining two had been convicted of robbery. the identification of the extra x - chromosome was carried out in the study by detecting the presence of bbs in pbs and bs. pbs was prepared by obtaining blood by pricking the finger (capillary blood) with all aspetic precautions. pbs and bs were stained using leishman 's stain and cresyl violet stain, respectively. a total of 100 cells were counted in each smear at 100 magnification for the detection of the bbs. in pbs, neutrophils were analyzed for bb as they are present on the nuclear lobes of the neutrophils as drumstick shaped structure [figure 1 ]. in the case of bss, bbs were found to be attached to the nuclear membrane of epithelial cells [figure 2 ]. statistical analysis was performed using t - test and mean number of bb was calculated using chi - square test and results were tabulated. photomicrograph showing barr bodies attached to nuclear lobe of neutrophil in peripheral blood smears (leishman 's stain, 1000) the presence of the barr bodies attached to the nuclear membrane of the epithelial cells of buccal smears (cresyl violet stain, 1000) smears prepared from peripheral blood, revealed positivity in thirty jail inmates for the presence of bb out of total fifty cases which contributed to an overall accuracy of 60%. whereas, in remaining 20 (40%) jail inmates pbs were negative for the same. similarly, detection of bbs was carried out in bss stained with cresyl violet stain in fifty jail inmates. which, revealed positivity for bbs in 18 jail inmates (36%) and depicting negativity in 32 (64%). the average number of bbs present per high power field in case of pbs of jail inmates were around 26 cells while ; it was reduced to 13 cells per high power field while studying bss [table 1 ]. positivity for barr bodies in peripheral blood smears and buccal smears of jail inmates similarly, the analysis performed on control group showed a decreased number of positivity for the presence of bbs in pbs contributing around 7 controls (14%), whereas none of the bss depicted the presence of bbs in controls. the average number of bbs in pbs of controls was 0.32 [table 2 ]. the comparison of the average number of bbs in the case of jail inmates and in controls showed insignificant results. positivity for barr bodies in peripheral blood smears and buccal smears of control group a comparison was performed between jail inmates and controls for detection of bbs using pbs and bs. in the case of pbs, an overall obtained positivity was 60% (thirty jail inmates) while for controls it was reduced to only 14% (7 controls) with an overall positivity of 37% [table 3 ]. comparison for positivity of barr bodies in jail inmates and controls (peripheral blood smears) similarly, the comparison between jail inmates and controls for detecting bbs using bs yielded 36% positivity in jail inmates (18 subjects), and none were positive in case of controls. the comparison between jail inmates and controls for detection of bb using bs showed statistically significant results [table 4 ]. comparison for the positivity of barr bodies in jail inmates and controls (buccal smears) ks is a genetic disorder which results due to the presence of extra x - chromosome in men. many times ks goes undiagnosed or is diagnosed late due to its varied clinical presentation. studies have revealed a correlation between ks and criminal behavior and have determined the presence of 47 chromosomes, xxy disorder by karyotyping which is an expensive method and can not be performed as a routine screening procedure on a large population. xxy men are found in about 0.81% of males hospitalized with schizophrenia, a four - to - five - fold excess over general live birth rates. this may indicate that genes that are overexpressed in the brains of xxy males may also be relevant to schizophrenia and other psychiatric disorders. since the search for genes leading to susceptibility to major psychiatric disorders has produced inconsistent results. the xxy karyotype may serve as a naturally occurring genetic model to provide clues to the inherited abnormalities that cause these disorders in individuals with normal chromosomal numbers. it is now known that the xxy karyotype occurs in 1 in 500 live male births and is the most common type of human chromosome anomaly. unlike chromosomal duplications or translocations on autosomes this mildness is probably due to inactivation of most genes on the extra x - chromosome. however, there is a class of x - chromosome genes that have homologies on the y chromosome and tend to escape the normal extra x - chromosome inactivation process, as do some other x - specific genes. it is thought that the characteristic features of ks originate from genes that escape inactivation, and are expressed in excess. thus, the tall stature, testicular and sex hormone deficiencies, reduction of secondary sex characteristics, and in some cases, breast development in postpubertal klinefelter 's men may be due to expression of x y homologous genes. similarly, behavioral and cognitive symptoms of ks are likely to result from this class of genes as well. it has been suggested that at least one gene or genes in x y homologous regions of the sex chromosomes that escape normal x - inactivation are crucial for language functioning. thus, the aim of this study was to detect the presence of bbs in jail inmates and to co - relate with their criminal behavior. this is the first study to correlate the criminality of jail inmates by detecting their bbs using pbs and bs among the indian population. the study has obtained 60% positivity for bb in jail inmates as compared to normal controls which showed only 14% in pbs. the comparison between the groups was statistically significant. whereas the presence of bb in bs was less compared to pbs, but when it was compared within the group, it was statistically significant. a similar study using karyotyping was conducted by stochholm. and showed the presence of 47, xxy in a register - based cohort study found moderately increased crime rate in men with ks, whereas few other studies found no such correlation between 47, xxy and increase in crime in affected men. we found good correlation between the presences of bb in jail inmates which further confirms their psychological behavior to commit crime. bb can also be seen as hyper pyknotic, basophilic, intranuclear structure adjacent to the nuclear membrane in the resting stage of a cell in karyotypic female. it can also be seen adjacent to the nucleolus or be free in the nucleoplasm. it resembles the letter v, w, s or x under electron microscope and measures around 0.8 1.1 in its greatest diameters. it is commonly seen in buccal smear, pulp tissue, hair follicle and vaginal smears commonly used for determination of sex. in a smear only 30% of the cells any nuclear stain can be used for the demonstration of the bbs in bs ; most commonly used ones are hematoxylin and eosin (h and e), papanicolau stain, feulgen stains, guard stains, cresyl violet, carbol fuschin and fluorescent stains. in h and e and papanicolau stains, the bacteria stains heavily, hence the bbs are not noticed prominently. fluorescent stains being more confirmatory for the detection of the bbs but is expensive. in this study, cresyl violet was preferred as it stains bbs more prominently with fewer artifacts and is cost - effective. the comparison between two methods used for detection of bb (pbs and bs) was carried out. number of cases for the presence of bb was high in pbs method when compared with bs. it is suggested that pbs is the most accurate and reliable one for detection of bb. comparison between number of bb present in the case of jail inmates and normal healthy male groups revealed that there was no much difference between the two groups for the average number of bb but the number of positive cases for bb was less in controls compared to jail inmates. this suggests that probably the number of bb does not play an important role in the determination of extra x - chromosomes. even though presence of extra x - chromosome in men can be diagnosed using various methods but detection of bb in pbs presence of 60% of the bb in pbs in jail inmates has revealed definite association between the presences of extra x - chromosome in men with criminality. it is well accepted that x - chromosome bears around 1100 genes which are responsible for the normal functioning of brain and testes. with the adequate numbers of studies in large group of population one can find out the reason for the criminality in ks. the detection of bb in males and its correlation to criminality has proved that there is an association between genetic alteration and antisocial behavior of a person. in this study, the jail inmates showed increased number of positivity for bb, which is suggestive of extra x - chromosome. this preliminary study revealed presence of bb in jail inmates which can be correlated with their criminal behavior but further confirmation needs to be done for the presence of the extra x - chromosome by performing karyotyping. nevertheless, detection of bb using pbs and bs appears to be a simple, cost - effective and appropriate screening method to detect xxy syndrome in criminals. presence of bb in jail inmates will further confirm their criminal mind, which has been suspected to have committed crime and it also confirms the crime in victims who is already a convict. authors intend to further study and correlate the presence of bb with karyotyping to confirm our preliminary findings.	background : cytogenetic studies from past decades have shown that interphase cells of female cats contain a densely stained chromatin mass in their nuclei called as barr bodies (bbs) named after the scientist murray barr. bbs are unique chromatin structures formed due to the condensation of the x - chromosome. many psychopathic disorders originate from defective genes including the multiple x syndromes. males with extra x - chromosome generally present with severe personality disorder. the present study was conducted to determine the presence of extra x - chromosome in male jail inmates through the detection of bb in peripheral blood and buccal smear.materials and methods : study included 100 male subjects (fifty jail inmates and fifty controls), after obtaining the consent, peripheral blood smears (pbs) and buccal smears (bs) were prepared and stained using leishman 's and cresyl violet stain respectively. one hundred neutrophils in pbs and epithelial cells in bs were screened for detection of the bb ; accumulated data were tabulated and statistically analyzed using t - test and chi - square test.results:60% of cases in pbs and 36% in bs showed positivity for the presence of bb in jail inmates as compared to 14% of cases in pbs and none in bs were positive for bb in controls.conclusion:presence of bb in male suggests increased likelihood of criminal tendencies. further studies are to be carried out to compare the results with karyotyping.
in developing countries and/or areas inhabited by indigenous populations, plants and other natural sources constitute the solely source of bioactive molecules used for a variety of purposes. the use of medicinal plants throughout thousands of years by these populations allowed accumulation of empirical knowledge of their utility, which demands adequate evaluation of efficacy, safety, and action mechanisms. the therapeutic properties of certain medicinal plants are generally related to their content of secondary metabolites, such as polyphenols, terpenes, phytosteroids, and alkaloyds, among others, which are produced in considerable amounts and variable proportions. essential oils are concentrated volatile aromatic compounds produced by aromatic plants, such as cymbopogon winterianus jowitt (poaceae), cymbopogon citrates stapf (poaceae), lavandula multifida linnaeus (lamiaceae), and thymus pubescens boiss. et kotschy ex celak (lamiaceae) that have been found to exhibit a variety of biological properties [36 ]. monoterpenes are the main chemical constituents of the essential oils of these plants and are found as mixtures of odoriferous components that can be obtained by steam distillation or solvent extraction from a large variety of aromatic plants. they are found in edible as well as in medicinal plants with a therapeutic property [710 ]. recent works have demonstrated that monoterpenes may present important pharmacological properties including antimicrobial, antioxidant, analgesic, and antitumoral activities, as well as effects on cardiovascular system and central nervous system (cns). (+) -camphene, p - cymene, and geranyl acetate (figure 1) are monoterpenes present in the essential oils of various plant species, such as cypress, origanum, and eucalyptus oils [15, 16 ]. these substances are present at significant amounts in a wide variety of products derived from natural sources used as food, medicines, or other purposes in different countries. however, reports with reference to their therapeutic effects by studies aiming to establish their individual characteristics, as described in the present work, are scarce in literature. oxidative stress is the result of an unbalance in reactive species production and antioxidant defense and is a main component in cancer, infectious diseases, cardiovascular disorders, and neurodegenerative conditions. pharmacological agents showing therapeutic efficiency against some diseases may exert antioxidant properties in target tissues, which may be related to their mechanism of action. monoterpenes isolated from medicinal plants have been previously described as redox - active molecules, being able to scavenge specific reactive species such as hydroxyl radicals and nitric oxide (no), preventing oxidation of biomolecules and influencing pain and inflammation [3, 19, 20 ]. since reactive species and oxidative stress are linked to a wide array of pathological conditions, the evaluation of the redox action of monoterpenes with potential pharmacological activity may indicate new pharmacological agents for such diseases. in the present work, we performed a screening of redox activities and antinociceptive actions of the monoterpenes (+) -camphene, p - cymene, and geranyl acetate. these monoterpenes are active compounds isolated from several medicinal plants traditionally used in brazil and also in other countries to treat a wide range of chronic and/or infectious diseases related to pain, inflammation, and oxidative stress. studies with plant extracts or other products containing one or more of these substances have been conducted previously, but conclusions on the potential properties of their isolated constituents are highly limited due the presence of several metabolites in these preparations. acetic acid, (+) -camphene (95% purity), p - cymene (97% purity), geranyl acetate (98% purity) (figure 1), polyoxyethylene - sorbitan monolate (tween 80), aaph (2,2-azobis(2-methylpropionamidine)dihydrochloride), luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), 2-deoxyribose, glycine, griess reagent, snp (sodium nitroprusside), tba (2-thiobarbituric acid), (4,6-dihydroxypyrimidine-2-thiol), h2o2 (hydrogen peroxide), adrenaline, catalase, and sod (superoxide dismutase) were purchased from sigma (usa). adult male albino swiss mice (2834 g) were randomly housed in appropriate cages at 21 2c with a 12/12-h light / dark cycle (light from 06:00 to 18:00), with free access to food (purina, brazil) and tap water. nociceptive tests were carried out by the same visual observer and all efforts were made to minimize the number of animals used as well as any discomfort. experimental protocols were approved by the animal care and use committee (cepa / ufs no. mice (n = 8, per group) were pretreated either by (+) -camphene, p - cymene, or geranyl acetate (50, 100 or 200 mg / kg), acetylsalicylic acid (aspirin, 200 mg / kg), and the vehicle (saline + tween-80 0.3%) by intraperitoneal route (i.p.). then, after 1 h, mice received the 0.65% acetic acid injection (i.p. each animal was placed in an individual observation chamber, and 15 minutes after acetic acid injection the cumulative number of writhing responses was recorded for 15 minute after a latency period of 5 minutes. the procedure described by hunskaar and hole was used. nociception was induced by injecting 20 l of 1% formalin in distilled water in the right hind paw subplantar. mice (n = 8, per group) previously received the same treatments described in the writhing test (1 h prior to injecting formalin). these mice were individually placed in a transparent plexiglass cage observation chamber (25 cm 15 cm 15 cm). the amount of time each animal spent licking the injected paw was indicative of pain. the number of lickings from 05 min (early phase) and 1530 min (late phase) were counted after formalin injection. this volume and percentage concentration of formalin were selected from our pilot studies that showed a pain - related biphasic behavioral response (face - rubbing) of great intensity at periods of 05 minutes (first phase) and 1540 minutes (second phase). pain was quantified at those periods by measuring the time (in seconds) that the animals spent facerubbing in the injected area with their fore- or hindpaws. the total reactive antioxidant potential (trap) is employed to estimate the nonenzymatic antioxidant capacity of samples in vitro. this method is based on the quenching of luminol - enhanced chemiluminescence (cl) derived from the thermolysis of aaph as the free radical source. briefly, we prepared aaph solution, added luminol (aaph + luminol, free radical - generating system) and then waited the system to stabilize for 2 h before the first reading. different concentrations of each substance were added and the luminescence produced by the free radical reaction was quantified in a luminescence plate reader (microbeta 1450, perkin elmer, boston, ma, usa) during 60 min. the results were transformed in percentage and area under curve (auc) and calculated in the graphpad prism version 5.01 software. the total antioxidant reactivity (tar) the tar results were calculated as the ratio of light intensity in absence of samples (i)/light intensity right after sample addition. although tar and trap evaluations are obtained in the same experiment, they represent different observations, since the tar is more related to the antioxidant quality (reactivity, the scavenging capacity in a short - term period) and trap is more related to the antioxidant amount and kinetic behavior. the formation of oh (hydroxyl radical) from fenton reaction was assessed using the 2-deoxyribose oxidative degradation assay. the principle of the assay is the incubation of 2-deoxyribose with a hydroxyl radical generation system, which produces malondialdehyde (mda). this system is then incubated with 2-thiobarbituric acid (tba), which reacts with mda and forms a chromophore quantifiable by spectrophotometry. briefly, typical reactions were started by the addition of fe (feso4 6 m final concentration) to solutions containing 5 mm 2-deoxyribose, 100 mm h2o2, and 20 mm phosphate buffer (ph 7.2). to measure the antioxidant of activity of each compound against hydroxyl radicals, different concentrations of (+) -camphene, p - cymene, and geranyl acetate reactions were carried out for 15 min at room temperature and were stopped by the addition of 4% phosphoric acid (v / v) followed by 1% tba (w / v, in 50 mm naoh). solutions were boiled for 15 min at 95c and then cooled at room temperature. the absorbance was measured at 532 nm and results were expressed as percentage of tbars formed. no was generated from spontaneous decomposition of sodium nitroprusside (snp) in 20 mm phosphate buffer (ph 7.4). once generated, no interacts with oxygen to produce nitrite ions, which were measured by the griess reaction. the reaction mixture (1 ml) containing 10 mm snp and either (+) -camphene, p - cymene, or geranyl acetate at different concentrations were incubated at 37c for 1 h. an aliquot of 0.5 ml was taken and homogenized with 0.5 ml griess reagent. thiobarbituric acid - reactive substances (tbars) assay was employed to quantify lipid peroxidation and an adapted tbars method was used to measure the antioxidant capacity of the monoterpenes using egg yolk homogenate as lipid rich substrate. the principle of the method is based on spectrophotometric measurement of the color produced during the reaction of thiobarbituric acid (tba) with lipoperoxidation products, such as malondialdehyde and 4-hydroxynonenal. briefly, egg yolk was homogenized (1% w / v) in 20 mm phosphate buffer (ph 7.4), 1 ml of homogenate was sonicated and then homogenized with 0.1 ml of (+) -camphene, p - cymene, or geranyl acetate at different concentrations. lipid peroxidation was induced by addition of 0.1 ml of aaph solution (0.12 m). samples (0.5 ml) were centrifuged with 0.5 ml of tca 15% at 1200 g for 10 min. an aliquot of 0.5 ml from the supernatant was mixed with 0.5 ml tba (0.67%) and heated at 95c for 30 min. after cooling, samples absorbance was measured using a spectrophotometer (uv-1800 shimadzu) at 532 nm. the results were expressed as percentage of tbars formed by aaph alone induced control. the ability of (+) -camphene, p - cymene, and geranyl acetate to scavenge superoxide anion (superoxide dismutase - like activity or sod - like activity) was measured as previously described. samples were mixed with 200 l glycine buffer (50 mm, ph 10.2) and 5 l of native catalase 100 u / ml. superoxide generation was initiated by addition of adrenaline 2 mm and adrenochrome formation was monitored at 480 nm for 5 minutes at 32c. superoxide production was determined by monitoring the reaction curves of samples and measured as percentage of the rate of adrenaline autooxidation into adrenochrome. the capacity of (+) -camphene, p - cymene, and geranyl acetate to degrade hydrogen peroxide (h2o2) added in the incubation medium (catalase - like or cat - like activity) was measured as previously described. briefly, h2o2 diluted in 0.02 m phosphate buffer (ph 7.0), to obtain a 5 mm final concentration, was added to microplate wells, in which different concentrations of (+) -camphene, p - cymene, and geranyl acetate were present. readings were made in a spectrophotometric plate reader (spectramax 190-molecular devices) at 240 nm every 15 seconds for 5 minutes at 37c. data were evaluated using graphpad prism version 5.01 (graph pad prism software inc., san diego, ca, usa), through analysis of variance (anova) followed by tukey 's test. all tested doses of p - cymene produced significantly (p < 0.05 or p < 0.001) antinociceptive effect in this test compared to control group (vehicle) (table 1). pretreatment with (+) -camphene or geranyl acetate, at higher doses, significantly reduced nociceptive behavior compared with control group. aspirin (200 mg / kg), used as positive control, also produced a significant antinociceptive effect (p < 0.001). the highest doses of either (+) -camphene or geranyl acetate caused a significant inhibition of the licking response to the injected paw in mice, compared with the control group, only in the second phase of the formalin test (table 1). however, p - cymene, in all doses, significantly inhibited (p < 0.01 or p < 0.001) both phases of formalin test when compared with control group. additionally, p - cymene - treated mice were more significantly protected when compared with (+) -camphene or geranyl acetate - treated animals. as expected, aspirin (200 mg / kg) reduced the licking time only in second phase. monoterpene - treated mice did not show any significant motor performance alterations with the doses of 200 mg / kg (figure 2). as might be expected, the cns standard depressant diazepam (5 mg / kg, i.p.) reduced the time of treated animals on the rotarod apparatus (p < 0.001) compared with the control group. to evaluate the antioxidant properties of three monoterpenes, we first assessed the ability of each compound to prevent lipid peroxidation in an in vitro peroxyl - generating system. geranyl acetate also presented antioxidant activity (figure 4(a)). on the other hand, p - cymene had no effect on aaph - induced lipoperoxidation (figure 5(a)). to further explore the redox profile of these compounds, the trap / tar parameters were evaluated, which indicate the capacity of a given sample to act as a general antioxidant or prooxidant agent in a constant reactive species generating system. we observed that (+) -camphene presents a significant antioxidant activity, which was indicated by both trap and tar parameters (figures 3(b) and 3(c)). on the other hand, geranyl acetate did not present antioxidant activity ; in fact, a significant prooxidant effect was observed in the trap assay (figures 4(b) and 4(c)). p - cymene had no effect towards antioxidant or prooxidant activity in both trap and tar measurements (figures 5(b) and 5(c)). (+) -camphene, geranyl acetate, and p - cymene all presented significant antioxidant activity against hydroxyl radicals generated in vitro, although at varying degrees (figures 3(d), 4(d) and 5(d)). besides, (+) -camphene presented a dose - dependent activity of no generation in relation to control (figure 3(e)), while geranyl acetate (figure 4(e)) and p - cymene (figure 5(e)) had no effect towards no formation or scavenging activity. (+) -camphene also presented increased sod - like activity, indicating ability to scavenge or inhibit superoxide radicals (figure 3(f)). geranyl acetate, on the other hand, enhanced superoxide - mediated adrenaline oxidation (figure 4(f)), while p - cymene had no statistically significant effect (figure 5(f)). finally, the cat - like assay demonstrated that (+) -camphene (figure 3(g)) and p - cymene (figure 5(g)) have a modest, although significant, activity against h2o2, while geranyl acetate had no effect (figure 4(g)). our results showed that the monoterpenes here evaluated are able to inhibit the nociceptive behavior in mice, as determined by a significant reduction in acetic acid - induced abdominal writhing. acetic acid - induced abdominal constriction is a standard, simple, and sensitive test for measuring analgesia induced by both central and peripherally acting analgesics [22, 27 ]. in acetic acid - induced abdominal writhing, pain is elicited by the injection of an irritant such as acetic acid into the peritoneal cavity, which produces episodes of characteristic stretching (writhing) movements, and inhibition of the number of episodes by analgesics is easily quantifiable. to investigate if the treatments with (+) -camphene, p - cymene, or geranyl acetate could influence the motor activity of the animals and consequently impair the assessment of the nociceptive behavior in the experimental models, the motor activity of the animals was evaluated with a rotarod apparatus. monoterpenes - treated mice did not show any significant motor performance change when evaluated in rotarod test. results showed in the present work support the hypothesis of (+) -camphene, p - cymene, and geranyl acetate participation in the inhibition of prostaglandin (pge) synthesis, as the nociceptive mechanism involves the process or release of arachidonic acid metabolites via cyclooxygenase (cox) and pge biosynthesis during abdominal writhing induced by acetic acid. the formalin test is a very useful method for not only assessing antinociceptive drugs but also helping in the elucidation of their action mechanisms. the neurogenic phase, commonly denominated first phase, is probably a direct result of paw stimulation and reflects centrally mediated pain with release of substance p while the late phase is due to the release of histamine, serotonin, bradykynin and prostaglandins. only p - cymene was able to reduce nociceptive behavior in both phases of the formalin test. the second phase, denominated inflammatory phase, depends on a combination of ongoing inputs from nociceptive afferents, due to the release of excitatory amino acids, pge2, no, tachykinin, and kinins among other peptides and, at least in part, of central sensitization [30, 31 ]. additionally, intraplanar injection of formalin has been described to induce the production and release of no, which in turn is suggested to be an essential component of the proinflammatory / nociceptive response by the stimulation of the production and release of cytokines, reactive species, and prostanoids. on the other hand, it is generally agreed that n - methyl - d - aspartate (nmda) receptors contribute to the persistent chemical stimulus during the late phase of central sensitization of dorsal horn neurons. our results show that monoterpenes produced an inhibition of the inflammatory pain, later phase, in mice as determined by a significant reduction of nociceptive behavior in second phase of formalin test. apparently (+) -camphene and geranyl acetate demonstrated a discrete analgesic profile when compared with p - cymene. in fact, some analgesic effects of p - cymene were previously demonstrated by our group. we here compared this activity with other monoterpenes and attempted to establish a correlation of anti - inflammatory and analgesic effects of these compounds with their redox properties, as it is suggested for other monoterpenes. monoterpenes with oxygen in their structure constitute a wide group of antioxidant molecules, largely due to their functional groups (alcohols). we performed a detailed screening of the redox properties of these monoterpenes to establish their antioxidant properties, as reactive oxygen / nitrogen species (ros / rns) mediate inflammatory processes and are involved in the molecular mechanisms of several pathologies. terpenoids exhibiting antioxidant properties have been considered potential candidates for new therapeutic agents, especially when found in medicinal plants traditionally used to treat ros / rns - related diseases. although p - cymene had a stronger antinociceptive effect, when compared to the other terpenes, this compound exhibited a poor antioxidant potential in vitro. p - cymene did not prevent aaph - induced lipoperoxidation, suggesting it is not able to act as a membrane antioxidant ; besides, trap and tar parameters did not show any effect of p - cymene on the in vitro free radical - generating system. high concentrations of p - cymene had a modest effect against h2o2 and hydroxyl radicals ; however, it did not prevent no and superoxide radicals formation, which are reactive species more related to pain and inflammation. hydroxyl radical has a high oxidant power and it is probably the most reactive radical. it is able to join dna nucleotides and cause strand breakage, which contributes to carcinogenesis, mutagenesis, and cytotoxicity. nevertheless, (+) -camphene had a pronounced antioxidant effect at tbars assay, which was confirmed at trap / tar measurements, cat - like activity and hydroxyl - scavenging assays. furthermore, (+) -camphene presented significant superoxide degradation activity but enhanced no formation. geranyl acetate had a mixed redox profile, with antioxidant activity in tbars and hydroxyl - scavenging assays and prooxidant activity at trap / tar measurements and sod - like activity assay, while the no - scavenging assay showed no activity. no is a dual molecule playing major roles in both cell signaling and oxidative / nitrosative stress in a concentration and time - dependent manner [37, 38 ]. this molecule may be physiologically generated through no synthases activity, triggering anti - inflammatory and cytoprotective pathways. however, no also may increase the synthesis / release of proinflammatory mediators such as cytokines and reactive oxygen species and prostanoids, thus promoting inflammatory reaction. we observed that (+) -camphene enhanced no production in vitro, and our in vivo results show that (+) -camphene has a modest antinociceptive activity. peripherally released no contributes to the development of oedema and hyperalgesia in tissue injury and inflammation. as mentioned above, expression of several inducible enzymes that contribute to the release of proinflammatory mediators such as no and pge2 are observed during inflammation, such as inducible nitric oxide synthase (inos) and cyclooxygenase-2 (cox-2). cox-2 is the inducible form of the enzyme, the synthesis of which is triggered by cytokines that also induce inos. the two pathways interact closely and no can stimulate cox-2 activity by combining with its heme component. the monoterpene p - cymene had no activity against no, but presented the highest antinociceptive effect. antioxidant properties are generally associated to beneficial effects, mainly due to the widespread association between free radicals with diseases and ageing. however, when a given substance presents little or none antioxidant activity at one or several in vitro assays, this may not be associated to lack of therapeutic properties. this apparent contradiction may rely on the fact that interaction of no with other reactive species results in loss of its regulatory properties ; for instance, no - mediated activation of cox and subsequent release of beneficial and anti - inflammatory prostaglandins is lost when superoxide production is also enhanced, since interaction between superoxide and no leads to in situ formation of peroxynitrite, a potent cytotoxic and proinflammatory reactive species [36, 37 ]. although p - cymene had no effect against no, we observed a mild activity against superoxide at sod - like activity assay, which may attenuate no deleterious effects and preserve its beneficial properties. we show here a screening of antinociceptive actions and redox properties of three monoterpenes isolated from medicinal plants. assays with animals demonstrated that p - cymene has the strongest antinociceptive effect, but (+) -camphene and geranyl acetate also present significant activity at higher doses. however, (+) -camphene had the strongest antioxidant effect at tbars and trap / tar assays, and also had the highest scavenging activities against different free radicals generated by in vitro systems. geranyl acetate and p - cymene also presented some antioxidant effect, but with a varying profile according the free radical - generating system studied. the results presented here suggest that (+) -camphene, p - cymene, and geranyl acetate may present pharmacological properties related to inflammation and pain - related processes, being potentially useful for development of new therapeutic strategies, with limited possibilities for p - cymene and geranyl acetate.	objective. to evaluate antinocicpetive and redox properties of the monoterpenes (+) -camphene, p - cymene, and geranyl acetate in in vivo and in vitro experimental models. methods. evaluation of the in vitro antioxidant activity of (+) -camphene, p - cymene, and geranyl acetate using different free radical - generating systems and evaluation of antinociceptive actions by acetic acid - induced writhing and formalin - induced nociception tests in mice. results. p - cymene has the strongest antinociceptive effect, but (+) -camphene and geranyl acetate also present significant activity at high doses (200 mg / kg). (+) -camphene had the strongest antioxidant effect in vitro at tbars and trap / tar assays and also had the highest scavenging activities against different free radicals, such as hydroxyl and superoxide radicals. sodium nitroprussiate - derived no production was enhanced by (+) -camphene. geranyl acetate and p - cymene also presented some antioxidant effects, but with a varying profile according the free radical - generating system studied. conclusion. (+) -camphene, p - cymene, and geranyl acetate may present pharmacological properties related to inflammation and pain - related processes, being potentially useful for development of new therapeutic strategies, with limited possibilities for p - cymene and geranyl acetate.
impaired thermoregulation is a known complication of high level spinal cord injury and side effects of hypothermia induced by such an injury may complicate trauma resuscitation. hypothermia defined as a core temperature of 10 meq / l, temperature < 12c, ph < 6.5 and a fibrinogen level < 50 mg / dl. the evidence of the patient 's paralysis was not apparent until he woke up following successful cardioversion and re - warming. no radiologic abnormalities were seen except the small 1-mm broad - based bulge seen at level c5 - 6 and c6 - 7. the mri done later that day demonstrated diffuse spinal cord edema which finally explained his injuries. this type of injury is most commonly seen in children and is termed a spinal cord injury without radiological abnormality or spinal cord injury without radiological abnormality (sciwora). sciwora is defined as the occurrence of acute traumatic myelopathy despite normal plain radiographs and normal computed tomographic (ct) studies. it is most described and discovered in the pediatric population and thought to be due to the mismatch in the elasticity of the tissue of the vertebral column and spinal cord. sciwora in adults is very rare and most of these injuries are often missed initially and diagnosis is often delayed because these injuries tend to occur in the multi - trauma patient. for the most part, sciwora in adolescents and adults are less severe because most of them present as incomplete paralysis with transient symptoms and a delayed onset and should always be suspected in patients that have a neurological deficits and apparently normal x - rays and/or ct scans. the use of steroids is controversial but has shown some long - term benefit and is recommended as a treatment option. the national acute spinal cord injury study (nascis) trials showed no statistical difference between methylprednisolone sodium succinate (mpss) and placebo in terms of neurological recovery at one year, but the post - hoc analyses of subgroups did. the nascis iii study found that in patients treated earlier than 3 hours after injury, the administration of methylprednisolone for 24 h was best. the dose, if considered, consists of a high dose methylprednisone bolus of 30 mg / kg iv within 8 h of the injury as well as an infusion of 5.4 mg / kg / h for the next 23 h. [1517 ] it is evident from this case report that even though hypothermia can occur from many different causes, it is still treated the same until its underlying cause can be identified. current trauma protocol, using warm saline and warm humidified air during mechanical ventilation, allows prompt treatment in those patients who are not initially discovered to be hypothermic. this case also highlights the complexity of the trauma patient and the multiple conditions that can present and mask other conditions. this case was ultimately caused by an occult spinal cord injury after the patient fell from his bicycle. this injury is classified as a sciwora and in adults is a condition that is rare. to have this condition causing severe hypothermia with cardiopulmonary arrest	we report a case of a 64-year - old caucasian male who was transported to the emergency department (ed) after being found unconscious on the side of the road. on arrival to the ed the patient went into ventricular fibrillation and advanced cardiac life support was started at that time. thirty minutes into the resuscitation, after multiple rounds of code drugs and defibrillation attempts, the patient was found to be severely hypothermic with a rectal temperature of 24.9c (76.9f). through the use of passive and active re - warming measures the patient 's temperature increased enough to allow successful cardioversion and stabilization. within minutes of cardiac stabilization the patient regained consciousness and was able to follow commands, but was found to be paralyzed from the neck down. subsequent ct scans revealed no acute fractures, subluxations or acute spinal cord injury. this case represents the rare finding of severe hypothermia secondary to occult spinal cord injury. case report was taken from case at bayfront hospital, st. petersburg, florida.
after the first report by diamond in 1939, juvenile polyps, one of the polyps found in the large intestine, have been reported to occur in relatively young people. the juvenile polyp is a type of harmatoma that develops in the connective tissue of the large intestinal submucosal layer, but its etiology is not clear. in a recent study, multiple juvenile polyposis was reported to have a cumulative malignant potential in 50% of the patients. on the other hand, a solitary lesion is thought to a benign disease with low malignant potential, and it can be treated sufficiently by using only an endoscopic polypectomy, with no follow - up observation being required. in a patient admitted for the chief complaint of anal bleeding, we detected polyps in the sigmoid colon and performed an endoscopic polypectomy. by histopathologic test, the polyp was diagnosed as a harmatoma with a focal signet ring cell carcinoma limited to the mucosal layer. this case is reported as a rare case of malignant degeneration of a solitary juvenile polyp. a 21-year - old female visited our hospital with a chief complaint of bleeding on defecation. in digital rectal examination, no hemorrhoid, anal fissure or other anal disease was found, but blood - tinged stool was detected. for several days, she had had symptoms of diarrhea, with no abdominal pain or febrile sense. none of her family had had inflammatory bowel diseases, multiple polyposis, malignant diseases in the digestive tract, except that her mother had undergone breast cancer surgery. the polyp had two branching heads, 20 mm and 27 mm in size respectively, and the surface was relatively smooth, but covered with mucous discharge. in addition, a lobulating change, different from that in typical juvenile polyps, was found (fig. after a detached snare had been applied, a snare polypectomy was done in the neck of the polyp. during the polypectomy, no hemorrhage or abdominal pain developed, and on pathologic examination, the polyp was diagnosed as a juvenile polyp that had several branches, multiple cystic changes, and interstitial edema with mucinous congestion and infiltration of acute and chronic inflammatory cells (fig. 2). in some areas of the polyp, local infiltration of a signet ring cell carcinoma into the mucosa was observed, but there was no adenomatous change (fig. performed after the polypectomy, no distant metastasis or infiltration to adjacent tissues was detected, and serum level of carcinoembryonic antigen or ca19 - 9 was not elevated. in the follow - up endoscopic examination performed 3 months later, no recurrence at the polypectomy site or residual polyps were found ; the patient is still under outpatient follow - up observation. horrilleno. termed pediatric polyps showing the formation of cysts by the epithelial grands with abundant mucoid, increased connective tissues, and chronic infiltration of eosinophils as juvenile polyposis, and morson referred to such polyps showing hyperproliferation of normal tissues in the upper part of interstitial tissue as a harmatoma. afterward, mccoll. reported multiple juvenile polyposis, which was considered to be a different disease from multiple adenomatosis. the most frequent symptom of juvenile polyposis is hemorrhage in the rectum, as shown in our case ; anal prolapse, abdominal pain, diarrhea, mucous stool, rectal prolapse, and other symptoms may also develop. in approximately 10% of the patients, juvenile polyposis in the large intestine is detected in the distal rectum in most cases. typical juvenile polyps have a round or an oval pedunculated form with a smooth continuous surface, and numerous microcysts filled with mucoid are detected in a cross section. in addition, in 20% of the cases, multicentric segments or branches are observed. in microscopic findings in the upper part of the mucularis mucosa, glandular epithelial ducts with columnar epithelial cells and goblet cells are found, and they show cystic dilatation and are filled with mucin and numorous inflammatory cells. these are benign lesions if cellular hyperproliferation, hypercchromatism, and excessive cell division of the epithelial cells covering the surface are not present, and they can be sufficiently treated by using only a colonoscopic polypectomy. since the malignant deterioration potential is very low, follow - up observation is not required. when such juvenile polyposis occurs in a familial form, it is inherited as an autosomal dominant trait. approximately 25 - 50% of the cases are associated with adenoma or dysplasia with a malignant potential ; thus, they are considered as precancerous lesions. in addition, in multiple polyposis, besides the large intestine, polyps may develop in the entire digestive system in some cases ; thus, if a family history is suspected or genetic mutation is confirmed, for the assessment of multiple lesions, an overall examination of the esophagus, stomach, small intestine and large intestine is required. the cause of the development of cancer in juvenile polyposis has not been elucidated yet. the surface ulcer observed in some polyps, infection and necrosis according to jass., the malignant potential of a solitary juvenile polyp is very low. nonetheless, jones. reported adenomatous changes and cases of signet ring cell carcinoma limited to the mucosal layer, but in our case, adenomatous changes were not found. the mechanism of malignant deterioration or carcinomatous changes in solitary polyps is thought not to be different from the mechanism in juvenile polyposis. although it is very rare, the possibility of malignant deterioration of solitary juvenile polyposis should be kept in mind, and in a polypectomy, attention should be paid to securing sufficient resection margins.	juvenile polyps are relatively common polyps that affect predominantly young patients and may occur in isolated, multiple, and/or familial forms. they have been considered to be benign lesions without neoplastic potential, but for patients with multiple juvenile polyposis, the cumulative malignant risk is greater than fifty percents. in patients with a solitary polyp, the risks are minimal, and only a few cases of malignant change from a solitary juvenile polyp have been reported. we describe the case of a twenty one year old female with one solitary juvenile polyp, which contained a signet ring cell carcinoma in the mucosal layer.
one of the major risk factors for chd are elevated serum cholesterol concentrations. lowering the level of serum cholesterol is an established clinical practice for chd treatment, intervention, and prevention. pharmacologically, circulating cholesterol concentrations are reduced by statins, which are 3-hydroxy-3-methylglutaryl coenzyme a (hmg - coa) reductase inhibitors, affecting biosynthesis of endogenous cholesterol. another approach is to block the absorption of dietary cholesterol, which is the other major contributor to serum cholesterol concentrations in the small intestine. ezetimibe 1 was originally discovered without a known molecular target through in vivo screening of cholesterol - fed hamsters. in 2004, researchers from the schering - plough research institute identified niemann pick c1-like1 (npc1l1) protein as a molecular target of ezetimibe 1. ezetimibe 1 acts by blocking the internalization of npc1l1, thereby preventing cholesterol from entering the cytoplasm of enterocytes. a thorough structure activity relationship (sar) study of the -lactam cholesterol absorption inhibitors in cholesterol - fed hamsters revealed that the 2-azetidinone backbone as well as the aryl group at the n-1 and c-4 position of the -lactam ring are required for activity. the aryl group at the c-4 position of the -lactam ring is optimally substituted with alkoxy or hydroxy groups at the para - position. the side chain at the c-3 position of the -lactam ring with three linking atoms bearing a pendent aryl group is optimal. preferred configuration at the c-4 chiral center of the -lactam ring is s and the c-3 atom tolerates s or r configurations. introduction of a heteroatom at the 1-position of the c-3 side chain can also contribute to the activity, whereas isosteric groups at the 3-position of the side chain decrease the activity. in continuation of our research in the field of -lactam chemistry [1115 ] and taking into consideration the requirements determined by sar studies, we synthesized bioisosteres 5 and 6 (fig. 2) of ezetimibe 1 bearing a nh group at the c-3 position of the -lactam ring. bioisosterism is a useful approach for lead compound modification that can result in improved pharmacological activity, decreased toxicity, and optimized pharmacokinetics. with the classical bioisosteric exchange of the ch2 with a nh group we aimed at investigating whether the change in polarity of the side chain, the ability of additional h - bond, and ammonium salt formations would affect their cholesterol absorption inhibition and cytotoxicity. here we show that our new ezetimibe analogs 5, 6 and their diastereoisomeric mixture 5/6 (70:30) are potent novel cholesterol absorption inhibitors. we synthesized two novel ezetimibe analogs 5 and 6 (fig. 2) and their diastereoisomeric mixtures 5/6 (70:30) and 6/5 (85:15) from enantiomerically pure trans-(3r,4r)-amino--lactam 2 (fig. 3) and determined their in vitro and in vivo activities as cholesterol absorption inhibitors. we extensively studied the stereoselectivity of the side chain keto group reduction with cbs - catalyst in proximity of the nh group at the c-3 position of 2-azetidinone. enantiomeric purity of 2 (> 99% ee) was determined by the mosher 's mtpa method on f nmr (67.79 ppm, s, 3f, cf3 and -116.22 ppm, s, 1f, n there are two possible approaches for the synthesis of ezetimibe analogs 5 and 6 and their diasteroisomeric mixtures 5/6 and 6/5 from trans - amino--lactam 2 : (i) n - alkylation of trans - amino--lactam 2 with commercially available 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 followed by stereoselective reduction of the side chain keto group at the c-3 of the -lactam ring with cbs - catalyst or (ii) stereoselective reduction of the keto group of 3 preceding n - alkylation reaction. in the present study we applied both approaches.(i)n - alkylation of trans - amino--lactam n - alkylation of trans - amino--lactam 2 was performed in mild conditions using nai for in situ generation of 2-iodo - derivative of 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 in the presence of et3n at room temperature and provided 4 in a moderate yield (46%) (scheme 1a). a mixture of thf and dmf in ratio 9:1 was found optimal for the reaction. the c-3 side chain hydroxy group was obtained by stereoselective reduction of the keto group with cbs catalyst (scheme 1b) [1921 ]. however, addition of cbs - catalyst (0.1 eq.) and bh3me2s (1 eq.) provided the diastereoisomeric mixture 5/6 (50:50), determined by h nmr. ratio) had no effect on the stereoselectivity, providing mixture 5/6 (50:50). the absence of stereoselectivity in the reduction of amino--lactam ketone 4 was probably due to nitrogen proximity to the keto group and the ability of borane to form a complex with amine, which allowed a direct hydrogen delivery to the keto group without participation of a chiral catalyst. addition of bh3me2s (2 eq.) to cbs - catalyst (0.1 eq.) did not result in improvement of stereoselectivity in keto group reduction of 4 either. improvement of stereoselectivity was accomplished with the complex formation between cbs - catalyst and bh3me2s, (1:1 eq. reaction with (r)-cbs - catalyst provided a diastereoisomeric mixture of amino alcohols 5/6 (70:30) at 20 c, whereas (s)-cbs - catalyst afforded 6/5 (85:15) (fig. 4). lowering the temperature to 80 c did not improve the stereoselectivity of the reduction. recrystallization of 6/5 (85:15) provided pure amino alcohol 6.(ii)stereoselective reduction of ketone 3 followed by n - alkylation. n - alkylation of trans - amino--lactam 2 with ketone 3 and subsequent reduction of the side chain keto group. n - alkylation of trans - amino--lactam 2 was performed in mild conditions using nai for in situ generation of 2-iodo - derivative of 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 in the presence of et3n at room temperature and provided 4 in a moderate yield (46%) (scheme 1a). a mixture of thf and dmf in ratio 9:1 was found optimal for the reaction. the c-3 side chain hydroxy group was obtained by stereoselective reduction of the keto group with cbs catalyst (scheme 1b) [1921 ]. however, addition of cbs - catalyst (0.1 eq.) and bh3me2s (1 eq.) provided the diastereoisomeric mixture 5/6 (50:50), determined by h nmr. ratio) had no effect on the stereoselectivity, providing mixture 5/6 (50:50). the absence of stereoselectivity in the reduction of amino--lactam ketone 4 was probably due to nitrogen proximity to the keto group and the ability of borane to form a complex with amine, which allowed a direct hydrogen delivery to the keto group without participation of a chiral catalyst. addition of bh3me2s (2 eq.) to cbs - catalyst (0.1 eq.) did not result in improvement of stereoselectivity in keto group reduction of 4 either. improvement of stereoselectivity was accomplished with the complex formation between cbs - catalyst and bh3me2s, (1:1 eq. reaction with (r)-cbs - catalyst provided a diastereoisomeric mixture of amino alcohols 5/6 (70:30) at 20 c, whereas (s)-cbs - catalyst afforded 6/5 (85:15) (fig. 4). lowering the temperature to 80 c did not improve the stereoselectivity of the reduction. commercially available ketone 3 was reduced following the original protocol for cbs - reduction developed by corey. and yielded alcohols 7a and 7b in > 99% ee (scheme 2). protection of the oh group in 7a, b with tbdmscl was carried out in dmf in the presence of imidazole to afford otbdms bromo derivatives 8a and 8b. exchange of bromine in 8a, b with iodine (in the presence of nai in acetone at 55 c) yielded otbdms iodo derivatives 9a and 9b. iodo - bromo exchange proceeded very slowly, providing the mixtures of 9a or 9b and unreacted 8a or 8b, respectively, in ratio 93:7 and 96% yield after 4 days. this mixture was used in the n - alkylation reaction of amino--lactam 2 without further purification. the reaction proceeded for 7 days in ch3cn to afford silyl intermediates 10a and 10b with a moderate yield (20%). silyl intermediates 10a, b were further deprotected with 3% hcl in ethanol to produce ezetimibe bioisosteres 5 and 6 (scheme 2). the crystal structure of (3r,4r)-3-[(2s)-2-(4-fluorophenyl)-2-hydroxyethylamino]-1-(4-fluorophenyl)-4-(4-hydroxyphenyl)azetidin-2-one (2s,3r,4r)-6 was determined to establish unambiguously both absolute and relative configurations at the stereogenic centres c17 and n2. two symmetry independent molecules of 6, related by a pseudo - twofold rotation axis, are present in the crystal structure designated as 6a and 6b (fig. 5) ; they are homochiral and of similar conformations (fig. 6). configuration at the stereogenic centre c17 was assigned to be s in both 6a and 6b, according to the known r - configurations at the stereogenic centres c2 and c3. 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 c hydrogen bonds into c2-symmetric dimers (fig. 7, each molecule has two hydroxy groups, which act as proton donors toward symmetry - equivalent molecules ; one toward a carbonyl and one toward another hydroxy group, giving a total of four o ho hydrogen bonds (table 1) that link the dimers into layers parallel to (011). we measured an increase of the absorbance intensity at 247 nm by the addition of naoh in the ph range 612 (fig. pka values calculated from the obtained sigmoidal curves for amino alcohol 5 (fig. we got the same pka value for amino alcohol 6 (data not shown), indicating that both compounds are present in the form of nh4 in the blood and small intestine. cytotoxicity of amino alcohols 5, 6 and their diastereoisomeric mixture 5/6 was analyzed using mtt cell proliferation assay and the lc50 values were determined in mdckii wild type, hnpc1l1/mdckii, and hepg2 cells (table 2). mdckii cells stably expressing human niemann - pick c1-like protein 1 (hnpc1l1/mdckii) (figure s1) are a pharmacologically validated system for investigating npc1l1-mediated cholesterol uptake. the lc50 values were higher than 100 m and considered nontoxic in all three cell lines. lc50 values for ezetimibe 1 were 62.29 m in hnpc1l1/mdckii and 69.74 m in hepg2 cells. in mdckii wild type cells, ezetimibe 1 showed no toxicity (table 2). in addition, we tested the in vitro cytotoxicity of ezetimibe 1 and compounds 5, 6, and 5/6 (70:30) in combination with micelles and found all lc50 values to be above 100 m (figure s2). first we tested ezetimibe analogs 5 and 6 and their diastereoisomeric mixtures 5/6 (70:30) and 6/5 (85:15) for their ability to inhibit cholesterol uptake. in mdckii wildtype cells, ezetimibe 1 had no effect, but inhibited cholesterol uptake in hnpc1l1/mdckii cells (figure s3). when hnpc1l1/mdckii were treated with 5 (fig. 10d), we observed 5055% inhibition of cholesterol uptake, reaching its maximum at 120 m concentration without a significant difference between the compounds. these results show that the novel ezetimibe bioisosteres 5, 6 and their diastereoisomeric mixtures 5/6 and 6/5 are potent inhibitors of cholesterol uptake in vitro. in vivo, we first determined the consequences of ezetimibe 1 and amino alcohols 5 and 6 on cholesterol absorption. we therefore gavaged mice with 2 ci [h]cholesterol and 10 mg / kg / day of each compound or vehicle, and radioactivity was measured in plasma, liver, and three equal parts of the intestine (duodenum, jejunum, ileum). both 5 and 6 showed significant inhibition of cholesterol absorption (table 3). treatment of mice with either 5 and 6 resulted in reductions of [h]cholesterol in plasma by 50% and 32%, respectively. radioactivities in the liver were decreased by 44% and 47%, respectively, and were comparable to ezetimibe 1. amino alcohol 6 markedly lowered [h]cholesterol in the small intestine with highest inhibition in the ileum by 5860%, whereas amino alcohol 5 reduced radioactivity in the small intestine by 22% without reaching statistical significance in either part of the intestine. we therefore increased the dose to 20 mg / kg / day and determined inhibition of cholesterol absorption. the inhibition in duodenum and jejunum was similar to the lower dose, whereas in ileum it raised from 21% to 41% (table 3). decreased cholesterol absorption of 5 in the small intestine compared to ezetimibe 1 might be explained by the fact that 2-azetidinones are moderate acyl - coenzyme a : cholesterol acyltransferase inhibitors and their level of activity is highly structure - dependent. it might therefore be speculated that acyl - coenzyme a : cholesterol acyltransferase activity of amino alcohols 5 and 6 and their diastereoisomeric mixtures is altered compared to ezetimibe 1. the effect of the diastereoisomeric mixture 5/6 (70:30) on cholesterol absorption was comparable to ezetimibe 1 in plasma and ileum, lower in duodenum and jejunum, and higher in the liver (table 3). this report demonstrates that we have successfully synthesized two novel ezetimibe bioisosteres 5, 6 and their diastereoisomeric mixtures 5/6 (70:30) and 6/5 (85:15) from enantiomerically pure trans-(3r,4r)-amino--lactam 2. crystal structure of 6 was determined to establish unambiguously both absolute and relative configurations at the new stereogenic centre c17 and were assigned to be s. both diastereoisomeres 5 and 6 as well as their diastereoisomeric mixture 5/6 showed significant cholesterol absorption inhibitory activity both in vitro and in vivo. based on our data and the pka value for 5 and 6 being 9.35, indicating that both compounds are present in the form of nh4 in the blood and small intestine, other diastereoisomeric mixtures (e.g. 6/5 (85:15)) may exhibit similar in vivo results as 5/6 (70:30). results from this study implicate a therapeutic potential of these novel compounds to reduce cholesterol plasma concentrations and improve chd. the ir spectra were recorded on a perkinelmer spectrum rx i ft - ir system spectrometer (kbr pellets technique) (perkinelmer instruments, norwalk, ct, usa). the h and c nmr spectra (in cdcl3 and dmso - d6 at rt) were measured on a bruker av 300 and/or av 600 spectrometer (bruker biospin gmbh., rheinstetten, germany), is given in ppm relative to tetramethylsilane as an internal reference. microanalysis was performed on a pe 2400 series ii chns / o analyzer (perkinelmer instruments, shelton, ct, usa). optical rotations : optical activity automatic polarimeter aa-10 in 1 dm cell ; c in g/100 ml (optical activity ltd., ramsey, england). column chromatography on silica gel, 70230 mesh, 60 (sigma aldrich, st. louis, mo, usa or acros - organics, new jersey, usa) was performed at rt. thin layer chromatography was carried out on tlc aluminium sheets, 20 20 cm, silica gel 60 f254 (merck, darmstadt, germany). uv / vis measurements were performed on a t80 + spectrophotometer (pg instruments limited, england). samples for hr - ms analysis were resuspended in 5 l of thap / dac matrix and 1 l was spotted onto a maldi plate. mass spectra were obtained on a matrix - assisted laser desorption / ionization - time - of - flight maldi - tof / tof mass spectrometer (4800 plus maldi - tof / tof analyzer, applied biosystems, foster city, ca, usa) equipped with nd : yag laser operating at 355 nm with firing rate 200 hz in the positive ion reflector mode. 1600 shots per spectrum were taken covering mass range 1001000 da, focus mass 500 da and delay time 100 ns. crystals of amino alcohol (2s,3r,4r)-6 suitable for data collection were grown from ethyl acetate and hexane by vapour diffusion. the selected crystal was a needle with dimensions 0.10 0.03 0.02 mm. single crystal measurement was performed on an oxford diffraction xcalibur nova r (microfocus cu tube, oxford diffraction, u.k.) at room temperature [293(2) k ]. the model was refined using the full - matrix least squares refinement ; all non - hydrogen atoms were refined anisotropically. hydrogen atoms bound to c and o were modelled as riding entities using the afix command. hydrogen atoms bound to n2a and n2b could not be located from the electron density map due to the data poor quality. however, it can be assumed that they are directed towards the nearest proton acceptor : the carbonyl oxygen of the neighbouring molecule. therefore, they were generated at the appropriate positions and refined using appropriate restraints (table 1). configuration of novel stereogenic centre c17 was established relative to the configurations of the known stereogenic centres c2 and c3. the structure comprises relatively large cavities filled by disordered water molecules. due to a very low electron density and low data quality (due to small size of the crystal) the disordered water was tentatively modelled as four isotropic oxygen atoms (hydrogens could not have been located) with occupancy of 0.5. molecular geometry calculations were performed by platon, and molecular graphics were prepared using ortep-3, and ccdc - mercury. crystallographic and refinement data for amino alcohol (2s,3r,4r)-6 reported in this paper are shown in table 4. supplementary crystallographic data for this paper can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the cambridge crystallographic data centre, 12, union road, cambridge cb2 1ez, uk ; fax : (+) 44 1223 336033 ; or [email protected]). amino--lactam 2 was synthesized following the procedure described by ojima. and habu. and purified by a silica gel column chromatography (hexane / ethyl acetate 9:1, gradually increasing the content of ethyl acetate in eluent to pure ethyl acetate) and obtained as yellow oil (1.3 g, 43%). []d = 26 (c = 1, etoac). ft - ir (kbr) cm : 2931, 2858, 1739, 1609, 1510, 1390, 1266, 913, 834, 513. h nmr (300 mhz, cdcl3) : 0.19 (s, 6h, si-(ch3)2), 0.97 (s, 9h, c-(ch3)3), 1.81 (bs, 2h, nh2), 4.05 (d, 1h, j = 2.2 hz, c4 -lactam), 4.58 (d, 1h, j = 2.1 hz, c3 -lactam), 6.83 (d, 2h, j = 8.5 hz, ar h), 6.92 (t, 2h, j1,2 = 8.7 hz, ar h), 7.18 (d, 2h, j = 8.5 hz, ar h), 7.237.26 (m, 2h, ar h) ; c nmr (150 mhz, cdcl3) : 4.27 (si-(ch3)2), 18.32 (c-(ch3)3), 25.75 (c-(ch3)3), 66.69 (c4 -lactam), 70.17 (c3 -lactam), 115.95 (d, j = 22.8 hz, 4-f - c6h4), 118.98 (d, j = 7.8 hz, 4-f - c6h4), 120.86 (4-otbdms - c6h4), 127.31 (4-otbdms - c6h4), 129.18 (4-otbdms - c6h4), 133.83 (d, j = 2.2 hz, 4-f - c6h4), 156.25 (4-otbdms - c6h4), 159.26 (d, j = 243.9 hz, 4-f - c6h4), 168.02 (co, -lactam). hrms for c21h27fn2o2si (mr = 386.53518) : calcd. m / z [m+h ] 387.1898, found 387.1901. to a solution of 2 (159 mg, 0.41 mmol) in a mixture of anhydrous thf and dmf (9:1, 7.8 ml), 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 (98 mg, 0.45 mmol), sodium iodide (68 mg, 0.45 mmol) and et3n (114 l, 0.82 mmol) were added. the reaction mixture was stirred at room temperature for 3 h after which solvent was evaporated to dryness. distilled water (10 ml) was added and the product extracted with ethyl acetate (3 20 ml). 4 was purified by a silica gel column chromatography (hexane / ethyl acetate 2:1) and obtained as light brown oil (99 mg, 46%). []d = + 20 (c = 1, etoac). ft - ir (kbr) cm : 3332, 2931, 1748, 1693, 1600, 1506, 1385, 1260, 1156, 913, 834, 515. h nmr (600 mhz, cdcl3) : 0.18 (s, 6h, si-(ch3)2), 0.96 (s, 9h, c-(ch3)3), 1.60 (bs, 1h, nh), 4.09 (d, 1h, j = 2.1 hz, c4 -lactam), 4.26 and 4.34 (abq, 2h, jab = 18.5 hz, c(=o)ch2nh), 4.80 (d, 1h, j = 1.9 hz, c3 -lactam), 6.80 (d, 2h, j = 8.5 hz, ar h), 6.93 (t, 2h, j1,2 = 8.6 hz, ar h), 7.127.16 (m, 4h, ar h) ; c nmr (75 mhz, cdcl3) : 4.28 (si-(ch3)2), 18.30 (c-(ch3)3), 25.74 (c-(ch3)3), 53.00 (c(o)ch2nh), 63.79 (c4 -lactam), 75.44 (c3 -lactam), 115.98 (d, j = 22.6 hz, 4-f - c6h4), 116.16 (d, j = 21.7 hz, 4-f - c6h4), 119.06 (d, j = 7.9 hz, 4-f - c6h4), 120.87 (4-otbdms - c6h4), 127.30 (4-otbdms - c6h4), 129.12 (4-otbdms - c6h4), 130.62 (d, j = 9.4 hz, 4-f - c6h4), 131.57 (d, j = 3.3 hz, 4-f - c6h4), 133.68 (d, j = 2.8 hz, 4-f - c6h4), 156.19 (4-otbdms - c6h4), 159.31 (d, j = 243.9 hz, 4-f - c6h4), 166.17 (co, -lactam), 166.23 (d, j = 254.8 hz, 4-f - c6h4), 195.58 (co). the reaction was carried out in dry conditions under argon atmosphere. to a solution of (r)-(+)-2-methyl - cbs - oxazaborolidine catalyst (251 mg, 0.91 mmol) in anhydrous thf (2 ml), 2 m thf solution of bh3me2s (454 l, 0.91 mmol) was added and the mixture stirred at room temperature for 10 min. the temperature was then lowered to 20 c and a solution of 4 (474 mg, 0.91 mmol) in anhydrous thf (5 ml) added dropwise. after 24 h the reaction mixture was warmed to room temperature and quenched with methanol. the solution was concentrated in vacuo, distilled water (15 ml) added and the product extracted with ethyl acetate (3 40 ml). collected organic layers were dried over na2so4 and solvent evaporated to dryness. crude product was dissolved in ethanol (22 ml) and 1 m hcl (5 ml) added. reaction mixture was stirred at room temperature overnight, solvent evaporated to dryness and crude product purified by a silica gel column chromatography (hexane / ethyl acetate 1:3) to afford diastereoisomeric mixture 5/6 (70:30) (283 mg, 76%). ft - ir (kbr) cm : 3341, 1736, 1615, 1601, 1509, 1390, 1223, 1154, 1101, 832. h nmr (600 mhz, dmso - d6) : 2.722.83 (m, 2h, ch(oh)ch2nh, 5/6), 2.93 (bs, 1h, ch(oh)ch2nh, 5/6), 3.97 (s, 1h, c4 -lactam, 5/6), 4.554.57 (m, 1h, ch(oh)ch2nh, 6), 4.594.62 (m, 1h, ch(oh)ch2nh, 5), 4.83 (d, 1h, j = 2.0 hz, c3 -lactam, 5/6), 5.30 (d, 1h, j = 4.3 hz, ch(oh)ch2nh, 5), 5.33 (d, 1h, j = 4.3 hz, ch(oh)ch2nh, 6), 6.736.75 (m, 2h, ar h, 5/6), 9.43 (s, 1h, ar oh, 5/6) ; c nmr (150 mhz, dmso - d6) : 54.53 (ch(oh)ch2nh, 5), 54.78 (ch(oh)ch2nh, 6), 62.36 (c4 -lactam, 5), 62.58 (c4 -lactam, 6), 71.43 (ch(oh)ch2nh, 5), 71.61 (ch(oh)ch2nh, 6), 75.43 (c3 -lactam, 5), 75.63 (c3 -lactam, 6), 114.60 (d, j = 20.9 hz, 4-f - c6h4, 5), 114.62 (d, j = 21.2 hz, 4-f - c6h4, 6), 115.68 (4-oh - c6h4, 5/6), 115.79 (d, j = 22.7 hz, 4-f - c6h4, 5/6), 118.68 (d, j = 7.7 hz, 4-f - c6h4, 5/6), 127.40 (4-oh - c6h4, 6), 127.48 (4-oh - c6h4, 5), 127.52 (4-oh - c6h4, 5), 127.58 (4-oh - c6h4, 6), 127.85 (d, j = 7.8 hz, 4-f - c6h4, 5/6), 133.86 (d, j = 1.9 hz, 4-f - c6h4, 5/6), 140.33 (d, j = 2.7 hz, 4-f - c6h4, 6), 140.36 (d, j = 2.8 hz, 4-f - c6h4, 5), 157.34 (4-oh - c6h4, 5/6), 158.12 (d, j = 240.3 hz, 4-f - c6h4, 5/6), 161.19 (d, j = 242.4 hz, 4-f - c6h4, 5/6), 167.06 (co, -lactam, 5), 167.08 (co, -lactam, 6). to a solution of (s)-()-2-methyl - cbs - oxazaborolidine catalyst (275 mg, 0.99 mmol) in anhydrous thf (2 ml), 2 m thf solution of bh3me2s (496 l, 0.99 mmol) and 4 (519 mg, 0.99 mmol) in anhydrous thf (5 ml) were added. diastereoisomeric mixture 6/5 (85:15) (278 mg, 68%) was obtained. amino alcohol 6 was obtained (111 mg, 25%) by recrystallization of diastereoisomeric mixture 6/5 (85:15) in ch2cl2. []d = + 9 (c = 1, etoac). ft - ir (kbr) cm : 3344, 1723, 1615, 1510, 1394, 1233, 1155, 829. h nmr (300 mhz, dmso - d6) : 2.702.86 (m, 2h, ch(oh)ch2nh), 3.08 (bs, 1h, ch(oh)ch2nh), 3.97 (d, 1h, j = 1.9 hz, c4 -lactam), 4.544.59 (m, 1h, ch(oh)ch2nh), 4.84 (d, 1h, j = 1.9 hz, c3 -lactam), 5.38 (d, 1h, j = 4.3 hz, ch(oh)ch2nh), 6.75 (d, 2h, j = 8.5 hz, ar oh) ; c nmr (75 mhz, dmso - d6) : 54.76 (ch(oh)ch2nh), 62.57 (c4 -lactam), 71.56 (ch(oh)ch2nh), 75.57 (c3 -lactam), 114.58 (d, j = 21.1 hz, 4-f - c6h4), 115.65 (4-oh - c6h4), 115.74 (d, j = 22.6 hz, 4-f - c6h4), 118.63 (d, j = 8.0 hz, 4-f - c6h4), 127.34 (4-oh - c6h4), 127.52 (4-oh - c6h4), 127.78 (d, j = 8.1 hz, 4-f - c6h4), 133.85 (d, j = 2.5 hz, 4-f - c6h4), 140.25 (d, j = 2.8 hz, 4-f - c6h4), 157.33 (4-oh - c6h4), 158.08 (d, j = 240.7 hz, 4-f - c6h4), 161.17 (d, j = 242.2 hz, 4-f - c6h4), 167.02 (co, -lactam). for the synthesis of 7a, 2 m thf solution of bh3me2s (1.15 ml, 2.30 mmol) was added to a solution of (r)-(+)-2-methyl - cbs - oxazaborolidine catalyst (64 mg, 0.23 mmol) in anhydrous thf (2 ml). the mixture was stirred for 10 min and a solution of 2-bromo-1-(4-fluorophenyl)ethan-1-one 3 (500 mg, 2.30 mmol) in anhydrous thf (4 ml) was added dropwise. reaction mixture was quenched with methanol, solvent evaporated to dryness and distilled water (15 ml) added. the product was extracted with ch2cl2 (3 30 ml), collected organic layers dried over na2so4 and solvent evaporated to dryness. 7a was purified by a silica gel column chromatography (hexane / ethyl acetate 6:1) and obtained as colourless oil (503 mg, 100%). the ee>99% of 7a was determined by chiral hplc (200/4 nucleodex beta - pm column, macherey nagel, germany, method : 0.1% teaa in h2o : methanol 55 : 45, 30 min, flow 0.7 ml / min, = 254 nm, retention time 19.5 min).. 7b was synthesized following the same procedure using (s)-()-2-methyl - cbs - oxazaborolidine catalyst. []d = + 28 (c = 1, etoac). ft - ir (kbr) cm : 3401, 2963, 2897, 1896, 1605, 1513, 1306, 1224, 1158, 1067, 992, 838, 779, 645, 556, 523. h nmr (300 mhz, cdcl3) : 2.75 (bs, 1h, ch(oh)ch2), 3.463.61 (m, 2h, ch(oh)ch2), 4.89 (dd, 1h, j1 = 8.7 hz, j2 = 3.5 hz, ch(oh)ch2), 7.05 (t, 2h, j1,2 = 8.6 hz, ar h) ; c nmr (150 mhz, cdcl3) : 40.23 (ch(oh)ch2), 73.33 (ch(oh)ch2), 115.76 (d, j = 21.6 hz, 4-f - c6h4), 127.88 (d, j = 8.2 hz, 4-f - c6h4), 136.25 (d, j = 3.1 hz, 4-f - c6h4), 162.86 (d, j = 247.0 hz, 4-f - c6h4). mr = 219.05) : c, 43.86 ; h, 3.68. found : c, 43.63 ; h 4.01. for the synthesis of 8a a solution of imidazole (254 mg, 3.74 mmol) in dmf (0.5 ml) was added to a solution of 7a (327 mg, 1.49 mmol) in dmf (1 ml) and stirred for 10 min, followed by dropwise addition of tbdmscl (293 mg, 1.94 mmol) solution in dmf (1.3 ml). solvent was evaporated to dryness, distilled water (15 ml) added and the resulting mixture extracted with ethyl acetate (3 30 ml). 8a was purified by a silica gel column chromatography (hexane / ethyl acetate 6:1) and obtained as colourless oil (452 mg, 91%). []d = 46 (c = 1, etoac). 8b was synthesized following the same procedure starting from 7b (424 mg, 1.94 mmol) and obtained as colourless oil (602 mg, 94%). []d = + 46 (c = 1, etoac). ft - ir (kbr) cm : 2957, 2930, 2887, 2858, 1606, 1509, 1463, 1417, 1362, 1257, 1226, 1155, 1113, 1012, 914, 834, 778, 724, 646. h nmr (300 mhz, cdcl3) : 0.09 (s, 3h, si ch3), 0.10 (s, 3h, si ch3), 0.88 (s, 9h, c-(ch3)3), 3.363.48 (m, 2h, ch(otbdms)-ch2), 4.82 (dd, 1h, j1 = 7.4 hz, j2 = 4.8 hz, ch(otbdms)-ch2), 7.02 (t, 2h, j1,2 = 8.7 hz, ar h), 7.287.33 (m, 2h, ar h) ; c nmr (75 mhz, cdcl3) : 4.75 (si ch3), 4.60 (si ch3), 18.35 (c-(ch3)3), 25.86 (c-(ch3)3), 39.48 (ch(otbdms)-ch2), 74.76 (ch(otbdms)-ch2), 115.39 (d, j = 21.6 hz, 4-f - c6h4), 127.98 (d, j = 8.1 hz, 4-f - c6h4), 138.20 (d, j = 3.2 hz, 4-f - c6h4), 162.59 (d, j = 245.8 hz, 4-f - c6h4). anal. calcd. for c14h22brfosi (mr = 333.31) : c, 50.45 ; h, 6.65. reaction was carried out in a reaction flask protected from light. to a solution of 8a (113 mg, 0.34 mmol) in acetone (3 ml), nai (254 mg, 1.68 mmol) was added. the reaction proceeded at 55 c for 4 days after which distilled water (15 ml) was added and product extracted with ethyl acetate (3 30 ml). collected organic layers were dried over na2so4 and solvent evaporated to dryness. thus obtained crude product was purified by a silicagel column chromatography (hexane). compound 9a was obtained as a mixture of 9a and unreacted 8a (93:7) as light brown oil (124 mg, 96%). 9b was synthesized following the same procedure starting from 8b (75 mg, 0.22 mmol) and nai (168 mg, 1.12 mmol) to give after a silicagel column chromatography (hexane) a mixture of 9b and unreacted 8b (94:6) as light brown oil (39 mg, 46%). ft - ir (kbr) cm : 3448, 2956, 2930, 2887, 2858, 1605, 1509, 1463, 1408, 1362, 1257, 1224, 1106, 997, 887, 837, 776. h nmr (300 mhz, cdcl3) : 0.13 (s, 3h, si ch3), 0.10 (s, 3h, si ch3), 0.89 (s, 9h, c-(ch3)3), 3.283.30 (m, 2h, ch(otbdms)-ch2), 4.73 (t, 1h, j1,2 = 6.0 hz, ch(otbdms)-ch2), 7.00 (t, 2h, j1,2 = 8.7 hz, ar 4.68 (si ch3), 4.56 (si ch3), 15.03 (ch(otbdms)-ch2), 18.36 (c-(ch3)3), 25.93 (c-(ch3)3), 74.65 (ch(otbdms)-ch2), 115.38 (d, j = 21.5 hz, 4-f - c6h4), 127.80 (d, j = 8.2 hz, 4-f - c6h4), 138.87 (d, j = 3.1 hz, 4-f - c6h4), 162.51 (d, j = 246.0 hz, 4-f - c6h4). reaction was carried out in a reaction flask protected from light. to a solution of 2 (154 mg, 0.40 mmol) in ch3cn (3 ml) 9a (151 mg, 0.40 mmol) was added. the reaction proceeded at 80 c for 7 days after which the solvent was evaporated to dryness. compound 10a was purified by a silica gel column chromatography (hexane / ethyl acetate 6:1) and obtained as light brown oil (52 mg, 20%). []d = 9 (c = 1, etoac). ft - ir (kbr) cm : 3480, 2931, 2858, 1748, 1607, 1505, 1464, 1385, 1259, 1226, 1101, 1006, 914, 834, 515. h nmr (300 mhz, cdcl3) : 0.15 (s, 3h, si ch3), 0.02 (s, 3h, si ch3), 0.18 (s, 6h, si-(ch3)2), 0.86 (s, 9h, c-(ch3)3), 0.96 (s, 9h, c-(ch3)3), 2.01 (bs, 1h, ch(otbdms)ch2nh), 2.782.99 (m, 2h, ch(otbdms)-ch2-nh), 4.00 (d, 1h, j = 2.1 hz, c4 -lactam), 4.61 (d, 1h, j = 1.8 hz, c3 -lactam), 4.77 (dd, 1h, j1 = 7.8 hz, j2 = 4.3 hz, ch(otbdms)ch2nh), 6.81 (d, 2h, j = 8.7 hz, ar h), 6.91 (t, 2h, j1,2 = 8.8 hz, ar h), 6.99 (t, 2h, j1,2 = 8.8 hz, ar h), 7.13 (d, 2h, j = 8.5 hz, ar h), 7.217.30 (m, 4h, ar 4.75 (si ch3), 4.41 (si ch3), 4.28 (si-(ch3)2), 18.29 (c-(ch3)3), 25.73 (c-(ch3)3), 25.96 (c-(ch3)3), 55.69 (ch(otbdms)-ch2-nh), 63.86 (c4 -lactam), 74,18 (ch(otbdms)ch2nh), 75.73 (c3 -lactam), 115.24 (d, j = 21.3 hz, 4-f - c6h4), 115.90 (d, j = 22.6 hz, 4-f - c6h4), 118.92 (d, j = 7.9 hz, 4-f - c6h4), 120.84 (4-otbdms - c6h4), 127.23 (4-otbdms - c6h4), 127.95 (d, j = 8.0 hz, 4-f - c6h4), 129.40 (4-otbdms - c6h4), 133.78 (d, j = 2.7 hz, 4-f - c6h4), 138.86 (d, j = 3.0 hz, 4-f - c6h4), 156.11 (4-otbdms - c6h4), 159.20 (d, j = 243.6 hz, 4-f - c6h4), 162.33 (d, j = 245.5 hz, 4-f - c6h4), 166.38 (co, -lactam). 10b was synthesized following the procedure for 10a and obtained as light brown oil (9 mg, 19%). []d = + 16 (c = 1, etoac). ft - ir (kbr) cm : 3439, 2929, 2857, 1749, 1608, 1506, 1472, 1385, 1259, 1101, 1012, 913, 833, 516. h nmr (300 mhz, cdcl3) : 0.14 (s, 3h, si ch3), 0.04 (s, 3h, si ch3), 0.18 (s, 6h, si-(ch3)2), 0.86 (s, 9h, c-(ch3)3), 0.96 (s, 9h, c-(ch3)3), 1.80 (bs, 1h, ch(otbdms)ch2nh), 2.802.96 (m, 2h, ch(otbdms)-ch2-nh), 4.02 (d, 1h, j = 2.0 hz, c4 -lactam), 4.62 (d, 1h, j = 2.0 hz, c3 -lactam), 4.81 (dd, 1h, j1 = 7.6 hz, j2 = 4.3 hz, ch(otbdms)ch2nh), 6.81 (d, 2h, j = 8.6 hz, ar h), 6.90 (t, 2h, j1,2 = 8.8 hz, ar h), 6.99 (t, 2h, j1,2 = 8.7 hz, ar h), 7.12 (d, 2h, j = 8.5 hz, ar h), 7.207.31 (m, 4h, ar 4.72 (si ch3), 4.39 (si ch3), 4.27 (si-(ch3)2), 18.30 (c-(ch3)3), 25.74 (c-(ch3)3), 25.97 (c-(ch3)3), 55.68 (ch(otbdms)-ch2-nh), 63.35 (c4 -lactam), 74,25 (ch(otbdms)ch2nh), 75.82 (c3 -lactam), 115.25 (d, j = 21.5 hz, 4-f - c6h4), 115.91 (d, j = 22.8 hz, 4-f - c6h4), 118.94 (d, j = 7.9 hz, 4-f - c6h4), 120.84 (4-otbdms - c6h4), 127.25 (4-otbdms - c6h4), 127.96 (d, j = 8.0 hz, 4-f - c6h4), 129.35 (4-otbdms - c6h4), 133.77 (d, j = 2.8 hz, 4-f - c6h4), 138.89 (d, j = 3.0 hz, 4-f - c6h4), 156.13 (4-otbdms - c6h4), 159.22 (d, j = 243.0 hz, 4-f - c6h4), 162.34 (d, j = 245.4 hz, 4-f - c6h4), 166.36 (co, -lactam). compound 10a (24 mg, 0.04 mmol) was dissolved in 3% hcl in ethanol (1.59 ml) and the mixture was stirred at room temperature overnight. solvent was evaporated to dryness and crude product purified by a silica gel column chromatography (hexane / ethyl acetate 1:3, 5% meoh). product 5 was obtained as light yellow powder (13 mg, 84%). []d = 18 (c = 1, etoac). ft - ir (kbr) cm : 3448, 1735, 1617, 1509, 1389, 1225, 1102, 833. h nmr (300 mhz, dmso - d6) : 2.752.77 (m, 2h, ch(oh)ch2nh), 2.95 (bs, 1h, ch(oh)ch2nh), 3.97 (bs, 1h, c4 -lactam) 4.584.63 (m, 1h, ch(oh)ch2nh), 4.84 (d, 1h, j = 2.0 hz, c3 -lactam), 5.33 (d, 1h, j = 4.4 hz, ch(oh)ch2nh), 6.74 (d, 2h, j = 8.5 hz, ar oh) ; c nmr (75 mhz, dmso - d6) : 54.55 (ch(oh)ch2nh), 62.34 (c4 -lactam), 71.45 (ch(oh)ch2nh), 75.47 (c3 -lactam), 114.60 (d, j = 21.0 hz, 4-f - c6h4), 115.66 (4-oh - c6h4), 115.80 (d, j = 21.7 hz, 4-f - c6h4), 118.65 (d, j = 8.0 hz, 4-f - c6h4), 127.49 (4-oh - c6h4), 127.52 (4-oh - c6h4), 127.84 (d, j = 8.1 hz, 4-f - c6h4), 133.87 (d, j = 2.3 hz, 4-f - c6h4), 140.40 (d, j = 2.7 hz, 4-f - c6h4), 157.31 (4-oh - c6h4), 158.08 (d, j = 240.7 hz, 4-f - c6h4), 161.17 (d, j = 241.6 hz, 4-f - c6h4), 167.10 (co, -lactam). stock solution of amino alcohols 5 and 6 (160 l, 2 mg / ml methanol) was added to nacl solution in water (20 ml, 0.1 mol / l), thus resulting in a solution of each amino alcohol 5 and 6 in concentration of 4 10 furthermore, solutions of naoh (0.1 mol / l, 1 mol / l and 2 mol / l) were prepared in nacl solution in water (100 ml, 0.1 mol / l). naoh solutions (1.520 l) were added to each solution of amino alcohol 5 or 6, ph values measured and uv spectra (200350 nm) recorded at ph 612. the titrations were performed at rt (20 c) and inflection points of sigmoid curves at 247 nm were calculated as pka values for both amino alcohols. darby canine kidney ii (mdckii) cells, mdckii cells stably expressing human npc1l1 (hnpc1l1/mdckii), and human hepg2 liver cells were maintained in dmem (dulbecco 's modified eagle medium, invitrogen, usa) containing 100 units / ml penicillin and 100 g / ml streptomycin supplemented with 10% fcs (sigma aldrich, germany). stable transfection of mdckii cells with human npc1l1 protein was performed using lipofectamine ltx (invitrogen, usa) according to the manufacturer 's protocol. selection with 1 mg / ml g418 (sigma aldrich, germany) was started 2 days after transfection. the concentration of g418 was decreased to 500 g / ml after 14 days. stably transfected cells were maintained in dmem containing 100 units / ml penicillin and 100 g / ml streptomycin supplemented with 10% fcs and 500 g / ml g418. mdckii, hnpc1l1/mdckii, and hepg2 cells were seeded in 96-well microtiter plates on day 0 at densities of 3000 cells / well. on day 1, tested compounds (73 mm stock solution in dmso) were added in five consecutive 10-fold dilutions (10 to 10 mol / l) and incubated for 72 h. cytotoxicity of the compounds was assessed on day 4 by the mtt cell proliferation assay (sigma aldrich, germany) which detects mitochondrial dehydrogenase activity in viable cells. the results are expressed as lc50 and the values for each compound were calculated from dose response curves using linear regression analysis. toxicity of the compounds in combination with micelles was also tested using the same method. mdckii and hnpc1l1/mdckii were seeded in 96-well microtiter plates (150 l medium / well) at a density of 2 10 cells / ml medium 24 h before the experiment. micelles were prepared according to the modified method reported by field.. in brief, 0.25 mm oleic acid, 50 m cholesterol, 10 m compactin, 50 m mevalonate, 5 mm na - taurocholate (sigma aldrich, germany) in dmem/10% fcs were mixed and sonicated. following the micellar formation, compounds 1, 5, 6, and 5/6 (70:30) (73 mm stock solution in dmso) were added to the medium and vortexed to obtain the final concentrations of the compounds (25, 50, 100, 150, 200 m) and applied to the cells for 1 h. thereafter, cytotoxicity was determined using the mtt assay. medium a contained dmem plus 100 units / ml penicillin and 100 g / ml streptomycin supplemented with 5% lipoprotein - deficient serum (density > 1.21 g / ml, prepared from fcs by ultracentrifugation). cholesterol replenishing medium (medium b) was prepared according to the modified method reported by field.. in brief, 0.25 mm oleic acid, 50 m free cholesterol, 10 m compactin, 50 m mevalonate, 5 mm na - taurocholate (sigma aldrich, germany), and [h]cholesterol (0.18 ci / ml medium, [1,23h(n) ] cholesterol, 1 mci / ml (arc inc., usa)) in medium a were mixed and sonicated. following the micellar formation, compounds 1, 5, 6, 5/6 (70:30), and 6/5 (85:15) (73 mm stock solution in dmso) were added to medium b and vortexed to obtain the final concentrations (10, 30, 60, 90, 120, 150, 200 m). mdckii wildtype and hnpc1l1/mdckii cells were seeded in 24-well plates at a density of 5 10 cells / ml medium (500 l medium / well) in medium a 24 h before the treatment. cells were washed once with pbs and incubated in medium b (500 l medium / well) containing micelles and tested compounds in indicated concentrations for 1 h. following the incubation, the medium was removed and cells were washed three times with ice - cold 0.2% free fatty acid - free bsa. cells were lysed and radioactivity in the lysate (100 l) was determined by liquid scintillation counting. protein concentrations of the lysates were determined with the dc protein assay (bio - rad, usa). the results were calculated as cpm / mg protein and expressed as percentage of inhibition compared to untreated cells. male c57bl/6 mice aged 2528 weeks were maintained under a 12 h light/12 h dark cycle in a temperature - controlled environment with free access to chow diet (ssniff, germany) and water. for 2 days, mice (n = 35) were fasted for 4 h and gavaged with corn oil (100 l) (vehicle) containing ezetimibe 1 (10 mg / kg / day) or compounds 5, 6, and 5/6 (70:30) (10 or 20 mg / kg / day). on day 2, 90 min post vehicle or compound treatment, mice were gavaged with corn oil (200 l) containing [h]cholesterol (2 ci) ([1,23h(n) ] cholesterol, 1 mci / ml, arc inc., st. plasma, liver, and small intestines were collected 4 h post - gavage. the results are presented as mean s.d. or mean s.e.m. statistical analyses were performed using unpaired two - tailed t - test or one - way analysis of variance (anova) followed by dunnett 's test for multiple comparisons using graphpad prism 5.0 software (san diego, ca, usa).	two new trans-(3r,4r)-amino--lactam derivatives and their diastereoisomeric mixtures were synthesized as ezetimibe bioisosteres and tested in in vitro and in vivo experiments as novel -lactam cholesterol absorption inhibitors. both compounds exhibited low cytotoxicity in mdckii, hnpc1l1/mdckii, and hepg2 cell lines and potent inhibitory effect in hnpc1l1/mdckii cells. in addition, these compounds markedly reduced cholesterol absorption in mice, resulting in reduced cholesterol concentrations in plasma, liver, and intestine. we determined the crystal structure of one amino--lactam derivative to establish unambiguously both the absolute and relative configuration at the new stereogenic centre c17, which was assigned to be s. the pka values for both compounds are 9.35, implying that the amino--lactam derivatives and their diastereoisomeric mixtures are in form of ammonium salt in blood and the intestine. the ic50 value for the diastereoisomeric mixture is 60 m. in vivo, it efficiently inhibited cholesterol absorption comparable to ezetimibe.
we retrospectively surveyed 128 students at a coeducational, state secondary school in an urban area of west midlands, uk, where attack rates for pandemic (h1n1) 2009 were high and (as of march 2010) levels of unemployment were among the highest in great britain (7). the head teacher selected 1 class from each of years 710 (equivalent to us grades 69, student ages 1115 years) to participate. the school had closed for 1 week in mid - june 2009, reopened for 2 days, then closed for another week. an electronic version of a similar questionnaire pilot tested at another school had been found comprehensible and acceptable to participants. the london school of hygiene and tropical medicine ethics committee approved the study ; the health protection agency approved it as part of wider outbreak investigations not requiring additional approval. students reported how many times they visited specified public places before the school closure and how many times they visited these places during closure (children had been advised to not visit public places only if they were symptomatic). students also provided information about persons who looked after them during closure. for typical school days before and during closure, students reported the number of different persons spoken to (contacted) in the following groups : contacts who attended their school (contacts from the same class [classmates ], the same year but a different class [yearmates ], and the same school but a different year [schoolmates ]) and others (age stratified to reflect the uk school system). students were asked whether they were ill during closure and whether being ill affected their contact patterns. approximately 100 students (range 99103, depending on place visited) stated how frequently they would visit public places while school was open, and 46 stated how many times they visited these places during school closure ; 45 (98%) of 46 visited > 1 place. fewer students visited shops, places of worship, parks, and playing fields at least 1/week when school was closed than when open (figure 1). for other places, visits to public places during open and closure periods of a uk secondary school, june percentage of students visiting public places > 1/week while the school was open (n = 99103, depending on the place) and while it was closed (n = 46). numbers after bars show percentages in each group ; p values are from fisher exact tests comparing the proportions during the open versus closed periods. among those who provided information about caregivers, 93 (95%) of 98 reported that > 1 adult looked after them during school closure ; 49% reported having 2 caregivers (range 15). among caregivers for whom further information was available, 125 (69%) of 182 would have seen the student on a typical school day, 54 (31%) of 173 typically worked outside the home, and 12 (34%) of 35 took time off work to care for the student during school closure. among students, 73 provided number of contacts on a typical day during school closure ; 35 also provided information for a typical school day, and another 6 only provided information for a typical school day. we therefore conducted unpaired and paired analyses on data from 79 and 35 respondents, respectively. students who provided contact data were most likely to be in years 7 or 9 but were otherwise similar to those who did not. the mean totals of reported contacts were 70.3 (sd 40.8) and 24.8 (sd 22.5) during typical school days and closure, respectively (figure 2). school closure was therefore associated with a reduction of 45.5 (95% confidence interval [ci ] 33.857.2) in students typical daily number of contacts, a 65% relative reduction (95% bootstrap ci 52%73%). the corresponding absolute and relative reductions in numbers of contacts with other students were 37.0 (95% ci 27.046.9) and 65% (95% bootstrap ci 52%74%), respectively. the absolute and relative reductions in the numbers of contacts made with adults (including teachers) were 8.5 (95% ci 4.912.1) and 63% (95% bootstrap ci 45%75%), respectively. no apparent change was found for number of contacts with adults outside school (34%, 95% bootstrap ci 6% to 63%). number of contacts made by students with persons in different categories and the changes associated with school closures. a) total contacts overall and with students and adults ; b) contacts with persons in different categories at school and in different age groups outside school ; c) absolute reductions in numbers of contacts with persons in different groups associated with school closure ; d) relative reductions in numbers of contacts with persons in different groups associated with school closure. in (a) and (b), large black markers indicate the mean number of contacts ; small gray markers indicate individual data points ; circles indicate data for when the school was open (n = 41), crosses indicate data for when the school was closed (n = 73). in (c) and (d), error bars indicate 95% confidence intervals. the greatest reductions in the numbers of contacts were for students from the same school (figure 2), e.g., 80% reduction in numbers of contacts with classmates and yearmates. absolute reductions in numbers of contacts with persons not attending the school were small ; the relative reductions had wide confidence intervals and rarely showed evidence of a genuine reduction (figure 2). paired analysis of data for 35 students with information for contacts during both periods produced similar results as unpaired analysis. among 40 respondents who reported illness during closure and self - assessed whether they consequently contacted fewer persons, 53% stated that their contacts were reduced, 33% stated that they were not, and 15% were unsure. closing this school was associated with a 65% reduction in face - to - face conversational contacts made by secondary school students, primarily because of reductions in contact with students from the same school. our estimated reductions exceed estimates from analyses of surveillance data for seasonal influenza - like illness in france (24% reduction in child - to - child transmission during school holidays compared with in - school days) (8) and a study conducted in belgium (19% reduction in total contacts made by children and adolescents during easter holidays) (9). our estimate of a 65% reduction in total contacts is similar to that from a survey at a primary school in germany, in which students reported 72% fewer contacts on sundays than on weekdays but in which all classmates were considered contacts (10). consistent with findings of other studies (1113), most students visited public places during closure, although certain places were visited less frequently while the school was closed than when open. our definition of contact excluded nonconversational contacts (e.g., passengers on public transport), which may enable transmission, and some conversations may not involve close contact. we did not collect data about duration or intensity of contact or whether persons were contacted multiple times. for logistical reasons, a 23 week delay occurred between school reopening and completion of questionnaires, providing potential for recall bias and underestimation of numbers of contacts during school closure (although closure was unusual). prospective data collection was impossible and has limitations, including greater effort required from participants and therefore potentially lower response rates. the data refer to a convenience sample from 1 secondary school during what was often perceived as a mild pandemic and may not be generalizable to other situations (e.g., primary schools, different socioeconomic settings, infections with high case - fatality rates, or different seasons). most students provided data only for the closure period, and few did so for a typical school day (probably because of the order of questions). paired analysis of 35 students who provided data for both periods produced similar results to the unpaired analysis. other issues must also be considered when deciding whether to close schools (14). subject to the limitations described above, reactive school closures may substantially reduce the numbers of contacts made by students and may potentially reduce transmission of infection in some settings.	to determine how school closure for pandemic (h1n1) 2009 affected students contact patterns, we conducted a retrospective questionnaire survey at a uk school 2 weeks after the school reopened. school closure was associated with a 65% reduction in the mean total number of contacts for each student.
diabetes mellitus (dm) and chronic periodontitis are common chronic diseases in adults in the world population. dm is a complex disease with both metabolic and vascular components characterized by hyperglycemia due to defects in insulin secretion, insulin action or both as well as dysregulation of protein and lipid metabolism. dm is associated with a wide range of complications such as retinopathy, nephropathy, neuropathy, micro and macrovascular disease and altered wound healing. also, as per of american diabetes association in 1993, periodontal disease is sixth most common complication in patients of diabetes mellitus. periodontitis is defined as an infectious disease resulting in inflammation within the supporting tissue of the teeth leading to progressive attachment and bone loss. periodontal infections are mixed infections characterized by a complex microbiodata. microscopic examination of sub - gingival plaque samples obtained from sites with periodontitis has revealed elevated levels of gram - negative aerobes and bacteriodes. these microbial species have even been shown to affect the endocrine metabolical status of diabetic patients. the incidence and severity of periodontal disease has shown to be influenced by dm and level of blood glucose levels. going by close relationship between diabetes and periodontitis, it can be assumed that the dental practitioners are extremely likely to encounter more number of patients having both, dm and periodontitis. the early diagnosis of diabetes, however, might help to prevent its long - term complications that are responsible for the high morbidity and mortality of diabetic patients. periodontitis is also likely to be more severe in diabetic individuals with advanced systemic complications because of poorly controlled, persistently high blood sugar levels known as sustained hyperglycemia. the release of bio - inflammatory cytokines in both the cases explains their bidirectional relationship. the mechanism associated with periodontitis found in diabetic patients is the accumulation of advanced glycation end products (ages), which would affect the migration and phagocytosis of polymorphonuclear cells, producing a sub - gingival flora with a predominance of gram - negative anaerobes. this would trigger the secretion of soluble mediators that facilitate connective tissue destruction and bone resorption and produce a state of insulin resistance in the tissues at the same time, the periodontal infection would also induce resistance to insulin in the tissues, which would contribute in turn to the accumulation of ages. a lot of diabetic patients undergoing dental treatment, needs to be first ascertained of their existing blood sugar level, so that appropriate treatment strategies can be provided to the individual patients. for this however, in such patients, this small amount of blood can also be obtained by simply probing the periodontium without any discomfort to the patient. therefore, the aim of the present study was to evaluate feasibility and accuracy of gingival cervicular blood when used as an aid to screen blood glucose in dental office as compared to the conventional method of peripheral blood sampling. the study was approved by the ethical committee of the institution and an informed consent was taken from all the subjects prior to start of the study. a total of 15 diabetic (type 2, previously undiagnosed) and 15 non - diabetic patients in the age group of 40 - 55 years with untreated moderate to severe periodontitis, having probing depth in the upper anterior teeth 5 mm, were selected from diabetes care and research centre, s. p. medical college, bikaner. patients requiring premeditation or prophylactic drug regime, suffering from any other systemic infection / diseases or sites with suppuration were excluded from the study. blood oozing from the gingival sulcus / pocket was collected by placing the glucometer diagnostic strip at the entrance of the gingival sulcus. in addition to it, regular finger stick capillary blood was collected. both samples, 3 l each were analyzed using a glucose self - monitoring device (quicktest, braun company, germany), according to the manufacturer 's recommendations. statistical analysis was performed by students (paired and unpaired) t - test or coefficient correlation test by spss (software package version) 10.0. statistical analysis was performed by students (paired and unpaired) t - test or coefficient correlation test by spss (software package version) 10.0. results of this study revealed a strong correlation (r = 0.715, p < 0.001) of blood glucose levels between gingival crevicular blood (gcb) and peripheral capillary blood (pfb). the mean blood glucose level from gcb of the subjects in diabetic group was 172.27 5.02 mg / dl while mean blood glucose level from pfb was 167.80 8.87 mg / dl. the correlation coefficient of diabetic and non - diabetic subjects were r = + 0.715 and r = + 0.619, respectively [table 1 ]. there was significant difference between peripheral capillary and crevicular blood glucose even with increasing blood glucose levels in diabetic and non - diabetic subjects [table 2 ]. the mean blood glucose level of diabetic and non - diabetic patients measure by gingival crevicular blood and peripheral blood the mean blood glucose level of diabetic and non - diabetic patients measure by gingival crevicular blood and peripheral blood the american diabetes association recommends that screening for diabetes should start at age of 45 years and be repeated every 3 years in persons without risk factors and earlier and more often in those with risk factor for diabetes. in our study correlation between pfb and gcb glucose readings from diabetic group participants was high i.e. r = 0.715. by contrast, for participants in non - diabetic group, correlation between the glucose readings was lower (r = 0.619). a lot of research has been done to develop painless and non - invasive methods to measure blood glucose levels. sites with periodontal inflammation have been shown to produce adequate amount of extavasate of blood during routine periodontal examination. therefore, it prevents the need for an extra procedure (finger puncture with sharp needle) to obtain blood sample for assessment of blood glucose levels. there is also an added advantage in cases with very low gingival crevicular bleeding as lesser amount of blood (3 l) is necessary to perform the analysis. although gingival bleeding is an indicator of inflammation, it may also be possible that vascular changes in dm result in increased gingival bleeding. the relation of the control of diabetes to development of vascular changes has been studied by tchobroutsky who observed less vascular changes in well - controlled diabetes. kjellman., came to the conclusion that diabetics with poor cooperation in the care of the disease had more gingivitis than those with good cooperation. the poor cooperating group also showed higher levels of blood glucose suggesting a poor control of the disease. according to our findings, there is a significant correlation between fingerstick capillary blood and gingival crevicular blood glucose levels. the present study is in agreement with another study which also demonstrated a strong correlation between gingival crevicular and fingerstick capillary blood. moreover, in this study, the correlation of fingerstick and crevicular gingival blood measured in a laboratory analyzer was found to be highly statistically significant (r = 0.715, p < 0.001). cohen., found that diabetics had an increased degree of inflammatory changes in their periodontium compared with health controls. in the same study it was found that the amount of soft deposits was smaller in the diabetic group while the amount of hard deposits was equal in both groups. sheridan., showed that pocket formation, presence of calculus, increased tooth mobility and tooth loss occurred with greater frequency in patients with a decreased glucose tolerance. patients with confirmed or possible diabetes also showed increased alveolar bone loss and marginal widening of periodontal membrane. so the results of present study indicate the gingival crevicular blood can provide an excellent source for measuring blood glucose level during diagnostic periodontal examination by glucometric analysis. however, study was carried out on a small group of population and the technique to obtain an acceptable blood sample from gingival crevices is not always feasible and reliable which would limit its application as a clinical practice. further studies should be carried out on a large group of subjects to arrive at a confirmatory diagnosis and to establish benchmark gcb glucose levels for both diabetic and non - diabetic patients. the result of this study suggests that gingival crevicular blood is a reliable and definitive indicator for analysis of glycemic status of an individual. however, further studies are required to isolate uncontaminated blood, ascertain ratio of contamination and large sample size for better comparison of gingival crevicular blood and variations with peripheral capillary blood values.	aim : to study the accessibility of chair side blood glucose non - invasive screening method for diabetes mellitus during routine periodontal examination.materials and methods : fifteen non - diabetics and 15 newly onset type 2 diabetics patients with moderate to severe periodontitis were selected after meeting inclusion and exclusion criteria. periodontal pocket probing was performed using a williams graduated periodontal probe. blood oozing from gingival sulcus of anterior teeth following periodontal pocket probing was collected with stick of a glucose self - monitoring device. as control, finger stick capillary blood was taken.results:a statistically significant correlation was observed between the blood glucose level of gingival crevicular blood (gcb) and peripheral fasting blood (pfb) of diabetic subjects. the mean gcb glucose level of the subjects in diabetic group was 172.27 5.02 mg / dl while mean pfb glucose was 167.80 8.87 mg / dl. the correlation coefficient of diabetic and non - diabetic subjects were r = + 0.715 and r = + 0.619, respectively.conclusion:the results suggested that blood oozing during routine periodontal examination may be used for diabetic mellitus screening in a dental office setting without the need for any extra procedure.
	real - time measurement of intracellular ph in live cells is of great importance for understanding physiological / pathological processes and developing intracellular drug delivery systems. we report here the first use of nanoscale metal organic frameworks (nmofs) for intracellular ph sensing in live cells. fluorescein isothiocyanate (fitc) was covalently conjugated to a uio nmof to afford f - uio nmofs with exceptionally high fitc loadings, efficient fluorescence, and excellent ratiometric ph - sensing properties. upon rapid and efficient endocytosis, f - uio remained structurally intact inside endosomes. live cell imaging studies revealed endo- and exocytosis of f - uio and endosome acidification in real time. fluorescently labeled nmofs thus represent a new class of nanosensors for intracellular ph sensing and provide an excellent tool for studying nmof cell interactions.
self - assembly is a remarkable process that nature uses to organize chemical systems composed of nonliving components into living, biological systems. nature accomplishes this incredible feat by adding information to matter and by guiding the self - assembly process to create functional structures. toward the goal of engineering biomimetic, bioinspired, or biokleptic components that can communicate, regulate, and actuate in artificial molecular networks, information - coding polymers such as dna, rna, and proteins have been used as ideal building blocks in the assembly of designer nanoarchitectures. this perspective will concentrate on the most recent and inspiring advances in structural dna nanotechnology and present an outlook of the future of this rapidly expanding field. more comprehensive reviews that provide very detailed descriptions of the state of the art in this field can be found elsewhere in the literature. dna, nature s molecule of choice for storing and transmitting genetic information, is an excellent nanoscale building block because of its specific three - dimensional (3d) conformation, chemical addressability, and predictable watson crick base - pairing. structural dna nanotechnology, derived from seeman s innovative proposal that dna could be used as a physical material for the self - assembly of nanoscale structures (figure 1), has developed with astounding speed over the past 30 years. the most significant underlying concept is the application of immobile, branched dna junctions, together with sequence - specific sticky end associations, to create self - assembling arrays, objects, and devices (figure 1a). structural foundations of structural dna nanotechnology and representative examples (each panel described left to right). original proposals to use immobile dna junctions to create self - assembling arrays (a) and self - assembled 3d dna lattices (b) as scaffolds to organize macromolecules into crystalline lattices. (c) dna nanostructure motifs used to create periodic 2d arrays and 3d crystal (top, helical structures of the motifs ; bottom, afm images of the assembled 2d arrays and optical image of the 3d crystal) : double - crossover dna tile, 44 dna tile, 64 dna tile, and tensegrity triangle dna tile. (d) polyhedral dna nanostructures : molecular models of a dna cube, dna tetrahedron, dna dodecahedron, and dna biprism. (e) algorithmic self - assembly based on double - crossover tiles : sierpinski triangles and binary counter. (f) dna origami nanostructures (top, schematic drawings of the structures ; bottom, corresponding afm or tem images) : 2d dna origami smiley face, 3d dna origami in the shape of a gear, curved single - layer 3d origami in the shape of a vase, and dna origami gridiron. (g) complex nanostructures produced using the single - stranded dna tile strategy. images reproduced with permission : (c) ref (13), 1998 nature publishing group (npg) ; ref (14), 2003 american association for the advancement of science (aaas) ; ref (15), 2006 american chemical society (acs) ; ref (16), 2009 npg ; (e) ref (28), courtesy of p. rothemund ; ref (29), 2005 acs ; (f) ref (32), 2006 npg ; ref (37), 2009 aaas ; ref (38), 2011 aaas ; ref (114), 2013 aaas ; and (g) ref (45), 2012 npg ; ref (46), 2012 aaas. over the past several decades, researchers have established a collection of convenient methods to construct dna nanostructures that exhibit significant geometric and topological complexity. designing and predicting the 3d conformation of these nanostructures is now routine thanks to several user - friendly software interfaces that have been developed. a number of 2d and 3d lattices assembled from small, repeating dna nanostructure motifs were produced (figure 1c), and several discrete polyhedral objects were constructed from fixed numbers of dna junction motifs (figure 1d). in 2009, seeman s group was the first to assemble 3d dna crystals from deliberately designed sticky - end connections (figure 1c, right) rather than through simple, nonspecific base stacking. they used self - assembling tensegrity triangle motifs to create 3d crystals with various unit dimensions. this work represents a milestone in fulfilling seeman s original vision of using 3d dna lattices as hosts to organize guest protein molecules and facilitate protein crystallography (figure 1b). several researchers encoded algorithms into dna nanostructure components to direct the assembly of particular 2d lattice arrays and had some initial success (figure 1e) ; however, scaling - up algorithmic assembly to realize more complex patterns remains a challenge, mainly because of the errors that accumulate during assembly. if error correction mechanisms could be implemented, it would represent a ground - breaking advance in this field. in 2006, the dna origami method uses a number of short single - stranded dna (ssdna) oligonucleotides to direct the folding path of a long ssdna scaffold strand. rothemund used genomic ssdna from the m13 phage as the scaffold strand (7249 nucleotides) and designed a set of short staple strands to selectively bind to distant regions of the scaffold and fold it into a predesigned shape. this assembly method results in near - quantitative yield for most 2d designs (figure 1f), even with unpurified staples. several groups successfully extended dna origami fabrication to 3d and to the assembly of twisted and curved 3d objects (figure 1f). other research groups have focused their attention on scaling - up dna origami using the following methods : edge - to - edge base - stacking interactions between individual origami units, sequence - specific sticky end cohesion between individual units, super origami, and use of longer scaffolds for origami construction. more recently, yin and co - workers synthesized a variety of 1d, 2d, and 3d dna nanostructures from single - stranded dna tiles (ssts). the platform that they developed is based on a series of interlocking local connections between ssts. collections of ssts form 2d sheet or 3d block canvases that can be selectively engraved to create different shapes and patterns by simply including or omitting specific ssts (figure 1 g). natural biological devices are designed to operate in dynamic conditions, responding to subtle biological cues to realize their functions. the structural properties of dna that allow it to serve as a versatile construction material have been exploited to create dynamic nanodevices (figure 2a) ranging from small switchable structures and reconfigurable systems to structures that display complex movements such as rolling, rotating, and walking. (d) dna assembly line : dna walker will transport gold nanoparticles to a different product formation station with instructions from dna strand displacement. protein molecular motors transform chemical energy into mechanical energy to facilitate a variety of biological functions from cell division, transport, and motility to enzymatic activity. dna nanotechnologists have long envisioned programming dna walker molecules to mimic the ability of natural motor proteins to walk along intracellular tracks and achieve controlled motion. imparting directionality to dna walkers could be realized by means of successively additng dna fuels, by coordinating conformational changes between different components of the walker, by leading the walker through selective track modifications, or by pairing their motion to unidirectional reaction cycles. researchers have already demonstrated unidirectional motion by dna walkers through prescribed tracks and landscapes (figure 2b, c). on the basis of this technology, it is possible to develop walkers that are programmed to travel a certain path by encoding the directions into the nucleotide sequences of the walker itself and into the corresponding landscape. for example, seeman s group reported a dna - based robot that manufactured structures on a nanoscale assembly line (figure 2d). their dna walker traveled through three fixed modules that were individually programmed to selectively incorporate a gold nanoparticle into the final product, resulting in eight possible outcomes. recently, researchers demonstrated that dna walkers can also be used to mediate multistep organic synthesis, pointing to the possibility of programming chemical reactions with dynamic dna nanodevices. as structural dna nanotechnology transitions from adolescence into adulthood, the need to demonstrate potential applications is of the utmost importance. we must improve our ability to engineer and program complex molecular systems and prove that designer dna nanostructures can be employed in real - world applications. if we continue to exploit the programmability of dna nanostructures to accurately template functional molecules, materials, and probes, we will be able to organize these external elements into practical devices and engineer molecular sensors, circuits, and actuators. inorganic nanomaterials such as quantum dots, nanowires and nanorods, and metal nanoparticles have attracted much attention because of their unique optical and electronic properties that can be used in solar cells, phototransistors, laser diodes, light - emitting diodes, and other optoelectronic devices. however, a better understanding of the photophysical behavior of these materials is necessary to use them in such devices. researchers have successfully used dna nanoscaffolds to organize metallic nanoparticles, semiconductor nanocrystals (figure 3a), and organic chromophores into well - defined architectures. these hybrid dna nanostructure complexes have enabled systematic investigation of distance - dependent interactions between photonic elements. in one example, liedl and co - workers constructed a spiral, nanoscale staircase on which gold nanoparticles were arranged at regular intervals and with chiral geometries (figure 3a). this work demonstrates how dna scaffolding can be used to control the precise structural arrangement of metal nanoparticles, enabling researchers to tailor surface plasmon resonance and the interaction with visible light. in another example, dna nanostructures were used to organize various organic chromophores into artificial light - harvesting complexes with control over cascading, unidirectional energy transfer. representative examples of dna nanostructure - directed assembly of inorganic and protein molecules (top, schematics ; bottom, corresponding tem or afm images). (a) left to right : gold nanoparticles organized by a 2d dna tile array, gold nanorod dimers with controlled angles between the nanorods organized by dna origami, dna origami - directed quantum dot architectures, and dna origami - directed gold nanoparticles in a chiral arrangement and the induced circular dichromic effect. (b) left to right : organization of streptavidin proteins by a 2d dna nanoarray, protein arrays templated by a 2d dna nanostructure through aptamer protein interactions, and orthogonal snap - tag- and his - tag - mediated decoration of dna origami. images reproduced with permission : (a) ref (69), 2006 acs ; ref (70), 2011 acs ; ref (71), 2012 acs ; ref (72), 2012 npg ; and (b) ref (14), 2003 aaas ; ref (79), 2007 acs ; ref (80), 2010 wiley. as we previously mentioned, one of the initial goals of structural dna nanotechnology was to use 3d dna lattices as hosts to organize guest protein molecules and facilitate protein crystallography. although this vision has yet to be realized, scientists have already begun to use dna nanostructures as chaperones to align and organize protein molecules using different strategies, including ligand streptavidin) interactions, aptamer target interactions, and ligand - engineered (tagged) protein interactions (figure 3b). shih and co - workers recently designed dna origami nanotube liquid crystals to provide the appropriate alignment environment for determining the previously unknown structure of a membrane protein by nuclear magnetic resonance (nmr). turberfield and co - workers used periodic 2d dna tile arrays as templates to arrange proteins and subsequently used cryo - em to solve their structures. proteins, some of nature s most powerful agents, are large macromolecules that perform a wide assortment of functions required to sustain life, including metabolic catalysis, dna replication, and molecular transport. in order to better understand the governing dynamics in complex protein systems, we need control over the number, orientation, and arrangement of the constituents. nucleic acid scaffolds afford this level of control, and researchers have already used rna and dna platforms to engineer a number of enzyme cascades (figure 4a). for example, silver and co - workers used a bacterial host to transcribe rna and assemble intracellular rna nanoscaffolds for spatial organization of metabolic elements for hydrogen production. willner and co - workers organized a glucose oxidase and horseradish peroxidase enzyme cascade by 2d dna lattices. more recently, yan and co - workers conducted substrate channeling in a multienzyme cascade by using an artificial dna swinging arm. representative examples of dna nanostructure - directed assembly of protein molecules for functional structures. (a) upper left, assembly and disassembly of holoenzymes mediated by dna strand displacement ; upper right, glucose oxidase (yellow) and horseradish peroxidase (red) enzyme cascade organized by 2d dna lattices ; lower left, substrate channeling in a multienzyme cascade by an artificial dna swinging arm ; and lower right, glucose oxidase (yellow) and horseradish peroxidase (red) enzyme cascade organized on dna origami with distance control. (b) rectangular dna origami travels on a cellular actin network through the binding and action of myosin lever arms. (c) molecular tug - of - war between two motor proteins displayed from a 12-helix dna bundle. dna origami scaffolds have also been used to organize motor proteins and study their spatially dependent motility (figure 4b). understanding how motors cooperate productively, and compete antagonistically, is important for understanding how intracellular transport is regulated. researchers recently demonstrated this molecular tug - of - war by displaying different numbers of dynein and kinesin motor proteins from a dna origami structure. by controlling the number, distance, and orientations of the two types of biological motors, they were able to systematically study coordinated motor behavior (figure 4c). structural dna nanotechnology has also emerged as a useful tool for biological and medicinal applications (figure 5). the intrinsic biocompatibility, nanoscale dimensions, programmability, and ability for functionalization of dna nanostructures are virtually unrivaled by existing techniques. in particular, the addressable configuration of dna origami lends itself to detection of gene expression and single nucleotide polymorphism. the sugiyama group developed dna origami frames and rulers to investigate biomolecular interactions such as protein dna binding events and homologous recombination processes in real time at the single molecule level (figure 5a). further, the spatial addressability and multivalent properties of dna nanostructures make them promising vehicles for targeted drug delivery. for example, douglas and co - workers demonstrated a barrel - shaped nanorobot that releases fab antibody fragments in the presence of target cells. in their system, two ssdna aptamer locks are opened by specific markers present on the surface of cells (figure 5b). after opening, the payload molecules inside the barrel are exposed, inducing a particular cellular signaling pathway. anderson and co - workers used a dna tetrahedron to deliver small interfering rna in vivo to target and suppress gene expression in a mouse model (figure 5c). yan, chang, and co - workers used a dna tetrahedron to coassemble model antigens and cpg adjuvants into nanoscale complexes with precise control of the valency and spatial arrangement of each component (figure 5d). dna complexes induced stronger and longer - lasting antibody responses against the antigen, without stimulating a reaction to the dna nanostructure itself, as compared to an unstructured mixture of antigen and cpg molecules. more recently, amir. showed that dna origami robots can dynamically interact with each other and perform logic computations in a living animal, opening up opportunities to develop smart theranostic nanodevices (figure 5e). (a) dna origami frames to investigate protein dna binding events in real time at the single molecule level. (b) barrel - like dna nanorobot programmed to be open in the presence of target cells and expose fab antibody fragment cargo. (c) six sirna duplexes and folic acid tags (gray) chaperoned by a dna tetrahedron are injected into mice ; the tetrahedra bind to tumor cells by targeting folate receptors expressed on the tumor cell surface. cpg odn adjuvant molecules (curved yellow ribbons) and the model streptavidin antigen (red) bind specifically to b cells and are subsequently presented to t cells to activate b cell response and antibody production. (e) three different drugs carried by a dna nanorobot can be released in a programmed fashion by undergoing complex dna computation in a living cockroach. image (e) reproduced with permission from ref (99), 2014 npg. the interdisciplinary nature of dna nanotechnology crosses the traditional boundaries of physics, chemistry, biology, and engineering and allows scientists to connect and integrate their unique perspectives in pursuit of solutions to the most pressing problems in medicine, technology, and more. from the earliest dna junction motifs to the most recently developed dna nanostructures of incredible complexity, the field has started to explore various novel applications, including directed material assembly, structural biology, biocatalysis, dna computing, nanorobotics, disease diagnosis, and drug delivery, as we mentioned briefly in the previous section. each of these applications is made possible by the ability of dna nanostructures to direct molecular species with nanoscale precision while maintaining the utmost structural integrity. dna nanotechnology is progressing with such incredible speed that it is becoming more and more difficult to predict from which areas the next breakthroughs will occur. next, we are merely providing our opinion about the critical challenges that the field faces and which directions we believe researchers should pursue to help structural dna nanotechnology reach its full potential. we have divided the remaining outlook into three main areas : design and assembly, which will include discussions of dynamic, developmental, quasicrystal lattice, 3d periodic crystal lattice, scaffolded, surface - mediated, algorithmic, and topological assembly ; future applications, which will include discussions of structural dna nanotechnology for molecular scaffolds, sensors, robotics, and computing ; and from nano to angstrom technology, in which we conceive of potential directions the field might explore over the longer term. george whitesides once wrote, although much of current understanding of self - assembly comes from the examination of static systems, the greatest challenges, and opportunities, lie in studying dynamic systems. perhaps the most important justification for studying self - assembly is its central role in life. dynamic self - assembly processes underlie many forms of adaptive and intelligent behaviors in natural systems ; however, very little is known about the principles that govern them. one of the most intriguing, dynamic self - assembly processes in living cells is the polymerization of cytoskeletal biopolymers such as microtubules. microtubule polymerization is characterized by two unique phenomena, referred to as tread - milling and dynamic instability. tread - milling is said to occur with the net addition of tubulin monomers at one end of the microtubule and simultaneous net loss of tubulin at the opposite end. dynamic instability is characterized by switching between phases of relatively slow and rapid shortening of the microtubules at their ends. although these phenomena were once thought to be incompatible, it is now known that both behaviors coexist in near - steady - state conditions in cells. it would be quite interesting if we could use the desirable properties of dna nanostructures to recapitulate these phenomena and ultimately dissect the governing dynamics of microtubule polymerization (figure 6a). dna tiles could be designed such that the rate of assembly equaled the rate of disassembly, resulting in steady - state tread - milling and fixed - length nanotubes. further, if tiles with bi- or tridirectional growth were utilized, the resulting arrays would have defined shapes. the intrinsic conformational flexibility and rigidity of different dna building blocks could be exploited to mimic dynamic instability, where polymerization of flexible dna tiles can be induced through seeded growth on a rigid tile and depolymerization of the flexible tiles can be initiated by removing the rigid tile protection cap. when the association and dissociation reactions reach equilibrium, the input of additional rigid tiles will catalyze the polymerization of released flexible tiles. studying the association and dissociation kinetics of model dna tile species with variable flexibility is absolutely essential to recreating this or similar dynamic self - assembling systems. schematic illustrations of (a) dynamic dna self - assembly with simultaneous joining in one end and dissociation in the other end, (b) developmental dna self - assembly, in which the assembly process may grow into different final products in response to different external cues, (c) an example of a possible quasicrystal (2d penrose tiling) using dna tile self - assembly, and (d) self - replication of dna nanostructures. the creation of new life depends on a set of extraordinary developmental processes including stem cell growth, differentiation, and morphogenesis. these processes rely on nature s ability to precisely control the spatial and temporal relationship between cellular components and signaling pathways. it would be extremely interesting if we could create synthetic dna systems that mimic this kind of spatiotemporal development. dna tiles have the potential to develop into unique patterns through instructions embedded in the building blocks or by external stimuli such as fuel strands that trigger new growth pathways (figure 6b). metastable dna nanostructures could be designed and used to serve as nucleation seeds and/or catalysts to increase the growth and development of particular pathways. multivalency and/or cooperativity within dna nanostructures could be exploited for nucleation and initiation of alternative assembly paths. for example, pierce and co - workers recently reported the dynamic assembly of dna nanostructures through a seeded cascade of hybridization chain reactions based on toehold - mediated strand displacement. strand displacement circuits have also been used to trigger dna tile assembly and control their growth into dna tubes. there are several key challenges to implementing toehold - mediated strand displacement in dynamic dna systems, including leakage, slow reaction rates, and the necessity for high salt conditions. zhang and co - workers reportedly designed toehold exchange probes and optimized the specificity of dna hybridization so that their system can detect single - base changes. designing robust self - assembling dna platforms to mimic developmental systems will also certainly require a thorough understanding of the thermodynamics and kinetics of dna self - assembly. in 2011, the nobel prize in chemistry was awarded to dan shechtman for his discovery of quasicrystals, a finding that fundamentally changed how chemists understand solid matter. prior to his report, scientists believed that the atoms in a crystal were always packed into symmetric patterns that repeated periodically. we have since come to understand that it is possible to form packed crystals from nonrepeating patterns, an arrangement of molecules now referred to as quasicrystalline. the distinctive properties of quasicrystals, as well as their unique structures, have intrigued scientists ever since their discovery ; however, very little is currently known about the properties exhibited by synthetic and naturally occurring quasicrystals. scientists have yet to determine what guides quasiperiodic rather than periodic growth and what factors result in the unique properties that quasicrystals display. one of the biggest challenges facing researchers today is the lack of plausible systems from which to assemble quasicrystals and enable further studies. dna platforms are promising candidates for the controlled, programmable growth of synthetic quasicrystals (figure 6c). interacting dna building blocks can potentially be programmed to assemble into 2d and 3d quasicrystal patterns, allowing us to investigate the still unknown mechanisms of quasicrystal growth and providing a means to organize other materials for engineering pursuits. realizing 3d dna lattices as hosts to organize guest protein molecules and facilitate protein crystallography necessitates that 3d dna crystals can themselves be reliably assembled and characterized. researchers successfully demonstrated the assembly of a 3d dna crystal in which the triangular unit tiles were connected by sticky ends and solved its structure to 4 resolution using x - ray crystallography. however, most dna crystals only diffract to 710, leaving scientists trying to determine why rationally designed dna crystals do not diffract with better resolution. there are several possible explanations, including defects that arise during crystallization, impurities in the synthetic dna, and the presence of bulky solvent molecules in the large cavities of the dna lattices. crystal defects may be caused by the limited rigidity of dna unit motifs, where any over- or under - twisting of the tiles causes inter - tile mismatches that are detrimental to the integrity of the crystal lattice. we surmise that imparting flexibility to certain domains of the dna building blocks may allow the unit tiles to more reliably accommodate their neighbors and reach a lower energy state for crystal lattice formation, thereby improving the overall quality of the crystal. the sleiman group pioneered dna junctions with metal complex modifications that combine rigidity within the core of the junction with intrinsic flexibility in the arms. this type of modified dna unit motif has the potential to improve the quality of dna crystals but has yet to be exploited for crystallization applications. reducing the volume of solvent present in the lattice cavities by inserting sequence - specific binding proteins may improve the diffraction quality, but sequence - independent methods to orient proteins within the dna cavities still need to be developed. this strategy is particularly attractive, as some have already demonstrated that rna - binding proteins are useful chaperones for rna crystallization. piccirilli and co - workers derived rna - specific antibodies using synthetic phage display libraries and showed that the antibody fragments promoted crystallization of rna molecules. similarly, dna tile binding antibodies could be identified through in vitro evolution and used for coassembly of the dna units and proteins into designed 3d crystals. recent developments in free electron laser (fel) x - ray nanocrystallography have the potential to revolutionize the field of structural biology by providing highly focused coherent x - ray beams with a peak brilliance that is 10 higher than the x - ray beams at the most powerful synchrotron facilities. obtaining high - quality diffraction patterns using fel x - ray requires micrometer - sized nanocrystals ; it might be possible to program the growth of 3d dna lattices into finite nanocrystals with suitable dimensions by designing a 3d box that acts as a scaffold to nucleate the growth of a periodic lattice of dna tiles. growing 3d crystals with designed crystal morphologies and dimensions is undoubtedly an interesting topic in itself. the development of scaffolded dna origami represents a milestone in structural dna nanotechnology. while the complexity and robustness of 2d and 3d dna origami objects has increased over the past few years, researchers still lack basic understanding of the thermodynamics and kinetics of scaffolded assembly. understanding the minutia of dna origami formation will allow us to guide the design of more complex dna nanostructures, optimize annealing protocols, and manipulate functionalized dna nanostructures more effectively. structurally speaking, we are still a long way from being able to weave a scaffold strand along arbitrary paths within a dna origami structure, although some progress has been made in this direction. recently, yan and co - workers developed a novel strategy to fold gridiron - like dna origami structures. in that work, interconnected four - arm junctions were used as vertices within a network of dna fragments, and measured distortion of the junctions from relaxed conformations allowed the scaffold strand to traverse through individual vertices in several directions. despite this initial success, interlacing the scaffold strand through the vertices of multiarm junctions remains a challenge that, if achieved, would dramatically improve our ability to form aperiodic tiling patterns and polyhedral 3d structures using the dna origami technique. besides increasing complexity, scaling up the size of dna origami and reducing the cost of staple strand synthesis are also important issues facing dna nanotechnologists. various strategies to address these limitations have been explored, including the use of longer single - stranded scaffolds, double - stranded scaffolds, origami of origami (super - origami), and enzymatic production of staple strands on microarray chips, which has the added benefit of greater fidelity than chemical synthesis. dna origami has shown great success in directing the assembly of nanoelectronic and photonic elements and has been used as a lithographic mask to etch nanoscale patterns on silicon and graphene substrates. for practical device applications, it is highly desirable to achieve robust patterning of self - assembled dna nanostructures on inorganic surfaces, and several groups have developed unique strategies to organize dna origami nanostructures on solid substrates. the next logical step is to generate chemically functional surface features to facilitate patterning of dna origami nanostructures into spatially addressable arrays. surface - mediated assembly may be the key to scaling - up dna nanostructure assemblies into wafer - size arrays. researchers have already shown that mica and silicon dioxide surfaces will mediate the assembly of small dna tiles into millimeter - range periodic 2d lattices. the buffer conditions, especially the concentration and species of the ions present, may play a critical role in surface - mediated diffusion of dna nanostructures, an important factor that remains to be explored. it would also be interesting to use fluidic 2d surfaces such as lipid bilayers to improve the surface - mediated diffusion of dna nanostructures. in mathematics and computer science however, if you look beyond their traditional context in mathematics, you will see that algorithms can be used to describe the process of self - assembly in the natural world. consider the self - assembly of lipids into membranes, or viral proteins into capsids, or even just amino acids into intricately folded protein structures, each process involves the spontaneous, or automatic, assembly of small components into larger, more complex structures. the process by which these structures grow can be described as algorithmic. in each example, a limited number of molecular building blocks grow into higher order structures by following the growth rules encoded into the building blocks themselves. dna tiles are information - rich building blocks ideally suited for implementing algorithmic self - assembly. originally proposed by winfree, algorithmically self - assembled dna nanostructure patterns have been experimentally demonstrated. for example, winfree and co - workers showed that dna double - crossover tiles could be programmed to compute and grow into sierpinski triangle and binary counter - assemblies. they also showed that prescribed dna origami displaying sticky - end - capture probes function as effective nucleation seeds to grow algorithmic arrays while suppressing spurious nucleation, which is a major source of errors during algorithmic assembly. the design of novel nucleation frames could improve the fidelity and robustness of algorithmic assemblies of dna tiles. other errors arise from sticky end mismatches between different tiles that share certain sticky end sequences. the kinetics of tile tile association between the algorithmic building blocks should be carefully investigated to promote the desired computations and reduce any undesirable mismatches. also, tile sets could be expanded beyond the typical double - crossover dna tiles to more complex or optimal geometries to facilitate multivalent and cooperative binding between the tiles and allow for improved understanding of the constraints that limit the scope of algorithmic assembly. in biological systems, there is a clear relationship between the specific structure of a biomolecule and its function. in particular, biopolymers are important molecules whose structure supports the organization and functionality of cells. the topology of biopolymers can be exploited to facilitate tasks such as packing information - bearing dna molecules into tiny compartments within cells. molecular topology is a fascinating and technically challenging topic that dna nanotechnology is ideally suited to examine. seeman and co - workers were the first to show that topological structures such as knots and borromean rings could be self - assembled from dna by combining right - handed b - form and left - handed z - form dna together to create positive and negative nodes. yan and co - workers later used the dna origami method to construct mbius strip topological structures that could be reconfigured into catenanes and twisted topological ribbons through toehold - mediated strand displacement. more recently, willner and co - workers developed strategies to interlock dna rings into multiring catenanes. weizmann and co - workers just reported the assembly of complex knots and links by specifically configuring four - way dna junctions. despite these interesting examples, the area of dna - based topological nanostructures is underdeveloped compared to the geometric structures that have been reported over the past decade. new construction strategies and topological targets should be identified to push the frontiers of dna - based molecular topology forward. self - replication is an astounding process by which a molecule in a dynamic system makes an identical copy of itself. biological cells, provided they have a suitable environment, reproduce by cell division. during cell division, linear dna autonomously undergoes replication by enzyme - mediated processes and is transmitted to offspring. it is a considerable challenge to design and construct autonomous structures that mimic the action of nucleic acid polymerases and are capable of replicating entire synthetic dna systems nonenzymatically (figure 6d). the first development in this direction was reported by seeman s group in 2011. they constructed a seven - tile seed and successfully generated several generations of progeny in a step - by - step manner. winfree and co - workers recently showed that mechanically induced scission of 2d dna crystals can accurately replicate self - assembled dna nanopatterns by creating new fronts of crystal growth. however, constructing autonomous self - replicating systems that do not require external manipulation remains a significant challenge. pierce and co - workers demonstrated autocatalytic dna duplex formation by way of a cross - catalytic circuit, yet extending this concept to independent formation of sophisticated dna nanopatterns needs additional development. the successful design and assembly of the dna nanosystems discussed above will undoubtedly lead to many new opportunities and innovative applications. the information - rich character of self - assembling dna nanostructures in particular will create many new frontiers for the application of designer dna nanostructures as molecular scaffolds, sensors, computers, and robots. in the following sections we will discuss the potential of dna nanostructures to serve as scaffold for functional nanoelectronic and nanophotonic devices, to regulate protein interactions, and to create sense compute actuate elements for molecular medicine. however, these examples are in no way limiting, and the field has already demonstrated a tendency to grow in unexpected directions, surprising even the sagest of researchers. one of the most obvious applications of a dna nanostructure is to direct the assembly of other, less controllable materials, as was discussed in the previous sections. we have seen several examples of spatially addressable dna origami structures being used to organize nanoelectronic and photonic components. however, we have not yet seen concrete examples of dna nanostructures in functional nanoelectronic and photonic devices, where bottom - up, dna - directed assembly is interfaced with top - down, lithographic methods of micro- and macroscale patterning. the latest developments in surface - mediated self - assembly and site - specific control of chemical properties could enable more precise arrangement of these nanophotonic or nanoelectronic elements into regular, large - scale patterns that can be integrated with macroscopic systems. dna - directed assembly of complex protein arrays is another area of development to watch for in the future. enzymes, marvels of natural evolution, are intramolecular organizations of proteins that are capable of recognition, capture, and activation of molecules and regulation of biochemical processes. these protein complexes act as the central functional components of metabolism and reproduction in living systems. the binding sites for substrates and cofactors are chemically specific, while the active sites are stereospecific and highly sensitive to conformational rearrangement. inspired by nature, researchers have pursued a variety of strategies to regulate and control the catalytic activities of enzymes, as well as to understand the mechanism of enzyme function and pathways. compared to most conventional techniques, dna nanotechnology is a highly efficient and controllable strategy to achieve structural programmability and reconfigurability through rational design and construction. assembling enzymes and cofactors on dna nanostructure scaffolds has already allowed researchers to probe the essential parameters for modulating catalysis, such as intermolecular distance and relative spatial position. one example of controlling the activity of an individual enzyme using dna was reported in 2013, where the authors achieved mechanical regulation of the enzyme luciferase by attaching a dna spring. in the same year an even loftier and more valuable goal is to engineer highly programmed cascading enzyme pathways on dna nanostructure platforms with control of input and output sequences. achieving this goal not only would allow researchers to mimic the elegant enzyme cascades found in nature and attempt to understand their underlying mechanisms of action but also would facilitate the construction of artificial cascades that do not exist in nature (figure 7a). (b) design and implementation of theranostic nanodevices on targeted cell surfaces that carry out functions such as compute, sense, release signal, trigger activation, and deliver therapeutic molecules across the cell membrane. one major challenge in integrating multiple proteins into dna nanostructures is to precisely define their relative orientation and position. a set of reliable and general methods for site - specific conjugation of proteins with oligonucleotides must be established in order to accommodate the diversity of proteins of interest. in an ideal system, a single protein with multiple coupling sites would be conjugated to unique dna sequences to enable absolute orientational control of the protein relative to the dna nanostructure. in this way, the active sites of the enzymes, in a multienzyme cascade for example, could be precisely oriented to facilitate substrate intermediate product transfer, and the overall enzymatic activity of the cascade could be optimized. a far - reaching goal of structural dna nanotechnology is to develop smart molecular machines that perform sense compute actuate mechanisms based on intrinsically information - rich dna molecules and structures (figure 7b). for example, the development of smart molecular doctors would revolutionize the field of personalized medicine. a smart molecular doctor would have the same responsibilities as a real (human) doctor, including diagnostic and therapeutic roles, but would operate entirely at the cellular level. directly treating individual diseased cells to cure them on the single - cell level offers improved therapeutic efficiency and fewer side effects since smaller drug doses are required compared to conventional therapies. other targeted drug delivery systems based on multifunctional liposomes, polymersomes, and nanoparticles have already been developed. dna is an attractive material for theranostic applications, not only because of its inherent design modularity, structural programmability, and biocompatibility but also because dna molecules of a particular sequence or with certain modifications can selectively bind, distinguish, and communicate with target cells to trigger drug release. researchers have made strides toward constructing dna - based drug containers and dna nanostructures that can be embedded into lipid bilayers, particularly after the establishment of the dna origami method. the first dna origami box with a responsive lid that recognized a specific oligonucleotide key and subsequently opened was reported in 2009. more recently, researchers developed a dna nanobarrel with two single - stranded aptamer locks that were opened by the presence of target cells in vitro. performing dna computation directly on the surface of cells, or in cellular environments, recently, rudchenko, stojanovic, and colleagues engineered dna strand displacement cascades that detected the presence of certain cell markers on the surface of cells. in another report, hemphill and deiters successfully engineered oligonucleotide logic gates to detect specific microrna inputs in live mammalian cells. as more complex and robust dna - based computing systems are developed, it may be possible to integrate them into cellular systems to control and trigger cellular functions such as gene expression or to interfere with the metabolic pathways. recently, researchers reported the construction of a consensus network that distinguishes between two different input signals and reports the majority signal. by combining dna computation - based target cell detection with reconfigurable dna - based drug containers, it may be possible to create a dna nanorobot that can interface and communicate with living cells (figure 7b). there are a number of critical issues that must be addressed before dna nanorobots can be used for drug delivery in vivo. researchers must find a way to protect dna nanostructures from degradation by the intra- and extracellular nucleases and liver metabolism over long periods. compact dna nanostructures generally display relative stability against dna nucleases for a short time (a few hours). in the future it will be important to increase resistance to biodegradation by using methods such as chemical cross - linking of selected dna strands or designated dna backbone modifications. identifying the mechanisms by which dna nanostructures enter cells without being damaged, and escape endosomal processing, is also a critical point. the biggest obstacles to transforming dna nanostructures from mere curiosities into real - world solutions are the cost of synthetic dna, small production scales, typically low yield of complex 3d structures, and sensitivity of dna to ionic strength, temperature, and nucleases. researchers have already begun to address these issues by optimizing origami design and folding strategies to increase assembly yields and shorten assembly times and by developing suitable purification strategies for large - scale synthesis. it is also important to develop biocompatible conditions for efficiently folding dna nanostructures, rather than by thermal annealing under high magnesium concentrations. living cells are information - rich, sophisticated machines that display angstrom - level organizational precision. although dna nanostructures are exquisitely programmable, they are only able to regulate biological molecules at a relatively coarse level compared to nature. if we want additional control, we must push the boundaries of nanoscale fabrication to the angstrom level. in contrast to dna, rna and proteins have more refined architectures with angstrom - level features. these aspects of their organization have attracted increasing attention in the past decade. methods for engineering designed proteins and nanostructured complexes using proteins have also begun to emerge. the progress of characterization techniques such as cryo - em, x - ray diffraction, and nmr supports the development of angstrom technology. in particular, the most recent developments in cryo - em techniques allow crystallization - free structural determination of large - sized proteins that is comparable to x - ray methods. using dna origami frames both as structural hosts and as references, the structure of dna and rna binding proteins may now be determined to angstrom - level resolution by cryo - em. this advance will provide researchers with atomic - level structural information (in conjunction with the structural solutions obtained from x - ray crystallography) that can be fed back into the design pipeline, elevating the field to unimaginable heights (figure 8). from nano- to angstrom - level control : engineering molecular toolboxes composed of dna, rna, peptide, and protein molecules and their unnatural derivatives to extract new design rules and create complex, self - assembling structures with angstrom - level spatial control. in summary, after more than 30 years of growth, structural dna nanotechnology is transitioning from adolescence into adulthood. the field is crossing the boundaries of physics, chemistry, biology, and engineering and is poised to generate unique approaches and solutions to real - world challenges in science and technology. in the next phase of structural dna nanotechnology, novel interactions between dna, rna, and proteins could be used to facilitate angstrom technology, representing the major challenges and opportunities in molecular design, assembly, computing, and programming. living cells are information - rich, sophisticated machines that display angstrom - level organizational precision. although dna nanostructures are exquisitely programmable, they are only able to regulate biological molecules at a relatively coarse level compared to nature. if we want additional control, we must push the boundaries of nanoscale fabrication to the angstrom level. in contrast to dna, rna and proteins have more refined architectures with angstrom - level features. these aspects of their organization have attracted increasing attention in the past decade. methods for engineering designed proteins and nanostructured complexes using proteins have also begun to emerge. the progress of characterization techniques such as cryo - em, x - ray diffraction, and nmr supports the development of angstrom technology. in particular, the most recent developments in cryo - em techniques allow crystallization - free structural determination of large - sized proteins that is comparable to x - ray methods. using dna origami frames both as structural hosts and as references, the structure of dna and rna binding proteins this advance will provide researchers with atomic - level structural information (in conjunction with the structural solutions obtained from x - ray crystallography) that can be fed back into the design pipeline, elevating the field to unimaginable heights (figure 8). from nano- to angstrom - level control : engineering molecular toolboxes composed of dna, rna, peptide, and protein molecules and their unnatural derivatives to extract new design rules and create complex, self - assembling structures with angstrom - level spatial control. in summary, after more than 30 years of growth, structural dna nanotechnology is transitioning from adolescence into adulthood. the field is crossing the boundaries of physics, chemistry, biology, and engineering and is poised to generate unique approaches and solutions to real - world challenges in science and technology. in the next phase of structural dna nanotechnology, novel interactions between dna, rna, and proteins could be used to facilitate angstrom technology, representing the major challenges and opportunities in molecular design, assembly, computing, and programming.	over the past three decades dna has emerged as an exceptional molecular building block for nanoconstruction due to its predictable conformation and programmable intra- and intermolecular watson crick base - pairing interactions. a variety of convenient design rules and reliable assembly methods have been developed to engineer dna nanostructures of increasing complexity. the ability to create designer dna architectures with accurate spatial control has allowed researchers to explore novel applications in many directions, such as directed material assembly, structural biology, biocatalysis, dna computing, nanorobotics, disease diagnosis, and drug delivery. this perspective discusses the state of the art in the field of structural dna nanotechnology and presents some of the challenges and opportunities that exist in dna - based molecular design and programming.
visual paraneoplastic syndromes associated with gynecologic malignancies include cancer - associated retinopathy (car) and diffuse uveal melanocytic proliferation (bdump). it has been hypothesized that in car visual loss occurs due to the presence of autoantibodies (aabs) specific to tumor antigens that cross - react with certain proteins existing in retinal photoreceptor cells. the published case reports on gynecological cancers associated with retinopathy, including an endometroid ovarian carcinoma, endometrial adenocarcinoma, undifferentiated cervical reserve cell carcinoma, ovarian papillary serous cystadenocarcinoma, primary epithelial ovarian cancers, except fallopian tube carcinoma have not reported on associated autoantibodies [3 - 5 ]. one case of car associated with primary ovarian carcinoma was linked with serum aabs against anti - carbonic anhydrase ii. however, the presence of aabs is the primary serological indicator of autoimmunity and should be studied to fully understand autoimmune - mediated pathogenic process in those diseases. the presence of serum aabs is thought to be the consequence of breakdown of immunological tolerance however aabs are not exclusive of car syndrome. aabs generated against self - antigens are also found in cancer during massive tissue damage but then of importance, they are also produced in healthy subjects without cancer or car and in the complete absence of known external antigenic stimulation. the potential roles of those natural antibodies could be prognostic and diagnostic in the monitoring of levels of natural igm antibodies to specific apoptosis - associated antigens to better predict future clinical manifestations. anti - retinal aabs that have been reported in car are diverse, cross - react with cancer antigens and often represent cytoplasmic proteins, including the 23-kda recoverin and the 46-kda -enolase proteins. in neurological paraneoplastic syndromes, the humoral response against cns intracellular proteins was usually correlated with poor prognosis, while aabs reactive with membrane components appeared to have a better prognosis. moreover, the repertoire of aabs found in cancer patients partly coincides with diverse repertoire of aabs found in autoimmune diseases. some cancer patients develop aabs that recognize new antigens whose expression is restricted to tumor cells, at the same time most tumor - associated aabs can also be detected in car patients, e.g. recoverin. retinal autoantigens associated with gynecological - car have not been studied because this is an uncommon syndrome. aabs are likely made as the initial body defense against the growing tumor, and when they cross - react with retinal antigens this may lead to car. taking into consideration the presence of anti - retinal aabs in both health and disease, the goal of this study was to examine the repertoire of serum anti - retinal aabs and identify distinct autoantibody biomarkers in car patients with different types of gynecological malignancies in comparison to healthy women and to patients with similar cancers but without visual symptoms of car at the time of testing. we hypothesized that aabs found in car patients might have unique disease - specific profiles that allow distinguishing between controls and cancer patients. sera were obtained from repositories of the md anderson cancer center and oregon health and science university (ohsu). the studies were approved by the irb from ohsu and md anderson cancer center and our research adhered to the tenets of the declaration of helsinki and is in accordance with hipaa regulations. the inclusion criteria for car diagnosis were unexplained and progressive visual loss, present photopsia, nyctalopia, abnormal erg, progressive worsening of visual acuity and visual fields, ring scotomas, suspected or diagnosed cancer. patients that have known genetic (familial) causes, ocular infection, ocular trauma, intraocular surgery (other than cataract surgery), drug toxicity, retinal detachment, and typical age - related macular degeneration were excluded from the studies. the subjects with symptoms of car represented 45 caucasian women and 1 asian woman of an average age of 5912.5 ranging from 25 to 89 years old and gynecological cancers, including endometrial, uterine, cervical, ovarian, and fallopian tubes cancers. women with signs of car presented with progressive loss of vision with photopsia (4 patients), reduced to undetectable responses in erg (19 patients), reduced visual acuity from 20/50 to complete loss (22 patients), color impairment (10 patients), constriction of visual fields, ring or central scotomas (17 patients), and night blindness (7 patients). in the ovarian cancer - car group, diffuse bilateral melanocytic proliferation (bdump) was found in 2 patients and another 2 patients had melanoma after ovarian cancer was detected. ocular inflammatory processes like uveitis, vitritis, cells in the vitreous and vasculitis were associated with car in 9 patients. also sera from 111 patients with related uterine and ovarian cancers without symptoms of car and 60 sera from age - matched healthy women were included for comparison. sera were tested for the presence of anti - retinal autoantibodies by western blotting as published before. briefly, human retinal protein extract was separated by sds - gel electrophoresis using 10% criterion gels (bio - rad) followed by transfer to a pvdf membrane using semi - dry apparatus. individual blots were then immunostained with 1:25 diluted patient s serum followed by incubation with anti - human igg (h and l chain) conjugated to alkaline phosphatase (invitrogen). as positive controls we used human anti - enolase reference serum (1:100), rat anti - enolase enol-1 mab (1:2000), and rabbit anti - recoverin antiserum (1:50,000). the differences in presence of antigens between the group with and without symptoms of car were tested by the fisher s exact test. sera were obtained from repositories of the md anderson cancer center and oregon health and science university (ohsu). the studies were approved by the irb from ohsu and md anderson cancer center and our research adhered to the tenets of the declaration of helsinki and is in accordance with hipaa regulations. the inclusion criteria for car diagnosis were unexplained and progressive visual loss, present photopsia, nyctalopia, abnormal erg, progressive worsening of visual acuity and visual fields, ring scotomas, suspected or diagnosed cancer. patients that have known genetic (familial) causes, ocular infection, ocular trauma, intraocular surgery (other than cataract surgery), drug toxicity, retinal detachment, and typical age - related macular degeneration were excluded from the studies. the subjects with symptoms of car represented 45 caucasian women and 1 asian woman of an average age of 5912.5 ranging from 25 to 89 years old and gynecological cancers, including endometrial, uterine, cervical, ovarian, and fallopian tubes cancers. women with signs of car presented with progressive loss of vision with photopsia (4 patients), reduced to undetectable responses in erg (19 patients), reduced visual acuity from 20/50 to complete loss (22 patients), color impairment (10 patients), constriction of visual fields, ring or central scotomas (17 patients), and night blindness (7 patients). in the ovarian cancer - car group, diffuse bilateral melanocytic proliferation (bdump) was found in 2 patients and another 2 patients had melanoma after ovarian cancer was detected. ocular inflammatory processes like uveitis, vitritis, cells in the vitreous and vasculitis were associated with car in 9 patients. also sera from 111 patients with related uterine and ovarian cancers without symptoms of car and 60 sera from age - matched healthy women were included for comparison. sera were tested for the presence of anti - retinal autoantibodies by western blotting as published before. briefly, human retinal protein extract was separated by sds - gel electrophoresis using 10% criterion gels (bio - rad) followed by transfer to a pvdf membrane using semi - dry apparatus. individual blots were then immunostained with 1:25 diluted patient s serum followed by incubation with anti - human igg (h and l chain) conjugated to alkaline phosphatase (invitrogen). as positive controls we used human anti - enolase reference serum (1:100), rat anti - enolase enol-1 mab (1:2000), and rabbit anti - recoverin antiserum (1:50,000). the differences in presence of antigens between the group with and without symptoms of car were tested by the fisher s exact test. to investigate the occurrence of anti - retinal aabs in gynecological car we analyzed serum samples from three groups for comparison : (i) car group - women with cancer presented with ocular symptoms of car, n=46 ; (ii) non - car group - women with cancer without clinical symptoms of car, n=111 ; and (iii) control group -age - matched healthy women subjects, n=60. car was diagnosed in patients with gynecological cancers, such as uterine, endometrial, cervical, ovarian, and fallopian tube and sera of those patients were tested for the presence of autoantibodies against human retinal proteins. then the identity of antibody reactive proteins was confirmed by additional western blotting using purified targeted proteins on the blot. our results showed that specific anti - retinal aabs occurred in car patients as well as in cancer patients without the visual symptoms of car but at a different percentage. as is presented in figure 1 and table 1 the patients with gynecological malignancies associated with car had the highest proportion of seropositivity (80%) than patients with gynecological cancers without car (61%) and healthy female subjects (58%). the analysis using the exact fisher test revealed the statistical significance between car and non - car groups (p=0.025) and car and control groups (p=0.021). all three groups of patients showed heterogeneity in their retinal protein recognition for 17 antigens but were focused on 5 proteins : enolase (46-kda), aldolase c (40-kda), carbonic anhydrase ii (30-kda), recoverin (23-kda) and gapdh (36-kda) as more frequently targeted than other retinal proteins (figure 2). while aabs against those retinal proteins were present in each of gynecological cancer groups their prevalence was different. as demonstrated in figure 2 an overlap occurs among specific aabs between groups of patients but their incidence varied. for example, the incidence of aabs specific to enolase, aldolase, and gapdh in controls subjects was significantly lower than in patients with cancer (figure 3). anti--enolase aabs were detected in 35% cancer patients and 13% control subjects (p=0.006, fisher s exact test). anti - gapdh antibodies were significantly higher in patients with car (13%) compared to 3% patients without retinopathy (p=0.028, fisher exact test). overall anti - carbonic anhydrase ii (caii) aabs were not significantly different between groups (p=0.682, exact fisher s test) and were detected in 13% car patients, 14% cancer patients and 18% healthy women and but there were greater differences within the individual cancer groups (figure 2). two patients with bilateral diffuse uveal melanocytic proliferation (bdump) had aabs against 35-kda and 46-kda proteins (one patient) and 30-kda, 50-kda and 70-kda proteins (second patient). our analysis showed that aabs against glycolytic enzymes such as enolase, aldolase, gapdh occurred almost 3 times more frequent in car patients than healthy individuals (figure 3). figure 4 shows anti - retinal aabs profiles in car and cancer without signs of car at the time of testing. remarkably, aabs against glycolytic enzymes were common, suggesting that they were generated in response to the upregulation of those proteins during intensive glycolysis in carcinogenesis, which in effect triggered abnormal antibody production. it has been reported that the increased expression of -enolase (eno1) on tumor cells is strictly related with the malignancy of those cells and their high metastatic ability. it is important to point out that in this cohort, all endometrial - car patients had metastatic tumor and had significantly increased aabs against enolase (p=0.00038) as well as recoverin (p= 0.00014), which make this a distinct antibody signature from non - car profile. although we observed an overlap in retinal protein recognition in all groups among different autoantibodies each gynecological car had its own antibody signature (figure 4). the striking difference occurred in anti - recoverin aabs that were present in car and almost completely absent in non - car women (p=0.00014, fisher s exact test). anti - recoverin aabs were present in 50% of endometrial - car, 11% uterus - car and 7% ovarian - car in contrast to none present in healthy subject sera. in the cancer group, only one woman with neuroendocrine fallopian tube cancer out of 21 subjects had anti - recoverin aabs (4.7%). this retinal calcium - binding protein has been recognized as a car antigen, which its aberrant expression is capable of triggering the generation of specific aabs in patients with malignant tumors and the subsequent development of the paraneoplastic syndrome, cancer - associated retinopathy. in the case of fallopian tube cancer without retinopathy, 12 out of 21 patients were seropositive with 19% of anti - enolase and 38% of anti - caii aabs. because we had only 2 patients with this rare fallopian - car it is not possible to conclude about the incidence of aabs in car in comparison to other groups. by combining antigens to a set of biomarkers, specificity and sensitivity of a disease signature increase. the maximum observed sensitivities with one marker were 0.4, 0.5 and 0.38 for ovarian, endometrium and cervix cancer, respectively. the sensitivities were 0.53 with 2 makers (30-kda and 46-kda), 1.00 with 3 markers (23-kda, 30-kda, 46-kda), and 0.64 with 4 markers (30-kda, 35-kda, 40-kda, and 45-kda) for each cancer. to determine whether the manifestation of visual symptoms is correlated with the time of cancer diagnosis, we examined the latency time from the cancer diagnosis to the discovery of car and the related antibodies. in this cohort, cancer was diagnosed in these women before diagnosis car with latency time from 2 months to 30 years with the longest interval in cervical malignancies (on average 21 years). such a long interval between the diagnoses of primary cancer in women with cervical cancers suggests that sudden manifestation of visual problems about 20 years later could be related to other problems although undiagnosed malignant tumor can not be excluded. on the other hand, the diagnosis of the ovarian and endometrial cancers and findings of car and autoantibodies often closely coincided in those patients and in some cases preceded the diagnosis of cancer by few months (figure 5). in addition, the ocular symptoms preceded the diagnosis fallopian tube carcinoma by 2 - 3 years in both of our patients. our studies showed that anti - retinal aabs were present in women with gynecologic malignancies with and without clinical symptoms of car as well as in healthy women controls however women with gynecologic - car had a higher proportion of seropositivity. five retinal proteins were targeted by aabs in car more frequently and these were : carbonic anhydrase ii, recoverin, -enolase, aldolase c, and gapdh. a high incidence of aabs specific to -enolase and other glycolytic enzymes in our patients suggests their causal relationship to car. the production of some of those autoantibodies may be related to metabolism of cancer cells that up - regulate a number of different pathways to keep up with demands for rapidly dividing cells. glycolysis is one such pathway, which is up - regulated in transformed cells regardless of the presence or absence of oxygen. studies showed that the overexpression of aldolase c by tumor cells was an indication of human endometrial cancer, like other tumor types, this cancer survival depends on glycolysis for its energy supply. other studies suggested that enolase might be a useful target for the therapy of cancer through the development of anti - enolase antibody - based therapy. however, an increased presence of anti - enolase antibodies could be harmful to the retina and could result in retinal degeneration. it has been demonstrated that all of these glycolytic enzymes (nse, nne, aldolase c, pyruvate kinase m1) are also membrane bound and expressed on the surface of neuronal cells. enolase existsas a monomer and also as dimers, including both - enolase dimer on the membrane surface. these are multifunctional proteins that are all expressed intracellularly and on the cell surface and such accessibility to these proteins on the membrane can stimulate the production of aabs. some patients with car have aabs against a single retinal protein, e.g. recoverin, and such aabs are considered to be specific for this entity. however, our current study shows that more than one antibody specificities may exist in a single cancer patient and even in healthy subject in a lower level. the antibody profile may be a potential signature of specific malignancy and among other factors can be considered as a diagnostic tool of car syndrome. of note, in some patients, visual symptoms of car may be present without the finding of anti - retinal autoantibodies, possible because aabs can have also tumor suppressing effects through the generation of immune complexes thus they may not be detected in the serum. overall car sera had higher incidence of aabs and each gynecological car had anti - retinal autoantibodies against more than one antigen, suggesting a trend towards an antibody signature associated with a specific gynecologic tumor. unfortunately, we do not have large enough numbers to make definitive statements about this. usefulness of anti - retinal aabs in recognition of the underlying tumor is also important, e.g. anti - recoverin aabs were frequent in endometrial car but absent in healthy subjects. the sensitivity can be improved by combining antigens to a set of biomarkers to increase specificity and sensitivity of a disease signature. for example, the maximum observed sensitivities with one marker were 0.4, 0.5 and 0.38 for ovarian, endometrium and cervix cancer, respectively. other proteins such as glycolytic enzymes that showed increased seroreactivity in cancer also showed reactivity in healthy individuals but in lower incidence. the fact that many of the antigens reacting with sera from cancer patients are also reactive with sera from patients without cancer diseases emphasizes the importance of combining different antigens to a set of biomarkers to increase specificity and sensitivity of a disease signature. the sensitivities can be enhanced to 0.53 with 2 makers (30-kda and 46-kda), 1.00 with 3 markers (23-kda, 30-kda, 46-kda), and 0.64 with 4 markers (30-kda, 35-kda, 40-kda, and 45-kda) for each cancer. also, antibody isotypes and titers may help in the diagnostic value of these autoantibodies as iggs seem more pathogenic then igms (found in normal subjects), and high concentrations of aabs are usually found in patients with cancer and car. the value of a biomarker depends not only on its occurrence in a given disease, but also in their specific use for early diagnosis, monitoring or prognosis. thus our findings suggest that anti - recoverin aabs testing should only be performed in the context of ocular presentation relevant to car that has a reasonable likelihood of having the disease for which the testing is appropriate. if not, the predictive value of a positive test maybe too low. it is worth to mention that the incidence and titer of autoantibodies in healthy individuals increases with age. our subjects average age is ~60 years old that predispose them to generation of various aabs. in addition, molecular mimicry and autoimmune cross - reactivity of autoantibodies has been suggested as a possible pathogenic mechanism in post - streptococcal disease. the etiology of car remains to be fully characterized but autoimmunity may occur when tumor antigens that mimic retinal proteins induce the immune system to lose tolerance for these self - proteins and accidentally begin targeting the retina. also, healthy people have natural antibodies that are involved in maintaining homeostasis by removing cell - debris or tumor cells, or by preventing inflammation by binding and neutralizing cytokines. therefore, it is important to consider that many aabs are found at low levels in healthy subjects and that aabs against retinal targets can be present years before disease onset and remain non - pathogenic if the blood - retina barrier is not breached. recent studies from the cancer field show that autoantibody signatures seem to be particularly relevant for detection of cancer at different stages because during cancer growth the immune system elicit serum autoantibodies that can be considered as relevant cancer biomarkers. novel autoantibody profiling will help to detect car for each cancer that may manifest months to years after the initial malignancy diagnosis and also before the cancer diagnosis. each gynecological - car has an own autoantibody profile different from non - car antibodies, which suggests that the complex autoantibody signatures may be more predictable than singular aab (figure 4). the diagnosis of these paraneoplastic syndromes associated with gynecologic malignancies is essential as they can be occasionally life - threatening. an additional benefit from our studies is that in some paraneoplastic manifestations anti - retinal aabs can be also used as markers of the underlying malignancy and aid in diagnosis of the primary cancer.	backgroundthe presence of autoantibodies (aabs) is the primary serological indicator of autoimmunity. cancer - associated retinopathy (car) is associated with aabs and different types of cancer. the goal of the study was to examine the profile of serum autoantibodies in women with gynecological cancers with and without paraneoplastic visual manifestation.methodsretrospective studies of a cohort of 46 women with symptoms of car and gynecological tumors, including endometrial, cervical, ovarian, and fallopian tubes, 111 women with similar tumors without symptoms of car, and 60 age - matched healthy controls. presence of serum aabs and the identity of targeted antigens were performed by western blotting and their significance was evaluated using an fisher s exact test.resultsthe patients with gynecological car had the highest proportion of seropositivity (80%), followed by patients with gynecological cancers without car (61%) and healthy controls (58%). differences in recognition frequencies were found for 17 antigens and 5 retinal antigens were frequently targeted : enolase, aldolase c, carbonic anhydrase ii, recoverin and gapdh. the occurrence of anti - glycolytic enzymes was 2 - 3 times more frequent in car and cancer patients than healthy controls. anti - recoverin aabs were prevalent in endometrial car. anti - caii antibodies were not significantly different between groups of women. in this cohort, cancer was diagnosed before the onset of retinopathy with latency from 2 months to 30 years. the discovery of the ovarian and endometrial cancers and manifestation of visual problems often coincided but fallopian tube carcinoma was found after visual onset.conclusionnew retinal targets were identified for gynecological car. each gynecological - car has its own autoantibody profile different from non - car profile, implying that a complex autoantibody signature may be more predictable for diagnosis than a singular aab. specific anti - retinal aabs were most prevalent in women with car but their profiles were not fully distinguished from cancer controls.
the worldwide number of long - term cancer survivors is growing fast with ongoing improvements in cancer therapies [1, 2 ]. however, long - term cancer survivors may suffer from late side effects of cancer therapy. one of these late side effects is radiation - induced heart disease (rihd), which may occur after radiotherapy of thoracic and chest wall tumors whenever all or part of the heart is situated in the radiation field. rihd has been described to occur, for instance, among survivors of hodgkin 's disease [3, 4 ] and breast cancer [5, 6 ]. radiotherapy planning has undergone many improvements over the last decades, with modalities such as intensity - modulated radiation therapy (imrt), image - guided radiation therapy, and proton therapy, leading to reduced exposures of the heart. for instance, patients with hodgkin 's disease, lung cancer, and esophageal and proximal gastric cancer may still receive either a high dose of radiation to a small part of the heart or a lower dose to the whole heart [711 ]. in addition, although there is increasing use of concomitant therapies, the extent to which these therapies affect radiotherapy side effects such as rihd is largely unknown. manifestations of rihd include accelerated atherosclerosis, pericardial and myocardial fibrosis, conduction abnormalities, and injury to cardiac valves [4, 12 ]. the disease is progressive and both incidence and severity increase with a higher radiation dose volume, younger age at the time of radiotherapy, a greater time elapsed since treatment, and concomitant use of cardiotoxic chemotherapeutic agents such as anthracyclines. although rihd is widely acknowledged as an impediment to quality of life for certain long - term cancer survivors, from a clinical perspective the only current way to reduce rihd is through efforts to improve radiotherapy treatment planning, as other methods to prevent or reverse rihd are not yet available. hence, pre - clinical studies seek to unravel basic mechanisms of rihd, with the ultimate goal to identify potential targets for intervention. pre - clinical animal models have long been used to study rihd [1318 ]. while transgenic mouse models are being used in investigations of radiation - accelerated atherosclerosis [19, 20 ], wild type rodents are usually not atherosclerosis prone. hence, studies that use rodents to investigate radiation - induced coronary artery disease are limited in number [21, 22 ]. on the other hand, many laboratory animals, including rodent, have been used successfully as models of radiation - induced cardiomyopathy [16, 2327 ]. common doses used in these pre - clinical models of localized heart irradiation are either a single dose between 5 gy and 25 gy, or fractionated schedules of, for instance, 5 daily fractions of 9 gy. some of the histopathological changes in pre - clinical models, such as myocardial degeneration and fibrosis, are also commonly described in human cases of rihd, mainly after exposure to doses of ~30 gy and above [3, 4, 2830 ]. although clinical and pre - clinical data on the cardiovascular effects of lower radiation doses are growing [11, 31 ], the focus of this review will be on myocardial injury and cardiac function changes after exposure to higher doses of radiation. previous paper indicate the important role of vascular injury and endothelial dysfunction (loss of thromboresistance and increased expression of adhesion molecules and cytokines) in the pathogenesis of normal tissue radiation injury [42, 43 ]. endothelial dysfunction may contribute to profibrotic and proinflammatory environments, which are common aspects of normal tissue radiation injury [42, 44 ]. although the role of endothelial dysfunction in rihd has not been studied in detail, experimental rihd is known to be associated with reduced myocardial capillary density [32, 33 ], focal loss of endothelial alkaline phosphatase [14, 34 ], and increased expression of von willebrand factor. hence, microvascular injury and the resulting local ischemic injury are considered to be some of the underlying mechanisms of rihd. radiation - induced vascular injury and endothelial dysfunction are mediated in part by transforming growth factor- (tgf-) [45, 46 ], a pluripotent growth factor that is part of many normal tissue radiation responses [4749 ]. previous studies have shown cardiac upregulation of tgf- in rat models of rihd after localized heart irradiation with 20 gy or 5 fractions of 9 gy [3638 ]. a tgf--inducing compound was used to investigate the role of tgf- in rihd in the rat. cardiac radiation fibrosis was more severe in animals that had been administered the tgf--inducing compound during the 6-month followup time after irradiation (unpublished data). mast cells, cells that belong to the hematopoietic myeloid lineage, reside in many organs and tissues including the heart. although best known for their role in hypersensitivity reactions, mast cells are also intimately involved in wound healing and tissue remodeling [5052 ]. mast cells store and release a wide range of cellular mediators, both via degranulation and via constitutive pathways that do not involve degranulation. increased mast cell numbers are commonly found in coronary atherosclerosis, myocardial fibrosis [54, 55 ], and also in animal models of rihd [40, 56 ], where mast cell numbers correlate with myocardial radiation injury. the development and maturation of mast cells depend on the c - kit receptor, which is specific for stem cell factor. several mast cell deficient animal models, based on a mutation in the c - kit receptor or stem cell factor, are available [5759 ]. our laboratories have made use of a rat model that is homozygous for a 12-base deletion in the c - kit receptor gene [60, 61 ]. both mast cell - deficient rats and their mast cell - competent wild type litter mates were exposed to localized heart irradiation with a single dose of 18 gy. although mast cell - deficiency was associated with reduced radiation - induced myocardial inflammation and degeneration, other manifestations of cardiac radiation injury such as myocardial fibrosis and ex vivo measures of myocardial stiffness were exacerbated in the absence of mast cells. these studies suggest that mast cells, in contrast to what had been the prevailing assumption but similar to what has been found in some other cardiac disease models [62, 63 ], play a predominantly protective role in rihd. mast cell - derived proteinases, for instance, have been shown to contribute to both the formation and degradation of endothelin-1 (et-1) [6468 ]. et-1 is a 21-amino acid peptide that was first discovered as a potent vasoconstrictor but also has proinflammatory and pro - fibrotic properties [69, 70 ]. the role of et-1 in cardiovascular pathology has been studied extensively [71, 72 ]. both et-1 receptors, eta and etb are expressed by a wide variety of cell types in the heart [70, 73 ]. short - term upregulation of et-1 and its receptors may serve as a mechanism to maintain cardiac function in certain cardiovascular diseases [74, 75 ]. long - term up - regulation of the endothelin system, on the other hand, may have detrimental effects due to the vasopressor, prohypertrophic, and pro - fibrotic properties of et-1 [73, 76 ]. mast cells express the receptor eta, which upon activation by et-1 induces mast cell degranulation, a pathway by which et-1 may enhance the activity of matrix metalloproteinases (mmps) in the heart [78, 79 ]. dual inhibition of eta and etb prevented mast cell degranulation and the associated increase in cardiac mmp levels, interstitial collagen degradation, and ventricular dilatation in a rat model of chronic volume overload. on the other hand, in a preliminary study of a rat model of rihd, dual inhibition of eta and etb did not alter radiation - induced functional or structural cardiac changes. moreover, vascular injury seemed aggravated by selective eta inhibition in a rat model of localized intestine irradiation. dosing of receptor antagonists and opposing cardiovascular effects of the eta and etb, receptors [83, 84 ] warrant further studies to clarify the role of et-1 and its two receptors in rihd. they are found in close proximity to nerve terminals or axons in many organs, including the heart [86, 87 ], and interact with nerves on the molecular level in many ways [85, 88, 89 ]. mast cells express - and -adrenergic receptors [90, 91 ]. in normal rat myocardium some sensory neuropeptides such as calcitonin gene- related peptide (cgrp), substance p, and neuropeptide y are able to induce or enhance mast cell degranulation [93100 ] while others have been shown to inhibit mast cell degranulation [101, 102 ]. mast cells, in turn, affect neuronal growth and function by producing nerve growth factor and by activating proteinase - activated receptor-2 on the surface of neurons [104, 105 ]. cardiac sensory nerves play a protective role in the heart via the release of nitric oxide and cgrp [106, 107 ]. for instance, cgrp plays a protective role in myocardial injury such as from ischemia reperfusion and the cardiotoxic chemotherapeutic agent doxorubicin [108, 109 ]. cgrp is a potent vasodilator but also has beneficial effects in the heart by local downregulation of tumor necrosis factor - alpha (tnf-) and upregulation of insulin - like growth factor-1 (igf-1) [110, 111 ]. interestingly, both downregulation of tnf- and upregulation of igf-1 are associated with reduced normal tissue radiation injury [112, 113 ]. in line with this evidence the role of the renin angiotensin system (ras) in normal tissue radiation injury has been well defined [115, 116 ]. inhibitors of angiotensin - converting enzyme (ace) and antagonists of angiotensin type 1 receptors reduce injury in animal models of localized kidney, lung, and brain irradiation [117119 ]. although the role of ras in rihd is less well defined, ras mediators may be upregulated in the heart after irradiation. however, while the ace inhibitor captopril reduced radiation injury in kidney, lung, and skin of rats [119121 ], captopril did not prevent cardiac function loss after localized heart irradiation with 20 gy in a rat model. captopril, on the other hand, did reduce myocardial fibrosis and prevented left ventricular capillary density loss after local heart irradiation. it is not known whether these effects were due to properties of captopril other than its inhibition of ace. inhibition of ace is considered to be cardioprotective in part by suppressing the breakdown of bradykinin by ace. bradykinin, a small peptide hormone that is sometimes considered to aggravate cardiac disease with a significant inflammatory component such as myocardial infarction, is also known to mediate cardioprotection via induction of nitric oxide and prostacyclin [124126 ]. bradykinin is formed in the kallikrein - kinin system by proteolytic cleavage of both high- and low - molecular weight kininogen by kallikrein enzymes, but also by the mast cell - derived enzyme tryptase [127, 128 ]. interestingly, the mast cell proteinases chymases are one of the main converters of angiotensin i into angiotensin ii. mast cells seem to hereby provide a particularly large contribution to local extravascular generation of ang ii. the roles of ras and bradykinin in cardiac radiation injury and the potential influence of mast cells herein need further investigation. radiotherapy planning has undergone many improvements over the last decades, leading to improved targeting and reduced normal tissue radiation exposure. nonetheless, some patients with hodgkin 's disease, lung cancer, and esophageal and proximal gastric cancer may still receive either a high dose of radiation to a small part of the heart or a lower dose to the whole heart, which may lead to late manifestations of rihd. some of the basic mechanisms of rihd have begun to emerge from recent pre - clinical studies and include the involvement of vascular injury, mast cells, and the ras. future studies will elucidate the significance of these mechanisms for clinical rihd and their usefulness as targets for intervention in rihd.	radiation - induced heart disease (rihd) is a potentially severe side effect of radiotherapy of thoracic and chest wall tumors if all or part of the heart was included in the radiation field. rihd presents clinically several years after irradiation and manifestations include accelerated atherosclerosis, pericardial and myocardial fibrosis, conduction abnormalities, and injury to cardiac valves. there is no method to prevent or reverse these injuries when the heart is exposed to ionizing radiation. this paper presents an overview of recent studies that address the role of microvascular injury, endothelial dysfunction, mast cells, and the renin angiotensin system in animal models of cardiac radiation injury. these insights into the basic mechanisms of rihd may lead to the identification of targets for intervention in this late radiotherapy side effect.
push and sonde enteroscopy were widely used for small - bowel endoscopy prior to the development of double - balloon enteroscopy (dbe). however, due to the insufficient depth of intubation into the small bowel and the high rate of complications with push and sonde enteroscopy, new techniques that overcame these issues were needed. moreover yamamoto. was the first to develop and use dbe in 2001. based on the length, channel width, and clinical use, dbe is categorized into three types : diagnostic, therapeutic, and biliary dbe. balloon enteroscopy can be used for all small - bowel pathologies including ileus and obstruction. dbe is also very useful for endoscopic retrograde cholangiopancreatography (ercp) in cases with postsurgical anatomy (roux - en - y gastric bypass or other types of gastric bypass surgery) and cases of difficult colonoscopy. the cecal intubation rate was 97% in cases of unsuccessful conventional colonoscopy, and the success rate of dbe - assisted ercp was 60% to 100%. dbe should not be used in patients who are at risk for impaired general condition and perforation. a watery diet and bowel cleansing should be implemented 1 day before the procedure to ensure a successful outcome. the most important advantage of dbe is the applicability of therapeutic interventions during the procedure. however, its disadvantages include a long learning curve, long processing time, and the requirement for patient sedation. the average processing time was 92.437.6 minutes, and this period was significantly longer in the first 10 patients (109.144.6 minutes ; p<0.005). the total rate of diagnosis has been reported as 43%, and the cause of bleeding was unidentified in 51% cases. however, in a study by yamamoto., who improved the technique, the mean procedure duration was 123 minutes and the oral, rectal, and all parts of small bowel could be investigated in 86% of patients over 178 enteroscopy procedures. in studies reported from europe (70 dbe procedures in 53 patients), the rate of observation of the entire small bowel was 8% and the average procedure time was 72 minutes. in the study conducted by fukumoto., the rate of observation of the entire small bowel was higher, although the difference was not statistically significant. the most important advantage of dbe is that it allows therapeutic approaches such as argon plasma coagulation (apc), polypectomy (fig. 1), dilatation, foreign body removal, and stent placement during the procedure. may. reported that dbe procedures were performed in 60% of 353 patients who were admitted due to bleeding. dbe was found to be diagnostically and therapeutically beneficial in 75% and 67% of these cases, respectively. although the complication rate for dbe is 3.4%, this rate is reported to be 10.8% in cases of polypectomy (bleeding - perforation) and 0.9% in cases of apc. in a multicenter study that included 2,362 dbe procedures, 709 cases of pancreatitis, five cases of perforation (three apc and two dilatation), and 18 cases of bleeding (13 after polypectomy) although the complication rate is greater in patients undergoing interventional procedures, the complication rate remains within acceptable limits. in a study comparing capsule endoscopy (ce) with dbe for gastrointestinal bleeding of unknown cause, it was found that both methods could identify the source of bleeding in some cases(table 1). in a meta - analysis study that compared ce with dbe, the diagnosis rates for vascular, inflammatory, and polypoid lesions were similar (figs. 2 - 4). the general diagnosis rate of these methods was also similar (ce, 60% ; dbe, 57%). in dbe, although the diagnosis rate of obstruction and bleeding of unknown origin are 81.3% and 80.6%, respectively, the diagnosis rates of diarrhea and abdominal pain are 36.5% and 37.7%, respectively. dbe has been found to be cost - effective for the diagnosis of bleeding of unknown origin. even though dbe is invasive and its duration of training and procedure time is long, it is usually the first choice as a therapeutic intervention in suitable patients. thus, under appropriate conditions, both procedures are good and complementary to each other. depending on the clinical experience and the available equipment, a different and specific approach for each patient if guidance can be obtained via radiologic methods, either dbe or direct surgery may serve as the first choice. enteroscopy via the oral route may also be preferred for patients with active bleeding when no radiologic guidance is available. dbe should not be performed in patients who have latex allergy. instead, single - balloon enteroscopy may be preferable in such cases. in disorders of the small intestine other than bleeding of unknown cause in addition, dbe permits anal entry during the same session. moreover, dbe can be used to exclude diagnoses in suspicious cases, obtain biopsy specimens from suspicious areas, and achieve dilatation of the stenosis in crohn disease. in a study that compared dbe with single - balloon enteroscopy, dbe was shown to be three times more effective than total enteroscopic examination. however, single - balloon enteroscopy was reported to be quicker and easier to learn. the use of an additional cannula system and co2 instead of indoor air has also shown to increase the technical achievement rate with dbe, and decompress air during the examination in order to make deep intubation. moreover, the placement of metal stents for malignant stricture of the small intestine is possible. clinical admissions and alarming symptoms in patients are the main factors through which a treatment method is selected. even if the result of ce is negative, enteroscopy or intraoperative enteroscopy should be performed if there is clinical suspicion.	advances in technology have facilitated the common use of small - bowel imaging. intraoperative enteroscopy was the gold standard method for small - bowel imaging. however, noninvasive capsule endoscopy and invasive balloon enteroscopy are currently the main endoscopic procedures that are routinely used for small - bowel pathologies, and the indications for both techniques are similar. although obstruction is a contraindication for capsule endoscopy, it is not considered to be problematic for double - balloon enteroscopy. the most important advantage of double - balloon enteroscopy is the applicability of therapeutic interventions during the procedure ; however, double - balloon enteroscopy has certain advantages as well as disadvantages.
ovarian torsion results from the rotation of the ovary about its pedicle, to an extent that the ovarian arteries and or veins are obstructed. this condition generally results from a benign tumor of the ovary and usually occurs in younger women. clinical signs include sudden onset of lower abdominal pain associated with other constituitional symptoms like nausea and vomiting. in 1896, fredrick krukenberg described a new type of ovarian tumor. this tumor was later identified as a malignancy in the ovary from a primary lesion in the gastrointestinal tract, and named presentation of a krukenberg tumor with ovarian torsion is a rare event which we will detail in this case report. a 43-year - old female, (gravida 2, para 2) presented to the emergency department (ed) with an acute onset of right lower quadrant abdominal pain, which was severe and progressive in intensity. the pain was episodic, with associated diarrhea. gynecological examination revealed a large fixed fluctuant abdominal mass within the pelvis, extending above the umbilicus. working diagnosis in the ed was cystic abdominal mass of unknown origin, likely ovarian. a contrast - enhanced ct scan of the abdomen and pelvis was ordered and revealed a large poorly enhancing, predominantly cystic mass arising from the left adnexa, measuring 21.6 cm craniocaudal 11.7 cm ap (anteroposterior) 22.8 cm transverse, with numerous thin non - enhancing septations and small vessels coursing throughout the lesion [figure 1 ]. the mean attenuation value of the mass ranged from 814 hounsfield units, confirming its predominantly cystic nature. contrast - enhanced coronal ct images of the abdomen and pelvis, demonstrate a large predominantly cystic left adnexal mass containing multiple septations almost completely occupying the abdomen (black arrow). the twisted vascular pedicle in the left lower quadrant demonstrates a whirlpool sign (white arrows) (similar to what is described in the ultrasound literature). the mass demonstrated a discrete vascular pedicle in the left hemi - pelvis in the vicinity of the round ligament of the ovary with enhancing engorged vessels in the pedicle. there was a discrete vascular swirl sign appreciated at the left ovarian vascular pedicle, suggesting that this represented a massively enlarged left ovary with a significant torsion of greater than 270 degrees. these findings helped clinch the diagnosis of an ovarian torsion [figure 4(a - d) ]. a second large mass measuring 5.3 cm craniocaudal 6.5 cm ap 5.4 cm transverse, originating from the contralateral right ovary with enhancing solid rind and central hypoattenuation, representing the right ovarian krukenberg tumor [figure 2 ] (a) contrast - enhanced axial and (b) coronal ct images through the pelvis demonstrate a solid heterogeneously enhancing right - adnexal mass, with a thick enhancing rind, consistent with a krukenberg tumor (arrow). the presence of bilateral ovarian masses strengthened the initial diagnosis of krukenberg tumors and an attempt was made to assess the primary lesion. there was an irregular plaque - like, mildly enhancing, mass - like area in the mid - gastric body, along the lesser curvature, relating to a primary gastric malignancy [figure 3 ]. other relevant findings included peritoneal / serosal implants and mild right hydroureteronephrosis due to extrinsic compression of the right ureter from the right adnexal mass. contrast - enhanced axial ct at the level of the liver reveals a focal nodular thickening of the gastric body along the greater curvature and antrum (arrow) suggestive of gastric carcinoma that was subsequently confirmed by histopathology. four contiguous sagittal images showing a discrete swirl sign at the vascular pedicle (white arrows) at the base of the large left ovarian mass, consistent with the torsion of ovarian krukenberg tumor at its pedicle. the patient underwent esophagogastroduodenoscopy / colonoscopy showing a large friable ulcer located in the posterior wall of the stomach measuring approximately 10 cm, extending to both the lesser and greater curvature, showing a concern for malignancy [figure 3 ]. the patient subsequently underwent an exploratory laparotomy, bilateral salpino - oophrectomy, total gastrectomy, and esophagojejunostomy. a gross pathological examination revealed a 26 cm left ovarian tumor, smaller right ovarian tumor, disseminated metastases in the peritoneum, and gastric tumor within the body of the stomach. microscopic pathology from the gastric specimen revealed a poorly differentiated gastric adenocarcinoma of the signet cell type, while ovarian pathology showed metastatic bilateral krukenberg tumors from the gastric carcinoma, with signet cell histology similar to the primary tumor [figure 5 ]. a gross image of bi - valved left ovary weighing 2475 g and measuring 26 21 6 cm. note the large hemorrhagic area on one end of the ovary (white arrow), measuring 11 5 2.5 cm, and other smaller areas of hemorrhage within the solid ovarian mass. (the deceptive cystic appearance on ct was due to a poor flow, with large areas of infarction). these tumors are relatively uncommon and account for only one to two percent of all ovarian tumors. these tumors primarily originate from carcinomas of the gastrointestinal tract and less commonly from the breast, gallbladder, colon, appendix, or pancreas. microscopically, these tumors are characterized by mucin - secreting signet ring cells in the ovarian tissue and in the primary tumor site. a review of literature indicates that 35 to 45% of the patients were younger than 40 years of age, with an average range of 40 to 46 years. although unanswered for a long time, it is now apparent that a retrograde lymphatic spread is the most common route for the spread of metastasis of gastric carcinoma to the ovaries. the common presenting symptoms are related to the ovary and include abdominal pain and distension due to the bilateral, often large ovarian masses. in addition, ascites is a common presentation in the krukenberg tumor and usually reveals malignant cells. however, there has been a reported case of bilateral krukenberg tumors with benign ascites and right hydrothorax that revealed no malignant cells. this is known as pseudo - meig syndrome, in contrast to the meig syndrome, which presents with a triad of benign ovarian tumor, ascites, and right - sided hydrothorax. in general, patients with a krukenberg tumor have a poor prognosis, with most patients averaging two years life expectancy, with a median survival time of 14 months. studies have shown that prognosis is poor when the primary tumor is identified after metastasis of the cancer to the ovaries. our case is unusual in that, the initial presentation of a gastric malignancy was acute ovarian torsion due to an ovarian krukenberg tumor acting as a lead point for torsion. ovarian torsion is the twisting of an ovary on its ligamentous supports and can result in a compromised blood supply. adnexal torsion is a term that is inclusive of either the ovary, fallopian tube, or both. concomitant ovarian and tubal torsion has been shown to occur in up to 67% of the cases of adnexal torsion. the main sonographic features of an ovarian torsion include a morphologically abnormal ovary, with or without ovarian blood flow. the presence of blood flow within the ovary does not exclude ovarian torsion, it suggests that the ovary is viable and can be salvaged with surgery. as this patient presented to the er with severe abdominal pain and a significant increase in abdominal girth, the first imaging study ordered in the ed was a contrast - enhanced ct of the abdomen and pelvis, due to suspected malignancy or any other acute abdominopelvic process, thus bypassing the typical first line abdominopelvic ultrasound, to save time. computed tomography is usually used as an adjunct modality when diagnosis is not straightforward on ultrasound and also when a wider field of view is needed, as in the case of our patient. computed tomography features commonly suggestive of ovarian torsion on magnetic resonance imaging (mri), the appearance of the krukenberg tumor is dependent on the degree of stromal overgrowth and mucin production. dense stromal reaction gives a hypointense signal and mucin material usually gives a hyperintense signal on t2w images. the presence of hypointense solid components within an ovarian solid mass is a characteristic, but a nonspecific finding for krukenberg tumors. although the patient was in the average age range for krukenberg tumors, benign ovarian neoplasms and benign pathologies are typically more common. currently, in radiology literature, there are no reported cases of krukenberg tumors presenting to the ed with acute ovarian torsion. although uncommon, krukenberg tumors should be considered in the differential diagnosis of ovarian torsion, in addition to benign entities such as mature cystic teratoma.	ovarian torsion is the fifth most common gynecological surgical emergency. ovarian torsion is usually associated with a cyst or a tumor, which is typically benign. the most common is mature cystic teratoma. we report the case of a 43-year - old woman who came to the emergency department with rare acute presentation of bilateral krukenberg tumors, due to unilateral ovarian torsion. in this case report, we highlight the specific computed tomography (ct) features of ovarian torsion and demonstrate the unique radiological findings on ct imaging. metastasis to the ovary is not rare and 5 to 10% of all ovarian malignancies are metastatic. the stomach is the common primary site in most krukenberg tumors (70%) ; an acute presentation of metastatic krukenberg tumors with ovarian torsion is rare and not previously reported in radiology literature.
as the majority of recent randomized controlled trials (rcts) have been unable to demonstrate a beneficial effect of various interventions aimed at improving outcome in end - stage renal disease (esrd), novel treatment strategies need to be tested in this high - risk patient group. among several novel risk factors, inflammation has attracted considerable interest in the last 10 years. although cytokine production is necessary to protect against pathogens and promote tissue repair, excessive release or decreased clearance (or both) can lead to organ failure and premature death. detailed reviews on causes of inflammation in the context of impaired renal function have recently been published. prospective studies in hemodialysis, peritoneal dialysis, and renal transplantation patients show that even a single measurement of inflammatory biomarkers independently predicts poor outcome. furthermore, inflammation has been identified as a strong prognosticator of sudden death in esrd patients, which indirectly supports a link between persistent inflammation and an imbalance between the sympathetic and parasympathetic nervous system. in 2008, a large genetic study of patients with ischemic heart disease (and controls) showed that c - reactive protein (crp) polymorphisms are not in themselves associated with an increased risk of ischemic vascular disease, therefore, available evidence suggests that although crp is a strong risk marker, it is not a risk factor of cardiovascular disease. in fact, among numerous studies that have shown that inflammatory biomarkers or cells involved in the inflammatory cascade predict outcome, interleukin (il)-6 seems to be the strongest predictor of cardiovascular morbidity and outcome. another study showed that by defining specific cytokine ratios related to the inflammatory and immunologic states, it seems possible to fine tune the prediction of death from cardiovascular and non - cardiovascular causes. based on the observation that persistent inflammation may serve as a catalyst and magnify the risk of poor outcome via mechanisms related to self - enhancement of the inflammatory cascade and exacerbation of the wasting and calcification processes, it can be argued that the effects of different interventions should be analyzed separately in inflamed and non - inflamed dialysis patients. although inflammatory markers persistently predict outcome in dialysis patients or following kidney transplantation, few studies have studied the effect of various anti - inflammatory interventions on outcome. however, as small studies have shown encouraging results after examining the levels of inflammatory biomarkers following intervention with gamma - tocopherol, soy, green tea, cholecalciferol and sevelamer, the renal community should conduct sufficiently powered rcts to prove the concept that treatment of persistent inflammation may provide benefit in reducing uremic morbidity and mortality. in a recent rct involving 2776 hemodialysis patients, the effects of rosuvastatin versus placebo were compared. no effect on mortality was observed despite a small reduction of crp following statin treatment. however, as the interaction between crp and the effect of rosuvastatin was not checked, we do not know if the effects of statins on the outcome differ between inflamed and uninflamed dialysis patients. no large study has yet tested if anti - cytokine therapy affects surrogate biomarkers of inflammation, morbidity, and mortality. however, as therapy targeting il-1-mediated inflammation not only reduces inflammation biomarkers but also holds promise for the treatment of glycemic control in type 2 diabetic patients, it can be speculated that targeted il-1 antagonism may also benefit uremic patients. in a recent pilot study of 10 dialysis patients that were randomly assigned to receive either the tumour necrosis factor - alpha (tnf-) antagonist etanercept or placebo twice weekly for 44 weeks, no significant effects on inflammatory biomarkers were observed. however, as a significant difference in the time - dependent effects of etanercept on prealbumin was observed (levels increased by 20% in the etanercept group and decreased in the placebo group), favorable effects on nutritional markers may be anticipated. as no adverse side effects were observed, administration of tnf- receptor antagonism seems safe in selected dialysis patients despite their documented increased risk of infectious complications. thus, larger studies are now needed to test the effects of selective anti - tnf, il-1, and il-6 therapies, respectively, not only on inflammatory and nutritional surrogate markers but also on morbidity and outcome. no doubt, the major problem in such a rct will be to select the patients with persistent non - infectious uremic inflammation. until adequately powered rcts have been performed with anti - inflammatory treatment strategies, no specific pharmacological treatment recommendations for uremic inflammation can be advocated. however, clinicians should be aware of the strong association between persistent inflammation and outcome before carefully evaluating (and if possible treating) the multiple putative causes of persistent inflammation in this patient group. although the clinician may minimize some aggravating factors that promote inflammation in the context of renal disease, it may not be possible to get rid of inflammation in a clinical situation. due to the fact that causes both unrelated to dialysis (such as intercurrent infectious and inflammatory complications, peridontitis, and ischemic heart disease) and related to dialysis (such as bioincompatibility, residual renal function, infections of vascular access, dialysis hypotension, impure dialysate, and volume overload) can contribute to uremic inflammation, the clinician needs to carefully evaluate all chronic kidney disease patients with signs of inflammation such as elevated crp levels. indeed, a recent study demonstrated lower cardiovascular mortality in dialysis units in which crp was regularly monitored.	since the first reports in the late 1990s connecting elevated circulating levels of c - reactive protein in patients with end - stage renal disease with an atherogenic, wasted phenotype and poor outcome, more than 3600 publications related to the subject have appeared on the medline bibliographic database. this reflects the exponential interest that this topic has evoked in the field of nephrology, and the possibility of treating this common uremic complication has been much discussed. several small studies have implied that various nutritional and pharmacological treatment strategies have beneficial effects on surrogate markers of inflammation. however, no randomized controlled trials on anti - inflammatory treatment have yet been performed to test the hypothesis that persistent low - grade inflammation contributes to uremic morbidity and mortality.
it has been used primarily by military, construction workers, fire fighters to see through smoke, and to spot heat leaks or the enemy. there are few reports and small - scale studies done in various disciplines of ophthalmology. it has been used in the diagnosis of dry eye and to evaluate bleb functionality after glaucoma surgery. it has also been used to demonstrate low ocular temperature in vascular occlusions and high temperature in ocular inflammation. this is a report on the study of thermographic images of ocular inflammatory and non - inflammatory conditions. a non - contact thermographic camera (flir p 620) was used to take thermal pictures of seven cases of ocular inflammation, two cases of non - inflammatory ocular pathology, and one healthy subject with mild refractive error only. ocular inflammatory cases included five cases of scleritis (three anterior and two posterior), one case of postoperative anterior uveitis, and a case of bilateral meibomian gland dysfunction with keratitis (mgd - keratitis). non - inflammatory conditions included a case of conjunctival benign reactive lymphoid hyperplasia (brlh) and a case of central serous chorio - retinopathy (cscr). thermal and non - thermal photographs were taken using same magnification and same distance from the eye at atmospheric temperature of 27c, humidity 60%, and with stable airflow. physiological blinking was allowed, no ocular medications were applied, and thermal barriers like eyeglasses or contact lenses were removed before photography. all images were transferred to a computer, and using analyzing software, average ocular surface temperature (ost) and temperature at the area of interest were calculated. the eye of the patient with a fresh episode of scleritis, which was not on treatment, showed hot colors on thermography [figure 1 ] indicating higher temperature in right eye compared to fellow healthy eye. eyes with scleritis on treatment showed slightly higher temperature compared to the fellow eye. a case of resolved necrotizing scleritis showed lower temperature at the site of the necrosis surrounded by normal tissue temperature [figure 2 ]. eye with postoperative anterior uveitis also revealed increased temperature, but paradoxically, eyes with mgd - keratitis depicted lower temperature in clinically more affected right eye (od) [figure 3 ]. eye with conjunctival brlh showed lower temperature at the site of the lesion [figure 4 ]. eyes with only refractive error but no other pathology showed no temperature difference between both the eyes. od showed higher temperature compared to the fellow healthy eye a 65-year - old female presented with necrotizing scleritis. temperature at the site of the necrosis (arrow) was 31.8c a 35-year - old male presented with bilateral mgd - keratitis. paradoxically, od shows low temperature as compared to os a 34-year - old male presented with conjunctival brlh. ocular thermography is a relatively newer imaging modality. attempts to calculate normal eye temperature were made way back in 1950s using contact thermometry. in 1968, r. mapstone introduced infrared thermometry using bolometer, a non - contact method of ocular thermometry. the normal corneal temperature measured by thermography ranges from 35.4 0.1c to 34.01 0.64c, and the mean inter - ocular difference of ost in normal individuals our first patient, a fresh case of scleritis who was not on any treatment, had higher ost difference (t) between the two eyes as compared to the eyes on treatment for more than a week [table 1 ]. this may suggest that ocular temperature rapidly decreases with anti - inflammatory treatment and has a positive correlation with the signs and symptoms. in this study, the case of mgd - keratitis (case 6) paradoxically showed low temperature in clinically more affected eye (od). rolando. also noted similar finding in dry eye and suggested that the ocular surface would be 0.10c lesser in dry eyes due to the increased rate of evaporation. hence, applying the same logic, it is clear that in case 6 in the present study, due to decreased tear film lipids owing to mgd, the worse eye (od) was subjected to rapid evaporation of the tear film which resulted in a relatively colder eye on thermography. detailed patient information and temperature difference between affected and unaffected eye thermography can also be used to distinguish between a cold lesion of non - inflammatory condition from a hot lesion of ocular inflammation. conjunctival brlh is a poorly vascularized tumor, and hence, case 8 in the present study showed low temperature at the site of the lesion. in contrast case 1 who came with similar clinical presentation showed high temperature, suggestive of inflammation due to scleritis. our cases of posterior scleritis had minimal increase in temperature as compared to the non - affected eye, as they were on treatment. this may suggest that posterior segment inflammation can also cause increase in ost, which can be picked up on thermography when posterior segment examination is difficult as in the case of mature cataract or when it is normal as in the case of mild posterior scleritis. ocular thermal imaging is an underutilized diagnostic tool which can be used to distinguish inflammatory ocular conditions from non - inflammatory conditions. it can also be utilized in the evaluation of tear film in dry eye syndrome and other corneal pathologies.	background and objectives : the purpose of this study was to evaluate ocular inflammatory and non - inflammatory conditions using commercially available thermal camera.materials and methods : a non - contact thermographic camera (flir p 620) was used to take thermal pictures of seven cases of ocular inflammation, two cases of non - inflammatory ocular pathology, and one healthy subject with mild refractive error only. ocular inflammatory cases included five cases of scleritis, one case of postoperative anterior uveitis, and a case of meibomian gland dysfunction with keratitis (mgd - keratitis). non - inflammatory conditions included a case of conjunctival benign reactive lymphoid hyperplasia (brlh) and a case of central serous chorio - retinopathy. thermal and non - thermal photographs were taken, and using analyzing software, the ocular surface temperature was calculated.results:patient with fresh episode of scleritis revealed high temperature. eyes with mgd - keratitis depicted lower temperature in clinically more affected eye. conjunctival brlh showed a cold lesion on thermography at the site of involvement, in contrast to cases of scleritis with similar clinical presentation.conclusion:ocular thermal imaging is an underutilized diagnostic tool which can be used to distinguish inflammatory ocular conditions from non - inflammatory conditions. it can also be utilized in the evaluation of tear film in dry eye syndrome. its applications should be further explored in uveitis and other ocular disorders. dedicated ocular thermographic camera is today 's need of the hour.
	we present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fu) and 5-hydroxymethyluracil (5-hmu), found naturally in dna. inherent reactivity differences have enabled us to tag 5-fu chemoselectively over its c modification counterpart, 5-formylcytosine (5-fc). we rationalized the enhanced reactivity of 5-fu compared to 5-fc via ab initio quantum mechanical calculations. we exploited this chemical tagging reaction to provide proof of concept for the enrichment of 5-fu containing dna from a pool that contains 5-fc or no modification. we further demonstrate that 5-hmu can be chemically oxidized to 5-fu, providing a strategy for the enrichment of 5-hmu. these methods will enable the mapping of 5-fu and 5-hmu in genomic dna, to provide insights into their functional role and dynamics in biology.
laparoscopic appendectomy, first performed by semm in 1983, has become increasingly popular. the surgical management of complicated pediatric appendicitis (gangrenous or perforated) has been more controversial, and the role of laparoscopy in its treatment has not yet been established. intraabdominal abscess (iaa) can form after laparoscopic appendectomy (la), especially in complicated cases and is associated with significant morbidity. the aim of our study was to establish the incidence of iaa formation in complicated pediatric appendicitis (cpa) treated with laparoscopy. pediatric patients presenting with acute appendicitis were identified from our prospectively collected database. between march 1996 and october 2001, a standard 3-trocar technique is used with two 5-mm trocars and one 12-mm trocar for the introduction of the stapling device (endogia, united states surgical corporation, norwalk, ct). the appendix is freed, and the mesoappendix is divided between applied clips or staples. the appendiceal base is divided with the stapling device ; the freed appendix is placed in an endoretrieval bag and removed. when present, a special effort is made to debride the fibrin peel from the peritoneum. patients with cpa have a jackson - pratt drain placed in the pelvis and right lower quadrant after completion of the appendectomy ; the drain exits through the suprapubic port site. after removal of the trocars, the fascia and the skin edges are approximated with absorbable sutures. postoperative analgesia and perioperative intravenous antibiotics are administered in the same fashion as in open appendectomy. noncomplicated cases received antibiotics only perioperatively, but patients with cpa received antibiotics during their entire hospital stay. an oral diet was resumed usually within 24 hours after admission. the diagnosis of acute appendicitis in all patients was based on clinical and computed tomography (ct) scan findings. patients with a positive ct scan underwent laparoscopy, and all of them had appendicitis. patients with vague symptoms and negative ct imaging were observed and did not undergo laparoscopy. thirty - five (67%) male and 17 (33%) female patients underwent surgery. five of the 52 patients (10%) had cpa. among the patients with cpa were 2 female (40%) and 3 (60%) male patients (table 1). one of the 5 (20%) patients with cpa developed postoperative intraabdominal abscess and underwent laparoscopic drainage during the same admission. the single postoperative abscess occurred early during our initial experience with laparoscopic appendectomy, and it was the first case of cpa performed laparoscopically. the average operative time for laparoscopic appendectomy has been 60 minutes (range, 30 to 120 minutes) and recently 40 minutes. in cases of cpa, the average operative time was 130 minutes (range, 120 to 200 minutes). at the same time, the average length of stay after laparoscopy in noncomplicated acute appendicitis was one day. the cost of the laparoscopic surgery was difficult to calculate because of the retrospective nature of our study. in recent cases, the specifics of the equipment used were not always available except for the standard stapling device. all these patients underwent laparoscopic appendectomy plus drainage if the appendix was gangrenous or perforated. all patients with noncomplicated appendicitis were effectively and definitively treated with laparoscopy, and almost all of them were discharged home within 24 hours. our findings corroborate previously established knowledge that appendicitis can be safely and effectively treated in children through laparoscopy. up to 21% of patients presenting with acute appendicitis can have a perforated appendix. in our series, 5 (10%) of the 52 patients presenting with acute appendicitis all of these 5 patients with cpa were treated with laparoscopy without conversion to open technique. although the patient underwent an extra laparoscopic procedure, it was well tolerated, and he was discharged home without other complications. this single complication occurred during the initial learning curve of laparoscopic appendectomy, was the first case of laparoscopic appendectomy performed for cpa at our institution, and was associated with a long operative time. since then, as experience has accumulated, no more complications have occurred after laparoscopic appendectomy for cpa.. a high degree of suspicion and liberal use of imaging helps in the diagnosis and early treatment of postoperative iaa. in a recent study, it was suggested that the placement of drains in the right lower quadrant might be beneficial in patients with perforated appendicitis and localized abscess cavities. in that study, it has been suggested in the past that laparoscopic appendectomy for cpa in children is not a safe procedure and should be avoided. our recent findings suggest that laparoscopy can be a reasonable therapeutic alternative to open appendicitis in these complicated cases. the incidence of postoperative iaa formation after laparoscopy in children with cpa varies anywhere between 5.8% and 41%. some of these abscesses are treated with antibiotics, but most of them require drainage. in another recent study, comparing laparoscopy to open appendectomy for cpa, no iaa occurred after la. it has been suggested that visualization of the abdominal cavity seems to be improved with laparoscopy. the pelvis and the abdominal cavity can be thoroughly irrigated, debrided, and drained. the surgery is less traumatic, and most patients return to their regular activities sooner. on the other hand, in cases of perforated appendicitis, anatomy can be obscure, and the operative time can be significantly prolonged. laparoscopy in complicated cases of acute appendicitis can be a challenging and technically demanding procedure that requires more than basic laparoscopic skills. there is limited tactile sensation, manipulation of inflamed tissues is difficult, and inadvertent injury to adjacent organs is possible. one should attempt laparoscopy in these complicated cases only after performance of many routine laparoscopic appendectomies. it can not be overemphasized that one should be prepared to convert early to an open appendectomy should a difficult case of cpa be encountered. that is especially true when the surgeon is on the initial part of the learning curve. our data were retrospectively collected, and the numbers are too small to draw safe conclusions. our findings and those of others suggest that laparoscopy might be a safe alternative even for cpa, but properly randomized, prospective studies will answer this question. complicated appendicitis in children can be effectively treated with laparoscopy by experienced laparoscopic pediatric surgeons. complicated appendicitis should not be considered a contraindication for laparoscopy, but more solid evidence is necessary before it becomes the standard treatment. close postoperative follow - up and a high index of suspicion for development of complications is essential. prospective randomized trials will be necessary to establish the role of laparoscopy in complicated appendicitis in the pediatric population.	background : complicated appendicitis (gangrenous or perforated) has been associated with increased risk for postoperative complications, especially intraabdominal abscess. caution has been advised when attempting laparoscopic appendectomy for complicated appendicitis in children. the objective of our study was to assess the incidence of intraabdominal abscess formation after laparoscopic appendectomy in pediatric patients presenting with complicated appendicitis.methods:this is a retrospective review of 52 pediatric patients presenting with acute appendicitis at a single teaching institution who underwent laparoscopic appendectomy by a single surgeon. all laparoscopic procedures were completed without conversion. treatment complications and outcomes were recorded for all cases.results:five of the 52 patients (10%) had complicated appendicitis. one of the 5 patients (20%) developed intraabdominal abscess postoperatively and underwent laparoscopic drainage during the same admission. no other complications were noted. none of these patients was readmitted for wound infections or intraabdominal abscesses. the single postoperative abscess occurred early during our initial experience with laparoscopic appendectomy.conclusion:laparoscopic appendectomy seems to be a safe alternative for the treatment of complicated appendicitis in children. caution is recommended during the initial experience of surgeons with this procedure, because the complication rate seems to be higher during the learning curve. close postoperative follow - up and a high index of suspicion for development of complications is recommended. as surgeons ' experience accumulates, the safety of the procedure seems to increase. a prospective, randomized trial is recommended to establish the role of laparoscopy in complicated appendicitis in the pediatric population.
after one year of preparatory work a model of cooperation for patients with chronic wounds was implemented in the hospital krankenhaus gttlicher heiland in june 2009. principals of the project are the vienna health insurance and vienna 's medical association, the practical implementation lies mainly in the responsibility of the krankenhaus gttlicher heiland. this project is arranged for the duration of two years ; within this time an integrative model of medical care involving hospital, doctors in the extramural sector and the medical mobile nursing care will be implemented. dr. viktor grablowiz (chief of the surgery department in krankenhaus gttlicher heiland) show, how such an approach of an integrative wound management can reduce treatment time by 50%.	description of projectafter one year of preparatory work a model of cooperation for patients with chronic wounds was implemented in the hospital krankenhaus gttlicher heiland in june 2009. principals of the project are the vienna health insurance and vienna 's medical association, the practical implementation lies mainly in the responsibility of the krankenhaus gttlicher heiland. this project is arranged for the duration of two years ; within this time an integrative model of medical care involving hospital, doctors in the extramural sector and the medical mobile nursing care will be implemented.resultsfirst experience reports of prim. dr. viktor grablowiz (chief of the surgery department in krankenhaus gttlicher heiland) show, how such an approach of an integrative wound management can reduce treatment time by 50%.
denitrification is an important process in biology that involves the sequential reduction of nitrate (no3) to nitrite (no2), nitric oxide (no), nitrous oxide (n2o), and finally to dinitrogen (n2), carried out by several different metalloenzymes. n2o + h2o) is a key step of this process and is catalyzed by nitric oxide reductases (nors). no is an important molecule in biology because it impacts events ranging from blood pressure regulation, neurotransmission, and immune response in mammalian cells to transcriptional regulation and biofilm formation in bacteria. the presence of nors in pathogenic bacteria such as pseudomonas aeruginosa helps to detoxify no and allow the bacteria to survive. furthermore, an increase in n2o production caused by the use of artificial fertilizers generated from artificial nitrogen fixation has disrupted the global nitrogen cycle, as well as highlighted n2o s potent ability to deplete ozone. despite the biochemical, biomedical, and environmental significance of nors, structural features responsible for its activity and a clear mechanistic understanding of its reaction, particularly the membrane - bound nors from bacteria, are not well understood. bacterial nor is a complex enzyme consisting of a c - type heme, a heme b, and a heme b3/nonheme iron (feb) center. electrons are delivered from heme c to heme b and then to the heme b3/feb active site, where no is reduced to n2o (figure 1). the active site of nor consists of a high - spin (hs) heme b3 and feb coordinated by three histidine and one glutamate residues (figure 1). three mechanisms of no reduction by nors have been proposed (scheme 1). briefly, the trans mechanism suggests that both heme b3 and feb sites bind no, one each, before n n bond formation, while in the cis heme b3 mechanism, a second no electrophilically attacks a heme - bound no. in the cis feb mechanism, representation of the electron - transfer pathway from cnor (left) and the active site structure including feb (orange sphere) pdb 3o0r (right). enzymatic and mechanistic studies of native bacterial nors are complicated by the presence of several metal sites (three hemes on a nonheme iron ; see figure 1), which makes spectroscopic studies difficult, as well as difficulties in purifying the protein in high yield and homogeneity because nors are membrane proteins. synthetic models of nor have been used to complement the study of the native enzyme to great success. the recent progress made in synthetic modeling has been summarized by other articles in this special forum and will not be duplicated here. to complement both native enzyme and synthetic modeling approaches, we have used small, stable, easy - to - purify, and well - characterized proteins such as myoglobin (mb) as scaffolds to make biosynthetic models of more complex metalloenzymes. while a great deal of effort has been put forth to understand both the structure and function of native enzymes and their variants using biochemical and biophysical methods, an ultimate test of our knowledge of this class of enzymes is creating functional models that mimic both the structure and function of native enzymes. in contrast to studying native proteins in a top - down approach, which can identify necessary structural features responsible for function, biosynthetic modeling is a bottom - up approach that elucidates structural features sufficient for activity. furthermore, the biosynthetic models may be amenable to investigation in ways that have not yet been developed for the native enzymes (e.g., replacement of the heme in the active site with a non - native cofactor, such as zinc(ii) protoporphyrin ix (ppix)). on the other hand, thanks to recent progress in molecular biology and protein biochemistry, protein models can now be more readily prepared than by the chemical synthesis of models of complex metalloenzymes such as nor because the latter method requires rigorous synthetic skills. for example, it normally takes about 1 week to construct, express, and purify protein models with a yield of 100 mg / l of escherichia coli culture ; it would take much longer to prepare heme feb models chemically with lower yield because the synthesis of porphyrin - containing models is quite challenging and requires multistep synthesis. despite challenges associated with preparing synthetic analogues of complex enzymes, remarkable progress has been made to understand the structure / function of complex enzymes using synthetic models. furthermore, it is becoming clear that noncovalent, secondary coordination sphere interactions around the primary coordination sphere, such as hydrophobicity and hydrogen - bonding interactions, often involving structurally well - defined water, can play a key role in enzymatic function. addressing these issues in modeling requires a rigid framework that allows the introduction of these elements at specific locations. biosynthetic modeling is an ideal choice in addressing this issue because such secondary coordination sphere interactions can be conveniently introduced at a specific location of the rigid protein scaffold, without elaborate synthesis of model compounds that may be more flexible. therefore, although biosynthetically designed protein models are intermediate in complexity compared to the complex native proteins and synthetic analogues, they contain many features of both the proteins and small - molecule models, providing us with unique constructs to understand the structure, function, and mechanism of complex metalloenzymes. in this article we will use the following nomenclature convention to designate various biosynthetic models and their corresponding metallated and nitrosylated derivatives. febmb1 and febmb2 represents the first and second generations of biosynthetic model proteins, respectively. the corresponding metallated derivatives are represented as m - febmb1(fe) where the m represents a metal ion with the designated oxidation state (ii) occupies the nonheme feb center and the fe represents fe - protoporphyrin ix (heme) in the heme - binding site. when the feb center is empty, it will be represented as e - febmb1(fe). when the fe - protoporphyrin ix (heme) is replaced with zn - protoporphyrin ix, it will be designated as m - febmb1(zn). based on this convention, fe - febmb1(fe) and fe - febmb1(fe) the corresponding nitrosyl derivatives, feno - febmb1(zn) and fe - febmb1(feno), indicate no binding to the nonheme feb center and heme fe center, respectively. in order to complement studies of native nors and its synthetic models, our group utilizes small, easy - to - purify, and well - characterized proteins like mb as the scaffold to prepare biosynthetic models of nors. this endeavor was built upon our initial success in using mb to prepare structural and functional models of heme copper oxidases (hcos) by introducing a cub center in the distal pocket of sperm whale myoglobin (swmb) through l29h / f43h mutations (called cubmb). because nors and hcos belong to the same superfamily with similar overall structural folds, and some hcos have been shown to exhibit nor activity, we first investigated the cross reactivity of this hco mimic cubmb to reduce no and found that the presence of cu at the cub site in cubmb indeed displayed nor activity with consumption of 2 mol of no / mol of cubmb / min, similar to that of hco from thermus thermophilus (3 mol of no / mol of hco / min). encouraged by the above success, we turned our attention to mimic both the structure and function of native nors. at the time of our pursuit, there was no crystal structure of nor available to guide the rational design of a nor model using mb. however, biochemical studies and sequence homology analysis have indicated that, in addition to the presence of fe in the feb center (instead of cu in the cub center), nors contain at least two conserved glu residues in the active site that are absent in the hcos. because cubmb did not bind fe and thus did not show any nor activity in the absence of a metal ion in the nonheme metal center, we decided to introduce a glu to the cubmb. after evaluation of several positions to introduce the glu through computer modeling and energy minimization, we found the best candidate to be v68e, called e - febmb1(fe) (l29h / f43h / v68e swmb). this protein binds fe readily (figure 2a) and the metal bound fe - febmb1(fe) displays nor activity, making it the first structural and functional model of nor. overlays of cnor (yellow) with (a) fe - febmb1(fe) (cyan), (b) fe - febmb2(fe) (green), and (c) fe - febmb1(zn) (magenta). feb sites are shown as brown spheres and amino acid residues as sticks, and the water molecule involved in hydrogen bonding is shown as a cyan sphere in part b. because there are at least two conserved glu in the active site of nors, we decided to investigate the role of the second glu in the biosynthetic models. while there is no room to introduce the second glu in the primary coordination sphere of the feb center, we evaluated introducing the second glu in the secondary coordination sphere of the feb center. we found i107e to be at an ideal location to provide an extended hydrogen - bonding network around the feb center ; thus, a second - generation model (i107e - febmb1(fe), called e - febmb2(fe) was prepared, which also bound fe(ii) in the feb site and the metallated derivative fe - febmb2(fe) improved the nor activity over fe - febmb1(fe) by nearly 100%. both fe - febmb1(fe) and fe - febmb2(fe) were prepared before the publication of the first crystal structure of cytochrome c dependent nor (cnor). after the crystal structure of cnor became available, we overlaid the crystal structures of fe - febmb1(fe) and fe - febmb2(fe) with that of native nor (figures 2a, b) and were pleased to see that, in addition to displaying nor activity, both biosynthetic models mimic native cnor structurally. this accomplishment, achieved through computational modeling guided by homology modeling with structurally related proteins and by activity that mimics those of native enzymes, demonstrated the immense potential of biosynthetic approach in making close structural and functional models of native enzymes. spectroscopic studies of fe - febmb1(fe) and fe - febmb2(fe) using fourier transform infrared (ftir) and resonance raman (rr) have shown that heme - bound no adopts a strong nitroxyl character through interactions with the nonheme iron, and time - resolved rapid - mixing experiments provided evidence for both heme and nonheme nitrosyl complexes, supporting the trans mechanism. additionally, electron paramagnetic resonance (epr) studies of fe - febmb2(fe) reacted with excess no showed the formation of a five - coordinate low - spin (5cls) ferrous heme species due to cleavage of the proximal histidine bond. epr measurements taken below 30 k of fe - febmb1(fe) and fe - febmb2(fe) upon the addition of 1 equiv of no show signals at g = 6.1, which likely arise from exchange coupling of an s = /2 6cls { feno } heme and s = 2 nonheme fe. heme nitrosyl species have been spectroscopically probed, but a more complete understanding of the nonheme nitrosyl was limited to only a few studies because the spectroscopic signals of the heme often dominate the spectra of nors over the nonheme iron center, hampering our understanding of the role of nonheme iron in nor reactivity. recently, we have replaced the native heme (iron protoporphyrin ix, feppix) in fe - febmb1(fe) with znppix, which also bound fe(ii), and the fe - febmb1(zn) derivative allowed for a thorough spectroscopic and computational investigation into the feb nitrosyl complex selectively, without interference from the heme nitrosyl. by using uv vis absorbance, epr, and mssbauer spectroscopies, as well as x - ray crystallography (figure 2c) and density functional theory (dft) calculations, the nonheme nitrosyl was characterized as an s = /2 { feno } complex, using the enemark feltham notation, best described as a hs ferrous iron antiferromagnetically coupled to an no radical. to further probe the interaction of our nor model with no, we report here uv vis and nuclear resonance vibrational spectroscopy (nrvs) measurements. using this information, we can systematically characterize the intermediates illustrated in scheme 1 because they are likely to have distinctly different spectroscopic signatures. nrvs gives a complete and quantitative vibrational frequency spectrum for fe - enriched nuclei. it offers a selectivity similar to that of rr spectroscopy but is not bound by the optical selection rules of rr or ir spectroscopy. this is especially important in the study of nors, given that the fe no stretching mode vibrations are ir - silent and decompose upon laser irradiation. unfortunately, performing nrvs requires extremely concentrated protein samples (> 5 mm) that are impractical when working with native enzymes such as nor. however, studies have already been performed on mb and its mutants, thus offering our nor mimics a unique opportunity to utilize this advanced spectroscopic technique that would otherwise be inaccessible to native proteins. vis spectroscopy was used to monitor no binding to fe - febmb1(fe) and zn - febmb1(fe) during nrvs sample preparation. representative spectra are shown in figure 3. upon the addition of 1 equiv of no to fe - febmb1(fe), where the nonheme site was reconstituted with fe, the soret peak at 433 nm (figure 3, black curve) underwent a blue shift to 419 nm (figure 3, red curve), corresponding to the formation of a 6cls { feno } species and another broad peak at 398 nm corresponding to a 5cls { feno } species (figure 3, red curve and inset), and this assignment is confirmed by nrvs results (vide infra). the addition of 1 equiv of no to the zn - febmb1(fe) sample also caused a blue shift of the soret peak from 434 nm (figure 3, cyan curve) to 403 nm (figure 3, purple curve), corresponding to the formation of a 5cls { feno } species. vis spectra of fe - febmb1(fe) (black curve), zn - febmb1(fe) (cyan curve), and the corresponding mononitrosyl derivatives fe - febmb1(feno) (red curve) and zn - febmb1(feno) (purple curve). the inset shows deconvolution of the soret region of fe - febmb1(feno) demonstrating two components : one peak at 419 nm corresponding to the 6cls { feno } species and a second broader peak at 398 nm corresponding to the 5cls { feno } species. the presence of both 6cls and 5cls { feno } species is consistent with the nrvs results (vide infra). in the case of the zn - febmb1(feno) sample, the presence of the soret peak at 403 nm is also consistent with the presence of 5cls { feno } also observed by nrvs (vide infra). nrvs exploits technology developed at third - generation synchrotron light sources to monitor the vibrational properties of mssbauer nuclei, including fe. tuning of a monochromatic x - ray beam in the vicinity of the nuclear resonance reveals vibrational sidebands displaced from the recoilless resonance observed in conventional mssbauer spectroscopy. a growing number of nrvs applications exploit its exclusive and quantitative sensitivity to vibrational motions of the probe nucleus. specifically, each vibrational mode contributes to the measured signal in direct proportion to the mean - squared displacement of the probe nucleus along the beam direction, and well - established data analysis methods directly extract a partial vibrational density of states (vdos) for that measurement. for a randomly oriented ensemble of molecules containing fe, each vibrational normal mode contributes to the fe vdos an area equal to the fraction efe2 of the mode s kinetic energy associated with motion of the fe nucleus. the information content of the vdos is quantitative, allowing direct comparison with vibrational predictions on an absolute scale. the vdos is comprehensive because all vibrations involving fe contribute, without the artificial restrictions imposed by selection rules in more familiar vibrational spectroscopies (ir and raman). finally, the vdos is uniquely site - selective because only motion of the fe will contribute, even in a macromolecule containing thousands of other atoms. on the basis of these characteristics, nrvs is a uniquely valuable probe of protein active sites containing iron. several investigations have reported the vibrational dynamics of iron in heme proteins and iron porphyrins using nrvs. nrvs measurements on oriented single crystals of iron porphyrins exploit the sensitivity to motion along the direction of the x - ray beam to provide additional insights into the interpretation of the results on heme proteins. proteins with nonheme iron sites are equally amenable to nrvs investigations, which have informed the structural characterization of reaction intermediates in nonheme iron enzymes. vibrational spectra resulting from previous nrvs measurements on proteins containing multiple iron atoms contain superposed contributions from all sites. here, we demonstrate for the first time that we can specifically label either the heme or nonheme iron sites of fe - febmb1(fe) with fe, allowing us to independently monitor vibrations of iron at each site. partial unfolding of the protein at low ph allows removal of the heme and reconstitution of the protein with fe - enriched ppix, following the same procedure as that used for previous nrvs investigations on native mb. heme vibrations will dominate the nrvs signal of the reconstituted protein, even after the incorporation of natural abundance iron (or another metal) into the nonheme site because the natural abundance of fe is only 2%. similarly, the incorporation of fe into the nonheme site of e - febmb1(fe) reconstituted with natural abundance heme should allow us to specifically monitor the vibrations of the nonheme iron. the vdos determined from nrvs measurements on reduced fe - febmb1(fe) (figure 4) demonstrate that specific labeling of the heme and nonheme iron sites with fe allows us to distinguish vibrations at distinct sites within the same protein. the vibrational signal from the heme iron strongly resembles that reported for native mb. the dominant feature of the vdos includes contributions from vibrations of the axial fe nhis bond to his 93 and of the equatorial fe npyr bonds to the four heme pyrrole nitrogen atoms at approximately 230 and 250 cm, respectively. the fe nhis frequency is well - known from rr measurements on heme proteins, where this vibration is strongly enhanced upon excitation into the soret band. the nrvs signal is determined by the relative amplitude of iron motion and also includes the fe npyr vibrations, which are not easily observable using other spectroscopies. site - selective enrichment of fe - febmb1(fe) with fe allows independent monitoring of iron vibrations at either the heme or nonheme site (fe - febmb1(fe)). the upper and lower traces present the partial vdos of the heme and nonheme iron of the reduced proteins, respectively, derived from such measurements and reflect the distinct coordination of iron at each site. the heme vdos is nearly identical with that reported for reduced native mb from horse heart, where contributions from the fe nhis vibration perpendicular to the heme and vibrations of the in - plane fe npyr bonds to the heme pyrrole nitrogen atoms were identified. although individual vibrations are not resolved for the less symmetric nonheme site, the stiffness derived from the vdos (table 1), nevertheless, reflects the lower coordination of iron in this environment. here and in subsequent figures, error bars reflect experimental uncertainties determined from counting statistics, while solid traces represent a five - point running average of the experimental vdos. the nonheme iron of reduced fe - febmb1(fe) displays a clearly distinct vibrational signal dominated by a broad feature with a peak near 230 cm. the crystallographic model includes his 29, his 43, his 64, glu 68, and a water molecule as ligands to the nonheme iron. the relatively featureless signal observed for the nonheme iron apparently masks the vibrational structure that one might expect in light of this diverse ligand field, and the reduced symmetry in comparison with the heme site may further increase the vibrational complexity. we identified multiple unresolved vibrational modes contributing to a similar broad vibrational feature in reduced cytochrome c, based on a quantitative comparison with fe / fe isotope shifts observed in rr measurements, and attributed this complexity to the reduced symmetry of iron coordination in the distorted heme. conformational heterogeneity is well - documented for native mb and may also broaden vibrational features. regardless of the reasons, the unresolved vibrational complexity may hinder the identification of well - defined iron - ligand vibrations. fortunately, the vibrational information revealed by the nrvs measurement yields a quantitative measure of the coordination strength even in the absence of detailed vibrational frequency assignments. the vdos d() determines the stiffness,1an effective force constant that directly measures the force required to displace the iron with its coordination environment held fixed. the stiffness for both sites is much lower than that for the ls heme iron in reduced cytochrome c, where the stiffness was more than 300 pn / pm. the stiffness is consistent with the presence of a hs iron at both sites in reduced fe - febmb1(fe) and fe - febmb1(fe). the stiffness of the heme iron in reduced fe - febmb1(fe) is the same as that in native mb (table 1), confirming the expectation that the introduction of the nonheme metal site does not significantly affect the coordination strength of the heme iron. however, the stiffness of the nonheme iron environment is significantly lower than that determined for the heme iron. the slightly lower force restraining the iron in the nonheme site presumably reflects its reduced coordination. similar conclusions follow from data recorded on oxidized fe - febmb1(fe) (figure 5). a feature near 270 cm dominates the heme iron vdos, which strongly resembles that previously reported for native swmb. because iron ligand vibrations are undoubtedly the primary contribution to this feature, this indicates that the ligation of the heme iron, to his 93 and to a neutral water molecule, is the same in fe - febmb1(fe) as it is in native mb. in particular, these data provide no indication for an oxo group bridging the two iron sites, as observed for the oxidized state of nor (scheme 1), and the hs nrvs signal from the heme iron contrasts with the ls heme iron reported for cnor from ps. the iron vdos of oxidized fe - febmb1(fe), shown in the upper trace, strongly resembles that reported for native mb from sperm whale, indicating that coordination of the heme iron is unaffected by the presence of the additional nonheme iron engineered in the distal pocket. in spite of the limited signal, the nonheme iron vdos in fe - febmb1(fe) (lower trace) clearly reports vibrations from a distinct iron site characterized by a reduced coordination strength. as seen above for reduced fe - febmb1(fe), the vdos for the nonheme iron is clearly distinct from that for the heme iron, in spite of a relatively low fe concentration and a consequently reduced signal in the fe - febmb1(fe) sample. this dual confirmation of successful site - specific labeling of each site illustrates the opportunity to probe the reactivity of each iron independently. the iron vdos determines an additional averaged force constant, the resilience2which provides information distinct from the stiffness. as defined more generally by zaccai, the resilience3measures the rate at which the mean - squared displacement xfe of the probe atom (here, iron) increases with temperature. nrvs lacks the energy resolution to capture highly anharmonic motions that contribute to temperature - dependent measurements of xfe using techniques such as inelastic neutron scattering or mssbauer spectroscopy above a dynamical transition near 200 k. on the other hand, at temperatures below 200 k, we have shown quantitative agreement between determinations of xfe from mssbauer measurements on oxidized cytochrome c at a series of temperatures and the values expected on the basis of the iron vdos determined using nrvs at a single temperature. the vibrational contribution to the resilience (eq 2) captures this temperature variation in a single parameter, with lower values of the resilience characterizing environments with large fluctuations of the fe probe atom. the resilience spectrum (eq 4) suppresses contributions from localized iron ligand vibrations and highlights low - frequency oscillations of the protein that drive translational motion of both iron sites in reduced fe - febmb1(fe) and fe - febmb1(fe). quantitative agreement between the areas determined for both sites yields values for the resilience that are identical, within experimental uncertainty (table 1). nevertheless, comparison as a function of frequency reveals subtle differences in the coupling of long - range protein fluctuations to these two sites. low - frequency vibrations play the primary role in determining the resilience, as we illustrate by directly plotting the integrand4 in eq 2 as a resilience spectrum in figure 6. the resilience is equal to the inverse of the area of this spectrum and is primarily determined by vibrations below 100 cm. molecular dynamics simulations on mb and cytochrome c show similar mean - squared displacements for all heme atoms including iron, supporting our suggestion that translation of the heme in response to fluctuations of the embedding protein matrix make the primary contribution to the nrvs signal below 100 cm. as a result, we interpret the resilience as a measure of the elastic properties of the protein environment. previously, we found a significant increase of the resilience in cytochrome c in comparison with mb. here, we find that the resiliences of reduced fe - febmb1(fe) and native mb are the same, within experimental uncertainty, suggesting that the introduction of the additional nonheme metal site does not seriously perturb the elastic properties of the protein. moreover, small differences in the coupling of the protein fluctuations to the heme and nonheme iron sites, apparent from examination of figure 6, average out to yield values for the resilience that agree quantitatively for the two distinct sites, within the experimental uncertainty. this further supports the notion that the resilience quantifies global properties of the embedding protein and contrasts with the sensitivity of the stiffness to differences in the coordination of the two iron sites. in short outer - sphere force constant that probes the elasticity of the embedding protein, in contrast with the probe of the immediate coordination environment, as quantified by the stiffness. the vibrational dynamics of the heme iron undergo noticeable changes upon exposure to no. the vibrational signal from fe - febmb1(feno) containing fe - enriched hemes covers a wider frequency range than that in the absence of no, with significant features resolved beyond 500 cm (figure 7). the experimentally determined stiffness for zn - febmb1(feno) exceeds 300 pn / pm (table 1), indicating a substantial increase in the coordination forces exerted on the iron. we observed stiffnesses exceeding 300 pn / pm for the ls heme iron in reduced cytochrome c. the heme iron vdos reveals that the presence of a second metal in the nonheme site influences no binding to fe - febmb1(fe) and zn - febmb1(fe). no stretching vibrations, clearly resolved above 400 cm, probe the axial ligation. for reference, dashed lines indicate fe no stretching frequencies reported for native horse heart mbno (452 cm), characteristic of a six - coordinate complex with no coordinated trans to a histidine ligand, and for fe(dpix)(no) (528 cm), a typical five - coordinate heme no complex. a substantial fraction of hemes exhibit an fe no stretching frequency characteristic of five - coordinate heme nitrosyls when either zn or fe is present in the nonheme site. this contrasts with previous measurements on native mbno, which revealed a nrvs signal consistent with six - coordinate heme no. the presence of well - resolved features yields more specific information on individual fe ligand bonds. in particular, previous nrvs measurements have identified an fe no stretching mode in the 520530 cm range in five - coordinate heme no complexes, binding of an imidazole ligand trans to no weakens the fe no bond, and we observe this mode at lower frequencies in the 450460 cm range for these six - coordinate heme no complexes. one well - characterized six - coordinate heme no complex is native mbno, where this fe no vibration appears at 452 cm and contributes to both the rr and nrvs signals. unlike native mbno, fe no stretching frequencies near 530 cm characteristic of five - coordinate heme no contribute to the experimental vdos of fe - febmb1(feno) and zn - febmb1(feno) upon the introduction of a divalent metal in the nonheme site (figure 7). this result supports previous evidence for formation of a five - coordinate heme no complex in fe - febmb1(feno) exposed to excess no, which was based on observation of the same fe no vibration at 522 cm using rr spectroscopy as well as the presence of a 1660 cm n o stretching frequency in ir measurements. the altered heme ligation in response to the neighboring nonheme metal contrasts with the insensitivity of the unligated heme to the nonheme metal noted above (figures 4 and 5) and demonstrates that the nonheme metal specifically influences the structure of the heme ligand complex. in the presence of 1 equiv of no, the fe - febmb1(feno) vdos (figure 7) also has a feature with a 480 cm frequency that we attribute to six - coordinate heme no. because the nrvs signal depends only on the mean - squared vibrational amplitude of the iron and on the relative population of contributing species, the fe - febmb1(feno) vdos suggest comparable amounts of five- and six - coordinate heme no (figure 7). interestingly, the fe no frequency is significantly increased with respect to that observed for native mbno, providing additional information on how the nonheme metal influences the electronic structure of the neighboring heme no complex. the contribution of a vibrational signal attributable to six - coordinate heme no is significantly reduced for zn - febmb1(feno) in the presence of 1 equiv of no. 560 cm in six - coordinate heme no complexes. experimental characterization of its kinetic energy distribution based on isotope shifts indicates that this vibrational mode primarily involved motion of the central nitrogen atom of the feno unit. on this basis, this n - centered vibration can be qualitatively described as an feno bending mode to distinguish it from the fe no stretching mode that contributes more strongly to the nrvs signal. however, it must be emphasized that neither mode can exhibit pure feno bending or fe no stretching character for the nonlinear feno unit. both modes exhibit rather modest soret enhancement in raman scattering from six - coordinate heme no complexes, but the feno bending frequency is more reliably detected in heme proteins because of its large sensitivity to n / n substitution and is thus more often reported. although the iron amplitude and thus the nrvs signal is necessarily smaller for the feno bending vibration, the fe - febmb1(feno) vdos includes minor features near 380 and 580 cm consistent with contributions from the feno bending vibration of five- and six - coordinate heme no complexes, respectively, supporting conclusions based on the stronger fe no stretching frequency discussed above. raman and ir measurements on fe - febmb1(feno) resulting from reaction with stoichiometric no also identify feno bending and n o stretching frequencies that are 1520 cm higher and 7080 cm lower, respectively, than those typically observed for six - coordinate heme no. together, the unusual values for all three vibrations of the feno fragment suggest that the nonheme fe strongly perturbs the electronic structure of heme no. in particular, it is conceivable that the fe cation electrostatically predisposes the heme - bound no to the electron transfer that will ultimately be required for reactivity. as found above, the nonheme iron influences the coordination of the heme, strengthening the fe no bond and weakening the fe in contrast, the vibrational dynamics of the nonheme iron in fe - febmb1(fe) exposed to 1 equiv of no do not differ significantly from those observed in the absence of no (figure 8). this indicates that the ligation and electronic structure of the nonheme iron is insensitive to the structural and electronic changes that take place upon no binding to the heme iron. moreover, it indicates that the nonheme iron has a much lower affinity for no than the heme iron does. the vdos of the nonheme iron atom reveals perturbations in fe - febmb1(zn) when the nonheme site is saturated with excess no (see materials and methods for details of sample preparation). when fe is present in the heme site, on the other hand, the nonheme iron vdos exhibits no significant change upon reaction with stoichiometric no, in contrast with the clear signatures for no binding to the heme iron seen in figure 7. replacement of the heme iron with znppix eliminates the possibility of no binding to the heme and allows the investigation of no binding to the nonheme iron selectively. the vdos of the nonheme iron in the resulting fe - febmb1(zn) (figure 8) in the absence of no strongly resembles that observed for fe - febmb1(fe) under the same conditions (figure 4), indicating that the structure of the nonheme iron site is insensitive to the substitution of the heme metal. however, noticeable changes in the nonheme vdos of fe - febmb1(zn) take place in the presence of excess no (figure 9), which we attribute to the binding of no to the nonheme iron forming the feno - febmb1(zn) under these conditions. computational models for the no - ligated nonheme feb site in feno - febmb1(zn) using differing functionals yield quantitative predictions for the iron vdos. comparison with the experimental vdos for feno - febmb1(zn) in the presence of excess no is consistent with a substantial contribution from no - ligated iron. the red trace indicates the contribution from iron motion along the fe no bond direction and highlights the variability of the predicted fe no stretching frequency, which shifts from 376 cm using b3lyp to 454 cm using m06l. one significant advantage of the nrvs method is the relative ease of quantitative comparison with dft predictions. overall, such comparisons provide useful guidance for interpreting experimental results, but we have found that predicted vibrational frequencies for the feno fragment exhibit significant dependence on the functional used for dft calculations. for five - coordinate heme no complexes, predicted fe no stretching frequencies vary by nearly 200 cm. dft investigations of nonheme feno complexes also reveal significant variability of the electronic structure predicted using different functionals. it remains to be established whether any currently available functional adequately accounts for electron correlation in iron nitrosyl complexes. examination of a wide variety of functionals found that m06l gave the best overall account of the iron vdos for the five - coordinate nitrosyl heme complex fe(oep)(no). the vdos predicted using this functional is presented both as the lower trace in figure 9 and, for comparison, as a filled area behind the experimental vdos in the upper trace of figure 9. the m06l prediction does exhibit significant correspondence with the experimental signal, supporting the conclusion that direct no ligation accounts for the observed vibrational changes. unfortunately, because of the limited fe concentration, the relatively low signal level from this sample precludes an experimental identification of the fe no stretching frequency. the biosynthetic models have allowed us to provide insight into nors that is otherwise difficult to obtain in studying native enzymes. for instance, to the best of our knowledge, the nonheme feb in native nors has not been replaced or removed, making it difficult to assess the role of feb in the activity of nors. in contrast, because the biosynthetic models are purified without a nonheme metal ion, investigations into the role of iron or other nonheme metals is greatly simplified by changing the nonheme metal source that is used (e.g., fecl2 vs zncl2). therefore, our biosynthetic model allowed us to answer the previously unaddressed question of what would happen if feb was replaced with cub. given the structural homology between hcos and nors and their known cross reactivity, a fascinating issue arises as to the role of each class of enzymes different nonheme metal. activity assays using iron or copper as the nonheme metal ion both demonstrated nor activity, while controls using redox - inactive zinc did not possess nor activity. this study demonstrated a critical insight that a redox - active metal ion was needed to confer nor activity, an insight that would not be possible when studying the native enzymes. in addition, we also demonstrated that the glutamate ligand to feb is essential for both iron binding at the nonheme site and nor activity. finally, an extended hydrogen - bonding network was shown as a critical component in improved nor activity in our biosynthetic models when a glutamate residue (i107e), was introduced at the secondary coordination sphere into our model to facilitate proton transfer to the active site. the ultimate goal of studying native enzymes and their models is to unravel the details of how they work and apply that understanding to other related enzymes as well as biomedical and biotechnological applications. this goal can best be achieved by thorough mechanistic characterization, which has been achieved to great success in our biosynthetic models of nors. for example, although progress has been made in elucidating the structural aspects of nors aided by recent success in solving the x - ray structure of cnor, understanding the mechanism of the enzyme continues to be problematic due to several technical barriers. to illustrate, even though the isolated enzyme cnor is reactive to no with a moderate turnover rate under steady - state conditions, in the reduced form the enzyme shows very slow turnover in pre - steady - state conditions because of the presence of an obscure structural form of the enzyme. flash - flow experiments with the carbonyl complex of cnor can result in fast reaction kinetics. vis changes of the protein, which is dominated by the signals from the high - affinity heme site and does not provide any information on the events occurring at the feb site. apart from these experimental challenges, the presence of multiple configurations of the oxidized cnor further hinders our understanding of the mechanistic aspects. in one such configuration where the enzyme exists as a -oxodiferric complex, strong magnetic coupling between the five - coordinate heme fe (his is dissociated from heme iron in this form) and nonheme feb is observed, while in other cases, only weak magnetic coupling was observed. in addition, there are no experimentally accessible methods to selectively probe the no complex of the feb site because the high - affinity heme site dominates spectroscopic signatures including uv vis, epr, mssbauer, and nrvs. owing to these practical problems, n bond formation from cleavage of the n o bond (scheme 1). in the first route,, one no molecule binds each of the heme iron and nonheme feb sites in a trans configuration, where both iron centers are present as { feno } complexes. this step is followed by the reductive activation of the dinitrosyl moiety, leading to the formation of a hyponitrite dianion intermediate, where both iron centers are now oxidized to fe. in the cis heme b3 mechanism, supported by theoretical studies, the first no binds to the heme fe, followed by reductive activation of the no complex, which is stabilized by electrostatic interactions with feb. next, a second no electrophilically attacks the first heme - bound no, leading to the formation of a hyponitrite dianion, which is electrostatically stabilized by feb. finally, in the cis feb mechanism, both no units bind to the feb site and the hyponitrite dianion form is stabilized by electrostatic interactions with the heme fe. in all of these proposed mechanisms, it is also unclear how the dianion leads to the product formation, e.g., whether this hyponitrite intermediate becomes protonated, followed by chemical rearrangement of the complex, is also not well understood. with these hurdles in understanding the mechanism of nor using native enzymes, simpler protein - based model systems that are stable, easy - to - prepare, and well - characterized are needed. to this end, engineered e - febmb1(fe) and e - febmb2(fe) and their corresponding metallated and nitrosyl derivatives have provided the much needed insight into the mechanistic aspects of nors, as summarized below. resonance raman studies have shown that in the reduced form both these models exist as 5chs heme in both the absence and presence of fe in the feb site. however, in the presence of 1 equiv of no, both proteins loaded with the feb site form stable 6cls { feno } complexes at the heme site. one important revelation from these studies was the presence of exceptionally low (no) stretching and high (feno) frequencies compared to all reported 6cls heme nitrosyl complexes. these results were attributed to ferric heme iron(iii) nitroxyl (feno) complex, where no was stabilized by electrostatic interactions with the feb site. strong back - donation from heme iron caused an increase in the (feno) frequency, while the negative charge on no resulted in a decrease in the (no) frequency. in the event of excess no addition, both proteins form a [{ feno}]2 trans nitrosyl dimer, leading to n2o formation, supporting the so - called trans mechanism. under single - turnover conditions, using ftir studies, no n2o production was observed in febmb1, suggesting that the presence of the feb site is not enough to reduce no to n2o. however, in fe - febmb2(fe), 50% n2o production was observed, suggesting that the presence of the second glutamate is critical for n2o formation, presumably by facilitating proton transfer via a hydrogen - bonding network during turnover. unproductive complexes in both the proteins are characterized by a trans dinitrosyl complex, where the heme iron is present as a 5cls { feno } species with a dissociated heme his bond and a second no bound to the feb site. surprisingly, from stopped - flow and rapid freeze quench experiments, no binding to the feb site was observed to be kinetically favored with a t1/2 of 1 ms, followed by binding of the second no to the heme iron, leading to the trans dinitrosyl 6cls { feno}-febno complex. this finding provided experimental evidence that feb binds no before it is bound to heme b3, which was suggested previously, but not confirmed, in a study of ps. nautica nor. in feno - febmb1(feno), the dinitrosyl complex leads to the formation of a dead - end 5cls { feno } species, where the heme his bond is dissociated, but in feno - febmb2(feno), the presence of the second glutamate residue leads to 50% effective turnover, which results in a decreased rate of dissociation of the proximal heme his bond, leading to the formation of the dead - end complex. during the decay of the trans dinitrosyl complex (6cls heme { feno}/feb - no), the hyponitrite intermediate was not observed in either protein derivative, in contrast to proposed mechanisms. furthermore, when excess no was added after formation of the 6cls { feno } complex, the same dinitrosyl complexes of feno - febmb1(feno) and feno - febmb2(feno) were observed (vide supra), ruling out the formation of any electrophilically attached second no. this observation, therefore, does not support the so - called cis heme b3, as proposed by theoretical studies. as stated above, a major barrier to understanding the nor mechanism is the difficulty associated with isolating pure feb - no complexes due to the presence of a high - affinity heme site. to circumvent this critical methodological barrier in native nors, in a recent effort we spectroscopically probed no binding to the feb site after replacing the high - affinity heme with znppix. such a strategy can not be easily applied with native nors since the heme can not be selectively replaced because of the complex nature of the enzyme. from epr, mssbauer, and quantum mechanics / molecular mechanics calculations on the no derivative of feno - febmb1(zn), the electronic state of feb - no could be best described as hs s = /2 fe - no having a high ferrous character and a radical nature on no. the radical nature on no would promote n n bond formation by radical coupling with a heme - bound no and thus would support the trans mechanism. these results highlight the usefulness of biosynthetic models of complex enzymes within easy - to - produce and well - characterized proteins. taken together, the engineered nor models have provided important insights into the reaction mechanism of nor and support the proposed trans mechanism of no reduction by nors. results presented here exploit the ability to replace metals at either site, illustrating an important opportunity enabled by the biosynthetic approach. selective substitution with fe provides an independent structural probe for either of the two metal sites in fe - febmb1(fe). nrvs measurements quantify the forces exerted on fe by its coordination environment and indicate the presence of hs iron at both sites in the absence of substrate. reduced and oxidized proteins serve to model the initial and final states, respectively, in scheme 1, although the vibrational signals provide no evidence for a solvent - derived ligand bridging the metals in the oxidized state. rather, vibrations of the heme iron are comparable to those reported for native mb, confirming that the heme is unperturbed by the engineered nonheme site. on the other hand, the observation of distinct vibrational dynamics for the nonheme iron confirms successful site - specific labeling with fe. substitution of a redox - inactive zn ion for fe allows the preparation of stable mononitrosylated intermediates that would precede the formation of the putative (and unstable) dinitrosylated intermediates depicted in scheme 1. the presence of zn in the nonheme site perturbs the vibrational properties of the adjacent heme no complex, in a manner consistent with electron transfer to the no ligand. this suggests that the electrostatic influence of the nonheme fe in nors could act to promote the enzymatic reaction in either the trans or cis heme mechanism. with zn in the heme site, alteration of the nonheme iron vibrations upon exposure to no confirms that no can bind to the nonheme fe if the heme site is unavailable, as would be required in the trans mechanism. considerable advances in the biosynthetic modeling of nors have been achieved recently, and given that both the resting state and ligand - bound and reduced forms of cnor have been crystallized, our understanding of nor modeling should only improve moving forward. importantly, models that can perform enzymatic turnover will be critical because we are unaware of any model, biosynthetic or otherwise, that is capable of reducing no with turnover numbers comparable to those of the native enzymes. in conjunction with this, new models that more closely replicate the secondary coordination sphere of native nors must be developed because these interactions are critical for improving activity. with improved structural information into the active site of nor, fine - tuning of factors such as the heme the creation of such models will provide further insights into the reaction mechanism and activity of nors. all samples were prepared in a 50 mm bis - tris ph 7.3 buffer after chelexing overnight, followed by ph adjustment and filtration to remove chelex beads. buffers were degassed in a schlenk line for 5 h by several cycles of freeze pump thaw prior to their transfer into an anaerobic chamber (coy laboratories, inc.) for sample preparations. dry sephadex g25 beads (ge healthcare) were suspended in a buffer solution and degassed in the schlenk line for several hours before transfer into the glovebag. all solid materials were kept under vacuum overnight in the antechamber prior to transfer into the glovebag. all protein solutions were exchanged from a 100 mm phosphate ph 7 buffer to a 50 mm bis - tris ph 7.3 buffer outside the glovebag using small size - exclusion columns (pd 10 columns, ge healthcare) preequilibrated in an exchangeable buffer. the protein solutions were then degassed by three cycles of freeze pump thaw in a schlenk line and brought into the glovebag. a diethylamine nonoate (dea - nonoate ; 250 nm = 6.5 mm cm ; cayman chemicals) solution prepared in 10 mm naoh and used as the no source was degassed by three cycles of freeze pump thaw before transfer into the glovebag. a stock solution of fecl2 was prepared inside the glovebag by dissolving solid fecl2 in degassed water. e - febmb1(fe) was purified using a known protocol, as reported previously. a similar protocol was employed for e - febmb1(fe) purification except that in this case fe - labeled heme (frontier scientific) was used during the protein refolding step. the identity of each of the purified proteins was confirmed by electrospray ionization mass spectrometry. the r / z of pure protein was > 4 in a 100 mm potassium phosphate ph 7 buffer. molar extinction coefficients 406 = 175 mm cm for met e - febmb1(fe), 433 = 143 mm cm for deoxy e - febmb1(fe), and 427 of 136.2 mm cm for e - febmb1(zn) were used to determine the concentrations of the corresponding proteins. a total of 300 ml of deionized water and 1 ml of 9.14% methanolic hcl (285.6 l of 32% hcl + 714.4 l of methanol) were degassed and transferred into the glovebag. a total of 25 mg of fe metal (0.44 mmol ; cambridge isotope lab) was taken into a small dry nmr tube and transferred into the glovebag. the degassed water was transferred into a small water bath and heated to 60 c using a hot plate equipped with a stir bar. the nmr tube containing fe was immersed into the water bath, and 350 l of 9.14% methanolic hcl (0.88 mmol) was added to the tube. the reaction was allowed to proceed for 34 h until the gas evolution ceased. the schlenk flask was removed from the bag, immersed in a dry ice / ethanol slush bath, and slowly opened to vacuum in a schlenk line. the flask was then slowly warmed to 100 c using a water bath while in vacuum. after the solvent evaporated and the solid turned from green to white, the water bath was replaced with an oil bath and heated to 160 c, allowing the residual methanol to evaporate. the product was cooled to room temperature slowly, purged with argon, sealed, and weighed. nrvs data were collected at the advanced photon source on beamline 3id - d, as described in detail elsewhere. briefly, the x - ray energy was scanned in the vicinity of the fe nuclear resonance at 14.4125 kev in steps of 0.25 mev. data were recorded on frozen solutions at 510 mm protein concentration at temperatures of 6080 k, and each measurement was the average of 1535 scans. a comparison of early and late scans confirmed the absence of spectroscopic changes during x - ray exposure. met e - febmb1(fe) was reduced inside the glovebag with excess dithionite and passed through a small hand - packed size - exclusion column (sephadex g25) preequilibrated with a 50 mm bis - tris ph 7.3 buffer to remove excess dithionite and eluted with the same buffer. the eluted protein was then concentrated to 1 mm (433 nm = 143 mm cm), and the nonheme site was reconstituted with 1.0 equiv of either fecl2 or zncl2 (prepared freshly in the inert - atmosphere bag) added in aliquots of 0.25 equiv with 15 min between each addition. e - febmb1(zn) was transferred into the glovebag after degassing and concentrated to 1 mm (427 nm = 136.2 mm cm), and the feb site was reconstituted with fe without reducing the protein because znppix is redox - inactive. reconstitution of the feb site with fe or zn, as desired, was ensured by checking the uv vis spectrum after metal addition, as evidenced by shifting of the soret peak from 427 nm in e - febmb1(zn) and 433 nm in e - febmb1(fe), in the absence of the nonheme metal to 429 nm in feii - febmb1(zn) and 434 nm in fe - febmb1(fe) or fe - febmb1(fe) in the reconstituted protein, respectively. when applicable, oxidation of the heme iron and nonheme iron was achieved after reconstitution by the addition of excess ferricyanide and then passage through a small size - exclusion column. the feb reconstituted proteins were then concentrated to 1015 mm before loading 15 l of the samples into the well of a high - density polyethylene block sample holder inside the glovebag, transferred outside, and frozen immediately where other components (sapphire window, copper block, brass screws) were assembled. nitrosyl derivatives were prepared using the following protocol. for e - febmb1(fe), 1 equiv of no was added to the fe or zn reconstituted proteins present at 1 mm concentration. at each step, 0.25 equiv of no was added to the reconstituted proteins in the form of dea - nonoate, allowing enough time to for no release between each addition (t1/2 = 16 min at ph 7.3). no binding to the proteins was confirmed by measuring uv vis spectra of no - bound samples. similarly, for fe reconstituted fe - febmb1(zn), the protein was kept at 1 mm concentration before no addition. excess no was added, in the form of dea - nonoate as described above, to fe - febmb1(zn) to saturate the feb site with no. after no binding, the samples were further purified by another pd10 column equilibrated in a 50 mm bis - tris ph 7.3 buffer to remove any trace impurities including the decay product of dea - nonoate. all of the nitrosyl complexes thus prepared were then concentrated to 1015 mm and loaded into nrvs cells described above. the protocol of adding no at 1 mm of the reconstituted proteins followed by concentrating to higher concentrations has proven to be a successful strategy in our studies, as we have recently reported. an aliquot of each of the above concentrated samples was diluted, and their uv vis spectra were checked inside the glovebag to ensure that no changes in no coordination occurred during the final step of sample preparation. the vtz basis set was used for the iron orbitals and 6 - 31 g for all other atoms. the computational model for the nitrosyl complex of the nonheme site of feno - febmb1(zn) used the atomic coordinates deposited in the protein data bank under access code 3k9z(39) for his 29, his 43, his 64, glu 68 (with -carbon atoms replaced by terminal methyl groups), the iron and a ligated water, and added no as a sixth ligand. energy optimization of the experimentally observed s = /2 state yielded nearly octahedral coordination for the iron, with an fe n o angle varying from 142.8 (b3lyp) to 143.2 (m06l). the atomic displacements of the vibrational normal modes were used to calculate the iron vdos as described previously.	this forum article focuses on recent advances in structural and spectroscopic studies of biosynthetic models of nitric oxide reductases (nors). nors are complex metalloenzymes found in the denitrification pathway of earth s nitrogen cycle where they catalyze the proton - dependent two - electron reduction of nitric oxide (no) to nitrous oxide (n2o). while much progress has been made in biochemical and biophysical studies of native nors and their variants, a clear mechanistic understanding of this important metalloenzyme related to its function is still elusive. we report herein uv vis and nuclear resonance vibrational spectroscopy (nrvs) studies of mononitrosylated intermediates of the nor reaction of a biosynthetic model. the ability to selectively substitute metals at either heme or nonheme metal sites allows the introduction of independent 57fe probe atoms at either site, as well as allowing the preparation of analogues of stable reaction intermediates by replacing either metal with a redox inactive metal. together with previous structural and spectroscopic results, we summarize insights gained from studying these biosynthetic models toward understanding structural features responsible for the nor activity and its mechanism. the outlook on nor modeling is also discussed, with an emphasis on the design of models capable of catalytic turnovers designed based on close mimics of the secondary coordination sphere of native nors.
the management of undescended testes is one of the most controversial aspects in pediatric urology. the problem is not, in principle, the time to bring the testis down to the scrotum, but whether and when to perform imaging studies such as ultrasound (us) and why we still get the patients with undescended testis at a later age than global recommendations for orchidopexy. cryptorchidism is the most common congenital anomaly in boys and occurs in 3% to 9% of all full - term male neonates. the incidence of cryptorchidism decreases, due to spontaneous descent, to 1% to 2% by 6 months of age. although the recommended age for orchidopexy is around 6 months to 1 year of age, later orchidopexies have been reported in many studies. after the age of 2 years, the degenerative process in an undescended testis can be observed histologically. it may also have internal structure changed, but such findings, their incidences and methods of evaluation have not yet been described. some practitioners agree that to evaluate undescended testes, precise physical examination complemented with appropriate imaging studies is advised. in fact, we need such a diagnostic tool that would allow the evaluation of the testis before or after surgery, informing the physicians, as well as the parents and patients about testis development and changes that may suggest deterioration or even malignancy. the wide availability, high repeatability, low costs, and noninvasive nature of us have made it the imaging modality of choice for examining the scrotum. in many cases recent advances in us have improved the resolution of gray scale images of pediatric testis, which is especially important in the evaluation of the internal structure of undescended and mobile testis. the aim of this study was to evaluate the testicular volume and structure using us before and up to 3 years after orchidopexy in children with different age. from january 2007 to december 2012, 128 patients between the ages of 2 and 10 years (mean 6.3 years) underwent orchidopexy for undescended testes. the patients in whom undescended testis was not visible during preoperative us and patients with retractile and abdominal testis were not included to the study. afterwards, patients were invited for annual follow - up and control scrotal us. among these patients, the total number of analyzed testes was 184 after orchidopexy and 72 scrotal testes in patients with unilateral cryptorchidisms. because of the broad age distribution, patients were divided according to age at the time of surgery into 3 groups : group i (24 years old, n = 56 testes), group ii (57 years old, n = 67), and group iii (810 years old, n = 61). the reference group was composed of 185 patients (n = 370 testes) between the ages of 2 and 13 years with normally descended testes with normal and homogeneous echogenicity of the testes. the institutional ethical committee approved our protocol and informed consent was received from all patients in this study. a phillips iu22 us scanner with an l17 - 5 mhz linear probe was used for the determination of testicular structure and volume. volume was calculated using the approximation for a prolate ellipsoid : v = 0.523 length thickness width. the testicular volume ratio was calculated by the equation of the operated testis volume to the scrotal (control) testis mean volume. the structure of the testis was assessed in gray scale with the same settings of the gain, focus, and depth. echogenicity was scored in 2 grades, normal (homogeneous) and abnormal (inhomogeneous). all us scans were performed by 1 radiologist with 15 years experience in the field of clinical us. spss statistical software version 14.0 was used, with wilcoxon signed ranks test for the analysis of differences between ratios. the mean testicular volumes of operated and scrotal testes measured by us in various age groups before and 1, 2, and 3 years after surgery are shown in fig. 1 and table 1. the age groups of patients from the reference group with normal testes are adjusted according to the increasing age of observed patients after orchidopexy. in patients with undescended testes from groups i iii, the testicular volume ratio of the operated testes on preoperative examination was 0.86 for group i, 0.82 for group ii, and 0.78 for group iii. on follow - up examination after 1 year of observation, the ratio in group i was still 0.86 and in groups ii and iii this increased to 0.87 and 0.86. after 2 years the ratios changed to 0.89, 0.88, and 0.84, respectively, and after 3 years of observation, the ratios showed an increase to 0.95, 0.92, and 0.90. testicular volume, in milliliters, of the undescended testes in various age groups, before and 1, 2, 3 years after orchidopexy and mean volume of the scrotal and reference testes. patient age at treatment, number of testes (n), and mean testicular volume measured by ultrasound of the undescended and scrotal testes in various age groups, before and 1, 2, 3 years after orchidopexy. testicular volume ratio of undescended testes and scrotal testes in patients with unilateral cryptorchidisms, at various age groups before and 1, 2, 3 years after orchidopexy and statistical significance of the ratios growth in relation to the ratio before orchidopexy (p), 2 to 1 year after orchidopexy and 3 to 2 years after orchidopexy (p) the testicular volume ratio was also calculated in scrotal testes in patients with unilateral cryptorchidisms. in most cases, the ratio was close to one, except for the preoperative examination in all groups and 1 year after orchidopexy in group ii (table 1). the structure of the testis upon preoperative examination was inhomogeneous in 38 patients (21.7%) (group i n = 17, group ii n = 15, and group iii n = 6). on follow - up exams, this type of structure remained in 13 (7.1%) patients (group i-9, ii-3, and iii-1) (table 3). the cryptorchid patients with inhomogeneous echo in the testis displayed a significant lower volume as compared to the testis with homogeneous echo (table 3). six testes (3.3% ; group i-3, ii-2, iii-1) with an initial ratio < 0.25 and the number and volume of testes with normal and abnormal testicular structure at various age groups before and 3 years after orchidopexy. microlithiasis was found in 9 patients with cryptorchidisms (4.9%), in 6 patients with inhomogeneous testis structure, and in 3 patients with homogeneous testis structure. there is a common consensus that cryptorchid testes should be brought down to the scrotum in early childhood to preserve function and mitigate cancer risk. however, in some pediatric centers, many of these procedures are performed later in life, even up to 10 years of age. this could be due to either lack of screening methods or unclear procedures in the case of undescended testes, as well as delayed diagnosis by medical practitioners. also, parental inattention and their unawareness of the necessity of this operation may affect the decision. other factors associated with the timing of orchidopexy include insurance status, and the hospital at which the surgery is performed. some authors suggest that us has no role in case of undescended testes, and a delayed urology consultation could be due to unnecessary us. although the possible consequences of cryptorchidism are well - known, there have been problems with the assessment of the influence of orchidopexy on the future function of the testis, as this requires a very long follow - up period from diagnosis and treatment in childhood until full testicular function in adulthood. to monitor testicular function, invasive testicular biopsies would be required, but such an invasive procedure in young boys would not be ethically acceptable. it has also been thought that repeated measurements of testicular volume could be translated to testicular function. another method of monitoring can be assessment of the serum hormone levels, like follicle - stimulating hormone, because of its role in spermatogenesis, but it does not give information about testis size and structure. in this study the use of us is emphasized, and indeed, abnormalities in the echostructure were seen in approximately 20% of patients in the present study (table 3). ultrasonography is known to be a reliable tool for the measurement of testicular volume with high reproducibility and is used to determine the volume of the cryptorchid testis before and after testis repositioning (fig. 2). in the case of unilaterally undescended testes, to compare the growth of the undescended testis to the scrotal testis, the testicular volume ratio can be calculated by using the operated to scrotal testis volume. this is an index of the degree of growth deficit of the cryptorchid testis and has been used in several studies concerning the growth of the undescended testis after surgery, as well as in this group of patients. in a study by kollin, there was an increase in the median ratio from 0.68 at 6 months to 0.81 at 4 years (p < 0.001) in the early treatment group operated on at 9 months of age. in contrast, a decrease in the median ratio was noted in the late treatment group (surgery at 3 years) from 0.68 at 6 months to 0.56 at 4 years (p < 0.001). at ages 2, 3, and 4 years, there were significant differences in this ratio between the early and late treatment groups, again demonstrating the partial catch - up growth of testes that are treated early (p < 0.001). this means that only an early orchidopexy is likely to result in significant recovery of testicular size and appearance. also, in a study by kim at the follow - up examination 2 years after orchidopexy, the testicular volume percentage (the ratio multiplied by 100) showed an increase : in the group of patients operated on before the 2nd year of life (i) from 46.6 to 90 ; in the group of patients operated between 2 and 5 years of life (ii), from 68.1 to 78.2 ; and in the group operated after the 5th year of life (iii), from 65.7 to 77.6. only group i, which received orchidopexy within 2 years of birth, showed a significant recovery of testicular volume at follow - up compared with groups ii and iii. however, unilateral cryptorchidism may have some effect on scrotal testis, thus contralateral testis should not be assumed as a reference (table 2). that is why independent control group was introduced giving possibility to the combined assessment of the unilateral and bilateral undescended testes. the measurements of the scrotal testis (rt) and undescended one (lt) before orchidopexy (upper images) and 3 years after orchidopexy (bottom images). lt = left testis, rt = right testis. in this study, there was an increase in the median ratio in all age groups (table 2), but the increase in the median ratio 3 years after surgery in group i (from 0.86 to 0.95) was statistically significant (p < 0.05). that is way these or earlier age should be addressed for orchidopexy. significant growth occurred mostly in the 3rd year of observation, which confirms the need for long - term follow - up after orchidopexy. despite quite good catch up growth, some of the testes had significantly changed volume at the time of diagnosis. a large difference in testis volume could be recommendation for early orchidopexy as well as finding that testicular volume ratio in undescended testes decreased with age if the testes were left untreated (from 0.86 in group i to 0.78 in group iii). this study also included patients who were elder at the time of orchidopexy than what is currently recommended (group iii). those children and children from the eldest reference group could enter into puberty during observation, which restrict the value of the results of this group and suggest its exclusion from future research. some abnormalities, like hypoechoic and inhomogeneous patterns, were seen in 20% of patients with cryptorchidism. in 5% to 10% of undescended testes, what is more microlithiasis can occur more frequently in testes with abnormal structure. however, the combination of assessing testis echostructure and its volume could have the strongest prognostic importance, especially in a group of testes with a very low volume ratio (< 0.25) and a hypoechoic, inhomogeneous structure, which was observed in 6 (3.3%) patients. this is too small group of patients for statistical evaluation, but because these testes did not show any recovery during observation in these patients more advanced methods of evaluation should be considered, like hormones levels or even biopsy. the symptoms of deterioration of the testis structure may also appear in later age, but such findings were not observed in our patients. some of abnormalities, which quickly resolved after orchidopexy, could be due to the inguinal position of the testis and the need for deeper penetration of a us and, consequently, a weaker signal. this could give some limitations for the initial exam, before the surgery, but only in the assessment of the testis echostructure. other information, such as the location and the volume of the testis, could be still very valuable. in fact, undescended testis is a predisposing factor for malignancy, as well as microlithiasis. this also applies to the patients after orchidopexy. since in the case of microlithiasis, us is an established method of observation, why can not be used also in the case of undescended testes and testes after orchidopexy. authors realize that the subjectivity of the evaluation of the echostructure could also be some disadvantage, but additional us tools can be used in the assessment of the testis condition, such as color and spectral doppler. also to monitor the stiffness or vascularity of the testes, beyond 2d us, modern us techniques could be applied, such as elastography or 3d us, but this requires additional study. potentially these are the tools we are looking for. in summary, scrotal us can provide an accurate comparative assessment of the growth of testes before and after orchidopexy. in group of the patients 2 to 4 years old the growth of the ratio 3 years after surgery was statistically significant and these or earlier age should be recommended for orchidopexy. abnormalities in the structure of the testes do not accurately reflect the testicular function, but can be used for identifying testes requiring more advanced methods of evaluation, like hormones levels or even biopsy.	abstractthe aim of this study was to evaluate the testicular volume and structure using ultrasound (us) before and up to 3 years after orchidopexy in children with different age.a total of 128 patients underwent orchidopexy for undescended testes. afterwards, patients were invited for annual follow - up and control scrotal us. the total number of analyzed testes after orchidopexy was 184. patients were divided according to age at the time of surgery : group i (24 years old), group ii (57), and group iii (810). in all patients, the testicular volume ratio was calculated as the operated testes volume versus the control testes mean volume.there was an increase in the median ratio in all age groups, from 0.86 to 0.95 in group i, 0.82 to 0.92 in group ii and 0.78 to 0.90 in group iii. in group of the patients 2 to 4 years old the growth of the ratio 3 years after surgery was statistically significant.abnormalities in the structure of the testes, which may indicate severe damage to the testis, were seen in approximately 20% of patients on initial exams. on follow - up exams, this type of structure remained in 7% of patients. testes with an initial ratio < 0.25 and inhomogeneous structure did not show any significant growth.scrotal us can be used for an accurate comparative assessment of the structure and growth of the testes before and after orchidopexy.abnormalities in the structure of the testes may identify testes requiring more advanced methods of evaluation.
regarding glucose levels in the brain, hypothalamic glucose levels are regulated from 0.7 to 4.5 when blood glucose levels fall to ~2.8 mmol / l during hypoglycemia, brain glucose levels also decline to ~0.3 mmol / l3. according to a longstanding dogma, sglt1 is a highaffinity transporter for glucose. in fact, panayotovaheiermann.4 determined that sglt1 in rats and humans had a similar km of ~0.4 mmol / l for glucose. based on this report, fan.1 concluded that sglt1 should be saturated under physiological blood glucose levels, and the amount of glucose entering neurons through sglt1 would start decreasing only under hypoglycemia. they developed and proved this hypothesis indirectly through their experiments in which the augmentation of counterregulatory responses to hypoglycemia was recognized in sglt1 knockeddown rats with acute or recurrent hypoglycemic bout(s). fan.1 knocked down the expression levels of sglt1 messenger ribonucleic acid in rat vmh using microinjections of an adenoassociated viral vector containing the sglt1 short hairpin ribonucleic acid. these rats were exposed to recurrent bouts or a single bout of hypoglycemia induced by hyperinsulinemichypoglycemic clamp procedures. during hypoglycemia, the glucose infusion rate decreased, glucagon and epinephrine levels increased, and hepatic glucose production increased in sglt1 knockeddown rats. their research design, consisting of an sglt1 knockeddown model and a recurrent hypoglycemia model, was elegant. first, they examined no transport capacity of glucose through sglt1 in the knockeddown vmh. in general, transport capacity depends on both the copy number and the turnover rate of the transporter proteins. direct verification of reduced electrical discharge frequency in the neurons infected with sglt1 short hairpin ribonucleic acid is expected in the future. second, a recent study4 showed that human sglt1 has a relatively lower affinity for dglucose, inconsistent with the previously reported value ; that is, k0.5 2 mmol / l versus km 0.4 mmol / l. as hummel.5 proved the kinetics of glucose transport using only human sglt1, the characteristics might vary in rat sglt1. readers should carefully interpret the report by fan.1, and consider the possible differences between humans and rodents. iatrogenic hypoglycemia in diabetic patients currently invites serious clinical attention because of its possible influence on quality of life and cardiovascular motility. impaired glucose counterregulation response is one of the most important factors that elicit severe and/or recurrent bouts of hypoglycemia. although the mechanisms of the impairment have not yet been fully expounded, it appears acceptable that glucose sensing and assembling metabolic information from the periphery in cns contributes to the deficit. in addition to ge neurons as aforementioned, other crucial participants, glucoseinhibited or glucoseinhibited neurons, have been defined, investigated and characterized as key players in the counterregulatory response to hypoglycemia. they increase the frequency of spontaneous action potential discharge through an interaction between adenosine monophosphate activated protein kinase (ampk) and nitric oxide. in recurrently hypoglycemic model rats or type 1 diabetic model rats, fan.1 also reported that the counterregulatory responses were amplified by the activation of ampk in the vmh with aminoimidazole carboxamide ribonucleotide. in contrast, the counterregulatory responses were lessened in neuronal nitric oxide synthase knockedout mice. when considering the recent finding from fan 's laboratory1, vmh glucosesensing neurons have three distinct mechanisms for detecting hypoglycemia : (i) the katp channel pathway in ge neurons ; (ii) the sglt1 pathway in ge neurons ; and (iii) the ampk nitric oxide pathway in glucoseinhibited neurons. in addition to these vmh neurons, there are several contributors to glucosesensing mechanisms, including orexin neurons in lateral hypothalamus, neuropeptide y neurons in arcuate nucleus and lateral hypothalamus, and hypothalamic glial cells. in particular, as fan.1 also specified in their recent study, numerous hypothalamic glial cells express various glucosesensing molecules. an increasing body of evidence suggests that hypothalamic glial cells might play a relevant role in glucose sensing and fuel homeostasis utilizing a glial metabolic substrate lactate. to develop applicable therapeutic strategies to prevent hypoglycemia, we must clarify the interaction and integration of inter/intracellular crosstalk among these glucosesensing cells mediated by various neurotransmitters and neuroendocrine hormones ; for example. for instance, it was recently shown that serotonin, a monoamine neurotransmitter, has an important role in regulating adrenomedullary responses to glucoprivation at the perifornical hypothalamus6. it has also been reported that the administration of selective serotonin reuptake inhibitors augments the counterregulatory responses to hypoglycemia. hence, as several types of cells throughout the cns have been proven to be glucosesensing cells, we must consider the vast heterogeneity among these cells, and carefully investigate this research field with welldesigned studies in the future. in conclusion, fan.1 proved a novel type of ge neurons, which detect hypoglycemia through decreasing glucose entry through sglt1. to conquer hypoglycemia unawareness, which limits an appropriate approach to tight control of the blood glucose level, the work achieved by fan.1 releases an alternative pathway to approach and resolve the problem.	in the central nervous system, especially in the hypothalamus, the energy status in the body is assessed by glucosesensing neurons since glucose is the major fuel in the central nervous system. glucose sensing mechanisms also have a significant role in prevention of hypoglycemic bouts. in a recent report, fan and colleagues proved a novel type of glucoseexited neurons which detect hypoglycemia through decreasing glucose entry via sglt1.
the study population was derived from the inhabitants of tianjin, which is the third largest city and located in the east coastal area in china. the city is composed of six urban districts, six suburbs, and three newly developed areas., a multiphase stratified cluster sampling method was implemented, and a random sample of the city was drawn for the study population. the strategy of sampling in this study was designed on the basis of socioeconomic status and was in line with the sampling scheme for the chinese national dietary and nutrition study in tianjin. in brief, a three - step randomized sampling procedure was performed as follows : first, two urban districts and one suburb were drawn as the first - stage sample ; second, three communities were selected as the second - level unit ; and, finally, three neighborhoods were chosen as third - level unit. thereafter, a total of 18 urban and nine suburb blocks were included in the study. all inhabitants (n = 8,540) who had lived in the selected blocks for > 5 years, who were aged 15 years, and who were type 1 diabetes free were initially invited to participate in the study. in july 2005, a two - phase survey consisting of a screening phase and further blood testing phase was implemented. the screening phase included a health interview, physical examination, and fasting peripheral blood glucose test for 8,109 (95.0%) participants. in the second phase, all subjects who had positive screening results (fasting peripheral blood glucose 5.4 data on age, sex, education, lifestyle, health status, and family history of chronic disease and major stressful events were collected from participants at the screening phase through the interview following a structured questionnaire. education was categorized by the maximum years of formal schooling and was dichotomized (9 vs. 20 cigarettes / day). experience of major stressful events was defined as job or close relatives lost, frequent spousal conflict, or accidents in the past 10 years. (two times or more per week for at least 1 year) and nonregular. fasting peripheral blood glucose was measured with a onetouch ultra2 meter (life scan) at the screening phase for all participants, and fasting and 2-h postprandial venous blood samples were taken for subjects who had fasting peripheral blood glucose 5.4 fasting plasma glucose and 2-h postprandial plasma glucose were measured using a glucose oxidase procedure. type 2 diabetes was ascertained as fasting plasma glucose 7.0 mmol / l or postprandial 2-h plasma glucose 11.1 lada was identified based on immunology of diabetes society criteria (16) as follows : 1) the presence of type 2 diabetes and age 35 years ; 2) a lack of requirement for insulin at least 6 months after the diagnosis of type 2 diabetes ; and 3) serum gada positivity as tested by radioligand assay (17). the characteristics of participants in different groups were compared using tests for categorical variables and one - way anova for continuous variables. the prevalence rates were calculated as the number of subjects with lada divided by number of subjects and standardized by the structure of total population in the city in 2005. multinomial logistic regression analyses were used to estimate the odds ratios (ors) and 95% ci of lada and type 2 diabetes in relation to age, sex, education, hypertension, family history of diabetes, smoking, alcohol drinking, physical activity, bmi, waist - to - hip ratio, and major stressful events. lada and type 2 diabetes were used as separate outcomes, and diabetes - free subjects were used as the referent group in the multinomial logistic regression models. the analyses were performed in participants aged 35 years (n = 6,137) because of the absence of lada in subjects aged 1534 years. all statistical analyses were performed using spss 17.0 (spss, chicago, il). data on age, sex, education, lifestyle, health status, and family history of chronic disease and major stressful events were collected from participants at the screening phase through the interview following a structured questionnaire. education was categorized by the maximum years of formal schooling and was dichotomized (9 vs. 20 cigarettes / day). experience of major stressful events was defined as job or close relatives lost, frequent spousal conflict, or accidents in the past 10 years. (two times or more per week for at least 1 year) and nonregular. fasting peripheral blood glucose was measured with a onetouch ultra2 meter (life scan) at the screening phase for all participants, and fasting and 2-h postprandial venous blood samples were taken for subjects who had fasting peripheral blood glucose 5.4 fasting plasma glucose and 2-h postprandial plasma glucose were measured using a glucose oxidase procedure. type 2 diabetes was ascertained as fasting plasma glucose 7.0 mmol / l or postprandial 2-h plasma glucose 11.1 lada was identified based on immunology of diabetes society criteria (16) as follows : 1) the presence of type 2 diabetes and age 35 years ; 2) a lack of requirement for insulin at least 6 months after the diagnosis of type 2 diabetes ; and 3) serum gada positivity as tested by radioligand assay (17). the characteristics of participants in different groups were compared using tests for categorical variables and one - way anova for continuous variables. the prevalence rates were calculated as the number of subjects with lada divided by number of subjects and standardized by the structure of total population in the city in 2005. multinomial logistic regression analyses were used to estimate the odds ratios (ors) and 95% ci of lada and type 2 diabetes in relation to age, sex, education, hypertension, family history of diabetes, smoking, alcohol drinking, physical activity, bmi, waist - to - hip ratio, and major stressful events. lada and type 2 diabetes were used as separate outcomes, and diabetes - free subjects were used as the referent group in the multinomial logistic regression models. the analyses were performed in participants aged 35 years (n = 6,137) because of the absence of lada in subjects aged 1534 years. all statistical analyses were performed using spss 17.0 (spss, chicago, il). the 8,109 participants consisted of 3,878 (47.8%) men and 4,231 (52.2%) women (= 15.37, p < 0.001). age and sex distributions in our study population were similar to the distributions in the source population in tianjin based on data from the annual statistic report, tianjin, 2005. among all subjects, 498 (6.1%) had type 2 diabetes, which included 268 (53.8%) with previously diagnosed diabetes and 230 (46.2%) with newly detected diabetes. of the 498 patients with type 2 diabetes, 46 (9.2%) were found to have lada. patients with lada were more likely to be older and obese, to have hypertension, and to have a family history of diabetes and stressful events but showed less alcohol drinking, compared with diabetes - free subjects. the three groups did not differ significantly in terms of education and smoking (table 1). characteristics of the study participants by type 2 diabetes and lada data are n (%) or means sd. numbers of subjects with missing values were 195 for alcohol drinking, 162 for hypertension, 803 for family history of diabetes, 43 for major stressful events, 5 for bmi, and 11 for waist - to - hip ratio. table 2 shows that the prevalence of lada was 9.2% (8.0% in women and 11.1% in men, p = 0.253) in individuals with type 2 diabetes. among all participants, the prevalence of lada was 0.6% for both men and women and was 0.7% in participants aged 35 years. the standardized prevalence of lada was 0.69% (0.68% in men and 0.70% in women), which was based on the source population aged 35 in tianjin in 2005. with age, the prevalence of lada slowly increased up to 5059 years and seemed to decline thereafter. however, the prevalence of type 2 diabetes was dramatically elevated with age and tended to continuously increase after age 60 years (fig. 1). prevalence of lada in patients with type 2 diabetes and all participants data are n (%) unless otherwise indicated. prevalence of lada and type 2 diabetes in all participants. in multinomial logistic regression, hypertension, family history of diabetes, high waist - to - hip ratio, and major stressful events were significantly associated with both lada and type 2 diabetes, compared with diabetes - free subjects. age, female sex, and high bmi (25 kg / m) were positively and alcohol drinking was negatively related to type 2 diabetes. although these factors seemed to be associated with lada, the association did not reach statistical significance because of the small number of patients with lada (table 3). regular physical activity (or 0.95 [95% ci 0.491.84 ] for lada and 0.91 [0.641.29 ] for type 2 diabetes) and education (9 years) (0.99 [0.531.83 ] for lada and 0.82 [0.671.01 ] for type 2 diabetes) were not significantly associated with either lada or type 2 diabetes. compared with subjects who never smoked, subjects who smoked low and high numbers of cigarettes had ors of 1.02 (95% ci 0.561.88) and 1.89 (0.448.02) for lada and 0.89 (0.721.10) and 1.50 (0.882.56) for type 2 diabetes, respectively. factors and ors of prevalent lada and type 2 diabetes adjusted for age, sex, alcohol drinking, bmi, waist - to - hip ratio, hypertension, family history of diabetes, and major stressful events if applicable. a standardized workup for the gada assay was performed in 50 patients with newly diagnosed type 1 diabetes and 100 control subjects (mean age 32, range 480 years). the results showed that the median area under the receiver operator characteristic curve was 0.95 (86% sensitivity and 95% specificity). in this large population - based cross - sectional study, we found that 1) the prevalence of lada is 9.2% among individuals with type 2 diabetes and 0.6% in all participants, 2) the prevalence slowly increased with age up to 60 years and was high in individuals aged 5059 years, and 3) hypertension, family history of diabetes, high waist - to - hip ratio, and experience of stressful events are related to the occurrence of lada. the prevalence of lada varies according to the populations involved, criteria used, and antibodies analyzed. it has become clear that lada accounts for 10% of initial diagnoses of type 2 diabetes (18). epidemiological studies have shown conflicting results on the differences in lada prevalence in terms of age and sex among people with type 2 diabetes. one study showed a male predominance of lada, whereas other studies found a higher prevalence among women (6,19). another study reported that the prevalence of gada positivity is 0.9% in a spanish population aged 1865 years (20). in this population - based cross - sectional study, we found that 1) the prevalence of lada is 9.2% among individuals with type 2 diabetes and 0.6% in all participants and 2) the prevalence is not sex specific but is high in individuals aged 5059 years. autoimmunity is the major cause of type 1 diabetes and is assumed to be the cause of lada, which shares the biochemical marker of -cell directed autoimmunity with classic type 1 diabetes (21). mild form of type 1 diabetes, which shows slow progression to insulin dependence. the fact that hla - dr4-dq8 antigens are more widespread among patients with classic type 1 diabetes than among those with lada may offer an explanation as to why patients with lada do not develop insulin dependence as quickly as patients with classic type 1 diabetes (22). the risk factors for lada are less well understood than those for type 1 and type 2 diabetes. previous studies based on cross - sectional data have shown that patients with lada were younger and less obese than type 2 diabetic subjects (5) or found these variables to be similar in those with type 2 diabetes. a population - based prospective study has shown the risk effect of age, overweight, and physical inactivity on lada (14). a cross - sectional study indicated that family history of diabetes is a strong risk factor for lada because of a combination of shared genetic and environmental factors (13). in addition, a recent clinical study reported patients with lada having more metabolic disorders such as hypertension and overweight (10). in the present study, we found that hypertension, family history of diabetes, high waist - to - hip ratio, and experience of major stressful events are related to both lada and type 2 diabetes, suggesting that lada and type 2 diabetes might share risk factors. however, the statistical power was limited for the analyses of other factors for lada because of the small number of patients with lada in our study. further population - based longitudinal studies are warranted to clarify the temporality for the relationships between these factors and lada risk. the main strengths of our study are the large population - based sample, the randomized sampling procedure, the diagnosis of diabetes based on both fasting and prandial plasma glucose level, and the identification of individuals with lada by gada positivity. first, we used fasting peripheral blood glucose rather than fasting plasma glucose as the screening test ; thus, patients with diabetes might have been misclassified in the nondiabetes group, which would attenuate our results. in addition, individuals with a severe or long - standing chronic disease, e.g., diabetes, might have refused to participate in this study because of their unhealthy condition. on the other hand, it is possible that individuals with mild type 2 diabetes might be more interested in participation than those without this condition. however, the participation rate was high (95%), and the prevalence of type 2 diabetes in this study is comparable to that in previous reports. second, the definition used for lada as age 35 years might be arbitrary. yet, in our study, all patients with type 2 diabetes who were aged < 35 years were gada negative. third, information on the presence of current and past disease and family history of diabetes was mainly based on self - report. subjects with diabetes are probably more likely to be aware of relatives with diabetes ; therefore, recall bias may not be ruled out. however, the prevalence of smoking and hypertension in our study was similar to the prevalence reported by other studies. in addition, information on several diabetes - related diseases such as cerebrovascular disease and coronary heart disease was not available. finally, the temporality of the given associations is unclear because of the cross - sectional design of this study. in summary, our results show that the prevalence of lada is 9% among individuals with type 2 diabetes and high in individuals aged 5059 years. hypertension, family history of diabetes, high waist - to - hip ratio, and experiences of stressful events are associated with the occurrence of lada, suggesting the involvement of both genetic and environmental factors in lada. given the high prevalence of type 2 diabetes worldwide, our findings highlight the need to identify patients with lada for proper treatment. further population - based longitudinal studies are required to verify the risk factors for the development of lada.	objectivedata on latent autoimmune diabetes in adults (lada) from population - based studies are sparse. we sought to investigate the prevalence and correlates of lada.research design and methodsa total of 8,109 participants, who were aged 15 years and living in tianjin, china, were assessed to identify individuals with type 2 diabetes (american diabetes association criteria, 1997) and further to detect patients with lada. lada was ascertained by 1) the presence of type 2 diabetes and age 35 years, 2) the lack of a requirement for insulin at least 6 months after the diagnosis of type 2 diabetes, and 3) serum gad antibody positivity. data were analyzed using multinomial logistic regression with adjustment for potential confounders.resultsof all participants, 498 (6.1%) were patients with type 2 diabetes. of them, 46 (9.2%) were found to have lada. the prevalence of lada was 0.6% (46 of 8,109), and tended to increase with age up to 5059 years in all participants. the odds ratios (95% ci) of lada related to hypertension, family history of diabetes, waist - to - hip ratio 0.85, and major stressful events were 1.93 (1.023.65), 17.59 (9.0834.06), 5.37 (2.3112.49), and 4.09 (1.759.52), respectively.conclusionsthe prevalence of lada is 9% in patients with type 2 diabetes. hypertension, family history of diabetes, central obesity, and major stressful events may be associated with the occurrence of lada.
seasonal influenza causes an estimated annual average of 226 000 hospitalizations and 36 000 deaths in the united states. the highest rates of influenzaassociated hospitalizations and death occur among the elderly, young children, and persons with certain highrisk medical conditions.,,,,,,, in october 2004, pediatric influenzaassociated deaths became a nationally notifiable condition to the u.s. national notifiable diseases surveillance system, following reports of death in children associated with influenza in the 20032004 season.,, during the 20062007 influenza season, an increase in influenzarelated pediatric deaths associated with staphylococcus aureus infection was first reported. this prompted collection of additional data on bacterial cultures from children with influenzarelated deaths during the 20072008 influenza season, including the culture results from any bacterial culture from a normally sterile site or specified nonsterile site, and information on timing of specimen collection relative to hospital admission. previously, information regarding cultures with negative results was not requested, and the timing for specimen collection relative to hospital admission was not collected. here, we describe the reports of children with influenzaassociated mortality and bacterial coinfection from the 20072008 influenza season. a case was defined as the death of a us resident aged < 18 from october 1, 2007september 30, 2008 with laboratory evidence of an influenza virus type a or b infection. a positive laboratory test for influenza type a or b could occur before or after death and may be determined by any of the following methods : rapid influenza diagnostic test, viral isolation, enzyme immunoassay, fluorescent antibody staining, immunohistochemical staining of tissue samples, or reverse transcription polymerase chain reaction. specimens for bacterial and viral culture were collected as part of routine clinical care and the postmortem examination, when applicable. cdc requested that respiratory specimens and postmortem lung specimens were sent to cdc laboratories if available. influenza virus isolates, other viral isolates, and bacterial isolates were characterized at local, hospital, state, or cdc laboratories. genotyping of available s. aureus isolates was performed at cdc by pulsedfield gel electrophoresis (pfge) using smaidigested dna. state or local health departments completed a standardized reporting form for each case of influenzaassociated pediatric mortality and transmitted the information to cdc via a webbased interface hosted on cdc s secure data network. through the standardized reporting form, information was collected on patient demographics, influenza laboratory test results, date and location of death, preexisting conditions, complications during the acute illness (including radiologically confirmed pneumonia), influenza vaccination history, and results of bacterial cultures obtained from normally sterile (blood, pleural fluid, chest tube fluid, or cerebral spinal fluid) and specified nonsterile (endotracheal tube aspirates, tracheal aspirates, and bronchial washes) sites. information was not collected on bacterial cultures obtained from nares or sputum, as positive cultures from these sites are more likely to represent colonization rather than infection. information regarding bacterial isolation from postmortem lung biopsies and fungal coinfections was not directly solicited ; however, this information could be reported in a notes section of the reporting form. for our analysis, bacterial isolation from postmortem lung biopsies were included only if the specimen was collected on the calendar day of death or the calendar day following death. dates of hospital admission and specimen collection were obtained for all patients from whom s. aureus was isolated from one of the designated sites. we focused our analyses on those patients from whom s. aureus was isolated from a specimen collected within three days of hospital admission, as we were attempting to identify patients in whom a bacterial coinfection may have contributed to their severe presentation (as opposed to having been acquired during hospitalization). data were stored in sql 2005 and microsoft access 2003 (redmond, wa, usa) and analyzed using sas 9.1 (sas institute, cary, nc, usa). during the 20072008 influenza season, a total of 88 influenzaassociated pediatric deaths were reported to cdc. of the 88 children, 47 (53%) were boys and the median age at death was 5. fortytwo (47%) of the children were < 5 years of age, and 14 (16%) were < 1 year of age (figure 1). frequency of age at death (years) among reported influenzaassociated pediatric deaths : usa october 1, 2007september 30, 2008. race / ethnicity, location of death, and influenza type among children with influenzaassociated mortality united states : 20072008 the median time from onset of symptoms to death was four days, with 40% dying within three days and 72% dying within seven days of symptom onset. about half of the children died in the intensive care unit, and 40% died in the emergency department or outside the hospital (table 1). ventilation status was known for 50 children who died in the hospital (excluding emergency department) of which 48 (96%) required mechanical ventilation. information on preexisting conditions was known for 81/88 of the children ; 39 (48%) had an advisory committee on immunization practices (acip)defined highrisk medical condition. of these, twenty children had one highrisk condition, ten had two highrisk conditions, five had three highrisk conditions, and four had four highrisk conditions. the highrisk medical conditions reported were asthma (n = 15), moderate to severe developmental delay (n = 13), seizure disorder (n = 11), cardiac disease (n = 9), neuromuscular disease (n = 9), chronic pulmonary disease (n = 6), immunosuppressive condition (n = 3), cystic fibrosis (n = 2), metabolic disorder (n = 2), and renal disease (n = 1). fiftysix (66%) of 85 children were recommended for vaccination by 20072008 acip criteria, and of these 48 had a known vaccination status for the 20072008 influenza season. eleven (23%) of 48 recommended children received at least one dose of influenza vaccine in the 20072008 season at least 14 days prior to illness onset. of these 11 children, six were completely vaccinated, one was partially vaccinated, and complete vaccination status could not be determined in four. one child who received the vaccine in the 20072008 season did not meet an agerelated or acipdefined highrisk criteria for vaccination. the complications most commonly reported were pneumonia (n = 34), acute respiratory distress syndrome (n = 25), sepsis (n = 15), seizures (n = 15), shock (n = 13), encephalopathy or encephalitis (n = 6), and bronchiolitis (n = 5). of the 57 children who had specimens for bacterial culture collected from one of the specified sterile or nonsterile sites and had known results, 29 (51%) had positive bacterial cultures. s. aureus was the most commonly identified organism, identified in 20 (69%) of the 29 children with any positive culture. in 17 (85%) of these 20 children, specimens yielding s. aureus were collected within three days of inpatient admission (table 2). children with bacteria isolated from specified sites among children with influenzaassociated mortality united states : 20072008 blood culture, pleural fluid, chest tube fluid, or cerebral spinal fluid. specimen collected on the day of death or the day after death. because specimens from nonsterile sites may represent colonization or contamination, we evaluated the relationship between nonsterile site cultures and radiologically confirmed pneumonia. of the 29 children with cultures collected from nonsterile sites, 13 of the 17 (76%) children with 1 isolate identified, and 10 of the 13 (77%) children with s. aureus identified had radiologically confirmed pneumonia. overall, 25 of 29 (86%) children who had 1 isolate identified from one of the specified sterile or nonsterile sites had radiologically confirmed pneumonia. the same species of bacteria was also isolated at one of the specified culture sites for three of these children. s. aureus and s. pneumoniae were the most common bacteria identified, each reported in four children (table 2). among the 23 children from whom s. aureus was isolated from one of the specified sterile or nonsterile sites or a postmortem lung specimen, 12 (52%) had a methicillinresistant staphlyococcus aureus (mrsa) isolate recovered, 9 (39%) had a methicillinsusceptible staphlyococcus aureus (mssa) isolate recovered, and 2 (9%) had a s. aureus isolate with an unknown methicillin susceptibility recovered. of the 12 mrsa isolates, six were available for genotyping, five were identified as pfge type usa300, and one was pfge type usa600. four of the 5 usa300 isolates and the usa600 isolate were collected within three days of hospital admission. two of the 9 mssa isolates were available for genotyping and were identified as pfge types usa200 and usa800 ; the usa200 isolate was collected within three days of admission while the usa800 isolate was not. the most common viral coinfections with influenza were adenovirus and respiratory syncytial virus (rsv). this child also had hemophagocytic syndrome that was believed to be secondary to influenza virus infection. two children had candida albicans isolates recovered : one by blood culture and the other by endotracheal tube fluid culture. the time periods from illness onset to death in the three children with fungal isolates were 23, 17, and 21 days, respectively. viral coinfections among children with influenzaassociated mortality united states : 20072008 the 17 children who had s. aureus isolated at a sterile or nonsterile site within three days of hospital admission were significantly older, were significantly less likely to have one or more highrisk medical condition, had significantly more time from symptom onset until death, were more likely to have radiologically confirmed pneumonia, were more likely to be infected with influenza b virus, and were less likely to be recommended for vaccination by 20072008 acip criteria than the 37 children who had cultures from the specified sites that did not grow s. aureus (table 4). factors associated with isolation of staphylococcus aureus within three days of hospital admission among children with influenzaassociated mortality who had bacterial culture results from specified sites united states : 20072008 acip, advisory committee on immunization practices. identified at a normally sterile site (blood, pleural fluid, chest tube fluid, cerebral spinal fluid) or specified nonsterile site (endotracheal tube aspirate, tracheal aspirate, bronchial wash). children receiving longterm aspirin therapy who might be at risk for experiencing reye syndrome after influenza virus infection or those with chronic pulmonary (including asthma), cardiovascular (except hypertension), renal, hepatic, hematologic, or metabolic disorders (including diabetes mellitus). children with immunosuppression or any condition (e.g., cognitive dysfunction, spinal cord injuries, seizure disorders, other neuromuscular disorders) that can compromise respiratory function or the handling of respiratory secretions or that can increase the risk for aspiration. to further evaluate those children with a likely s. aureus coinfection (as opposed to colonization), we analyzed data from the 14 children who had s. aureus isolates identified within three days of hospital admission from a sterile sites or specified nonsterile sites and had a radiologically confirmed pneumonia. as mentioned earlier, we compared them with the 37 children who had cultures from the specified sites that did not grow s. aureus. the findings were consistent with those mentioned earlier with the exception of the association with influenza b virus. this report describes the 88 reports of influenzaassociated deaths in children from the 20072008 influenza season and provides more detailed information about isolation of potentially pathogenic bacteria from specified sites in these children. among children for whom results of bacterial cultures from sterile sites or s. aureus was identified in 69% of these children, the majority (85%) of whom had specimens yielding s. aureus obtained within three days of inpatient admission, indicating that they may have been admitted with a bacterial coinfection or complication of influenza. as reported previously, young age and highrisk medical conditions were common among influenzaassociated pediatric deaths., healthcare providers should suspect influenza s. aureus coinfection among children with severe respiratory illness and a suspected or confirmed influenza infection, and treat with appropriate antimicrobial and antiviral agents. an increase in the number of influenzaassociated pediatric deaths in association with s. aureus coinfection was first recognized during the 20062007 influenza season, and s. aureus remained the most commonly identified bacteria among these children in the 20072008 season. comparisons with data from previous years can only be made from sterile sites because information about isolation of bacteria from nonsterile sites was not collected. one (2%) child with a sterilesite s. aureus isolate was identified among 47 influenzaassociated pediatric deaths in the 20042005 influenza season, compared with 2/46 (4%) in 20052006 and 18/72 (25%) in 20062007. in the 20072008 season, s. aureus was isolated from sterilesite cultures in 11 (125%) of 88 children. comparisons between these numbers should be made with caution because the proportion of children from whom bacterial cultures were obtained likely varied each year. those children from whom s. aureus was isolated within three days of admission from sterile or specified nonsterile specimens were more likely to be older and were less likely to have a highrisk preexisting condition than those children with cultures of the specified sites that did not grow s. aureus. the older median age among children with positive s. aureus cultures could be related to higher s. aureus nasal carriage rates among older children. in 20032004, a nationally representative carriage survey determined that children aged 511 and 1119 were significantly more likely to be colonized with s. aureus than children aged 15. whereas frequencies of s. aureus nasal carriage have not increased significantly, there is a potential for increasing coinfection with mrsa and influenza virus, resulting in severe morbidity and mortality in children. while one can not consider pathogens isolated from specified nonsterile sites (endotracheal tubes, tracheal aspirates, bronchial washes) to be definitively causative pathogens in pneumonia, we believe that there is some value in evaluating specimens from these sites. given the challenges of pneumonia diagnostics, these specimens, while not from sterile sites, but from the lower respiratory tract, can provide insights into the causative pathogens of pneumonia and the epidemiology of children with coinfection. the frequency of reported viral coinfections is consistent with reports from the 20032004 influenza season, when adenovirus and rsv infections were also the most commonly reported viral coinfections. information regarding viral coinfections, including the site of virus isolation, was limited, and not systematically collected in all children. the proportions of influenza types identified in these children were similar to the proportions circulating in the united states during the 20072008 season. nationally, 71% and 29% of viruses typed were influenza a and influenza b, respectively, in the 20072008 season. by comparison, among the 84 children with known virus type information that were infected with only one influenza type, 64% and 36% were infected with influenza a and b, respectively. vaccination remains the best way to prevent influenza and its complications. during the 20072008 influenza season, the acip recommended annual influenza vaccination for all children aged 659 months and children aged 518 years with an acipdefined highrisk condition. in february 2010, vaccination is now recommended for all persons 6 months, regardless of age or highrisk medical conditions. this new recommendation could make a strong impact on pediatric influenzaassociated deaths including those with s. aureus coinfection, because many of the deaths in the 20072008 season occurred among children who were older or previously healthy, and not recommended for vaccination. first, reports of influenzaassociated pediatric mortality are made passively by healthcare providers, and some deaths meeting the case definition were likely not reported. testing for influenza is at the discretion of the physician, and some children were likely never tested for influenza. additionally, the influenza rapid antigen test, a common diagnostic method for influenza has a relatively low sensitivity,,,, that could lead to undiagnosed cases. second, the children were not systematically tested for bacterial, viral, or fungal pathogens, or radiologically confirmed pneumonia, and thus our results are limited by the testing that was performed as part of routine clinical care and postmortem analysis. the children labeled as no lab evidence of s. aureus had culture(s) collected at 1 specified sterile or nonsterile site that did not grow s. aureus ; however, it is possible that these children had a s. aureus isolate present at a site that was not cultured. third, some bacterial cultures obtained from nonsterile sites could represent colonization rather than coinfections. however, we carefully selected only respiratory specimens that were collected in the trachea or bronchi to maximize identifying infecting versus colonizing bacterial isolates. fourth, not all bacterial cultures were obtained within three days of hospital admission, and therefore we may have misclassified some patients who were admitted with bacterial coinfections. influenzaassociated mortality among children is a rare event ; however, healthcare providers should be mindful of the severe outcomes associated with influenza in children, especially those with highrisk medical condition. critically ill children should be promptly treated with antiviral medications, unless contraindicated, upon influenza diagnosis or suspicion. because isolation of s. aureus potentially representing coinfection was identified in a substantial proportion (19%) of children with influenzaassociated mortality within three days of hospital admission, healthcare providers should consider administering antimicrobial agents active against locally circulating strains of s. aureus when empirically treating children with influenzalike illness and severe respiratory illness. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention.	please cite this paper as : peebles. (2010) influenzaassociated mortality among children united states : 20072008. influenza and other respiratory viruses 5(1), 2531. background since october 2004, pediatric influenzaassociated deaths have been a nationally notifiable condition. to further investigate the bacterial organisms that may have contributed to death, we systematically collected information about bacterial cultures collected at nonsterile sites and about the timing of staphylococcus aureus specimen collection relative to hospital admission. methods we performed a retrospective, descriptive study of all reported influenzaassociated pediatric deaths in 20072008 influenza season in the united states. results during the 20072008 influenza season, 88 influenzaassociated pediatric deaths were reported. the median age was 5 (range 29 days 17 years) ; 48% were < 5 years of age. the median time from symptom onset to death was 4 days (range 064 days). s. aureus was identified at a sterile site or at a nonsterile site in 20 (35%) of the 57 children with specimens collected from these sites ; in 17 (85%) of these children, specimens yielding s. aureus were obtained within three days of inpatient admission. these 17 children were older (10 versus 4 years, median ; p < 005) and less likely to have a highrisk medical condition (p < 005) than children with cultures from the designated sites that did not grow s. aureus. conclusions s. aureus continues to be the most common bacteria isolated from children with influenzaassociated mortality. s. aureus isolates were associated with older age and lack of highrisk medical conditions. healthcare providers should consider influenza coinfections with s. aureus when empirically treating children with influenza and severe respiratory illness.
in today 's obesogenic environment, losing weight through behavioral means can be a difficult task that requires a high level of self - monitoring, making healthy choices in the face of more desirable choices, and working against longstanding eating and physical activity habits. to overcome these barriers and successfully lose weight, high levels of motivation for weight loss and participation in a weight loss program are required. there is some evidence to suggest that this motivation drops during the course of a weight loss attempt. for example, adherence to weight loss recommendations such as self - monitoring, typically start at a high level and drop over time. one possible way to help encourage participants to continue the behaviors needed for weight loss after motivation has waned is to provide financial incentives for weight loss. financial incentives have been used as a way to encourage individuals to take part in preventative health behaviors, such as weight loss. a review by kane. found that for a variety of preventive health behaviors, introducing financial incentives led to an increase in positive health behaviors. looking specifically at weight loss, financial incentives have often been used in one of two ways. first, researchers have used behavioral deposit contracts. in these programs, participants are asked to deposit a set amount of money to participate in the program. they can earn the money back if they reach the study weight loss goal(s). the results from these studies have been generally positive in the short term (e.g.,). another approach for using financial incentives is to provide an incentive, such as money or entry into a lottery for money, to the participant for meeting a specified target or for each pound lost (i.e., there is no deposit required). finkelstein and colleagues used this approach and tested different levels of payment for weight loss ($ 5, $ 7, and $ 14 per percent of initial weight lost) as well as different payment schedules (consistent, early payment only, late payment only). the findings suggested that weight loss was associated with the magnitude of payment at the first follow - up visit and was associated with retention at the second follow - up. finally, a study published in 2008 compared the effect of behavioral contracts (deposits were matched by the study), to a lottery for a financial reward, to a no financial incentive condition. during the 16-week study, weight losses were greater in both of the financial incentive arms compared to the control arm. for a more comprehensive review of financial incentives and their role in weight loss, please see. despite the short - term positive outcomes when using financial incentives, controversy surrounding the long - term impacts of these incentives remains. much of this controversy stems from the cognitive evaluation theory (cet) by deci and ryan. this theory suggests that providing tangible external rewards for a behavior that is interesting will lead to a reduction in intrinsic motivation for the behavior. this theory was developed in response to a number of laboratory studies that compared the intrinsic motivation of individuals doing a task that is considering interesting, such as completing a word puzzle, in exchange for a reward to individuals completing the same task without the reward. a consistent finding in these studies was that when participants had prior knowledge that they would receive a reward for completing the activity, their intrinsic motivation for the task was lower than the comparison group 's who were not given rewards. this conclusion held in cases where rewards were task contingent (i.e., participants were rewarded for doing the task) as well as when rewards were performance contingent (i.e., participants had to complete the task at a certain level to receive the reward). deci and colleagues suggest that the decrease in intrinsic motivation is a reaction caused by shifting the focus from doing the activity for the purpose of self - improvement and because it is interesting to a focus on earning the reward. the proposition of rewards decreasing intrinsic motivation is a part of the meta theory developed by deci and ryan : self - determination theory. this broader theory suggests that for a behavior to be instigated and continued, an individual must feel that they are doing a behavior to better themselves, and they are inspired to carry out the behavior of their own will. in other words, the person is autonomously motivated. conversely, if an individual engages in a behavior in reaction to outside forces (i.e., they are demonstrating controlled motivation), the behavior is not likely to be continued. the authors suggest that practitioners who are interested in helping others to change behavior should do so in a manner that encourages participants to maintain high levels of autonomy. despite the popularity of cet, there are critics who believe the utility of this theory is limited to specific circumstances. for example, eisenberger and colleagues responded to deci and ryan 's theory suggesting that the meta - analysis published in 1999 overstated the reach of the undermining effect of rewards. specifically, they argue that undermining occurs mostly for task contingent rewards and that performance contingent rewards can actually increase intrinsic motivation. more recent work suggests the tenants of cet hold true but only for those who have control - oriented causal orientation (i.e., those who view their behavior as highly influenced by forces outside of themselves). these studies, along with the limited conditions under which the cet theory has been tested (viz., in laboratory settings), lead to a need to test cet in alternative contexts. specifically, it is important to test this theory in a situation where incentives may be used to promote long - term behavior change. if cet extends to health behaviors, use of financial incentives may be problematic because autonomous motivations for weight loss, exercise, and continuing in the weight loss program have all been found to be associated with weight loss success during weight loss programs. williams and colleagues found that autonomous motivation to remain in a weight loss program measured early in a weight reduction program was predictive of weight loss at 23-month follow - up. similarly, webber and colleagues found that autonomous motivation for participating in a weight loss program measured shortly after a weight loss program began was predictive of overall weight loss in a 16-week intervention. interestingly, neither autonomous motivation for participating in the weight loss program measured prior to the program beginning nor controlled motivation measured at any time were predictive of weight loss in this study. other studies have found that autonomous motivation for exercise is also associated with greater weight losses [14, 15 ]. finally, researchers found that a behavioral weight loss intervention developed to enhance autonomous motivation was more successful than a health education control group. as a result of autonomous motivation consistently predicting weight loss success, there is a need to understand whether the cet proposition regarding changes in motivation that occur after an external reward is given holds true in weight loss programs when financial incentives are used. some argue that intrinsic motivation, as described by sdt, is not relevant to health behaviors because these behaviors are not inherently interesting. however, exercise, a major predictor of weight loss, can be interesting, and intrinsic motivation for exercise has been found to be associated with weight loss. therefore, research is needed to understand which types of tasks cet can be applied to and in what contexts. despite the uncertainty regarding how interesting weight loss behaviors really are, offering financial incentives could still be construed by participants as controlling, therefore, leading to decreases in autonomous motivation. if this is the case, the shift in internalized motivation seen in studies of interesting behaviors could extend to health behaviors, making offering financial incentives detrimental once the incentives end. in other words, providing a financial incentive may undermine autonomous motivation for participating in a weight loss program and instead lead to increases in controlled motivation. this shift then may lead to limited maintenance of weight loss behaviors beyond the formal weight loss program. this paper tested the extension of cet and sdt to financial incentives for weight loss within a worksite weight loss program. first, it tested whether there were decreases in autonomous or increases in controlled motivation for participating in a weight loss program among participants randomized to receive an offer of a financial incentive as compared to those who were randomized not to receive an offer of a financial incentive. secondly, this study investigated whether there are decreases in autonomous motivation for participating in a weight loss program among individuals who lost weight and were randomized to receive an incentive as compared to those who lost weight and were randomized to an intervention that did not receive a financial incentive. this study also investigated whether there were differential increases in controlled motivation among the same groups. because the incentive in this study was performance contingent, not everyone who was offered an incentive ultimately received payment. presumably, if there is a negative effect of a financial incentive, it may be strongest for those who actually receive the incentive as opposed to those who only receive the offer. studying both the offer and the receipt of the incentive will provide maximal insight into the effects of the incentives on motivation for participating in a weight loss program. data for this analysis are from the way (worksite activities for you) to health this trial was designed to test the effects of two minimal intensity weight loss interventions compared to a usual care healthy dining program among overweight / obese employees at 17 community college worksites from the north carolina community college system. all participating campuses had access to the winner 's circle dining program (wc), a program focused on increasing access to healthier food options at work. overweight employees at campuses enrolled in the research study received one of the following interventions : winners circle (wc) only (not included in this analysis), wc + web - based weight loss program (web), or wc + web + cash incentives for weight loss (web plus incentives ; wpi). for colleges assigned to the web and wpi groups, the employees were offered the opportunity to access a self - directed study website which included behavioral weight control lessons, an online study progress tracking system, and weekly tips. this intervention was modeled after the self - directed weight loss intervention described by tate and colleagues but involved no ongoing professional e - mail support. for participants randomized to wpi, the website was identical to the web condition but also showed a personalized incentive chart showing exactly how much the participant would earn (cash incentive) for the weight loss achieved at each follow - up measurement when his / her baseline weight was compared to follow - up weight. participants were offered $ 5.00 for each 1% of their initial body weight lost at the 3-, 6-, and 12-month assessment visits, up to 10%. thus, a participant could earn a maximum cash incentive of $ 150 over the duration of the study if he / she lost 10% of baseline weight at 3 months and maintained that weight loss at the 6- and 12-month follow - ups. this level of incentive was chosen because it was identified during pilot work as being a feasible level of payment to be offered as part of an employer sponsored weight loss program. to maximize retention, all participants who completed follow - up assessment visits received a stipend of $ 5, $ 10, and $ 20 for the 3-, 6-, and 12-month assessments, regardless of weight loss status. using the language of deci and ryan, the incentives provided for weight loss would be considered performance - contingent rewards, while the stipends for completing the assessments would be considered task - contingent. the study protocol for way to health was approved by the irb at the university of north carolina chapel hill and research triangle institute. this analysis includes only data from participants from community colleges randomized to the web and wpi intervention arms of the way (worksite activities for you) to health trial (see figure 1). this decision was made because the focus of this study is on comparing the effect of the offer or receipt of incentives on motivation. because the web and wpi group vary only on the presence of incentives, comparing these two groups provides a clear comparison in which to test this study 's hypotheses. first, the impact of the offer of incentives was investigated using participants who were randomized into either the web or wpi intervention groups and attended the follow - up assessments at 3, 6, and/or 12 months. second, in order to investigate changes in motivation over time from receipt of an incentive apart from changes in motivation caused by weight loss, the second set of analyses will utilize data only from individuals who lost weight at either the 3-month or 6-month assessment. weight losers were defined as participants who lost a minimum of 0.5% of their initial body weight (the minimum weight loss that was eligible for an incentive within the wpi intervention). ten community colleges were randomized into the web and wpi study arms. within these groups, bivariate analyses were used to test for differences in baseline demographic and anthropomorphic characteristics and motivation variables between the web and wpi groups. the web and wpi were similar at baseline, although wpi contained more women than web (= 14.52, df = 1, p 0.06), with the exception of gender. similar to the overall group composition, there were more women in the returning wpi group than the web group (3 months : = 7.23, df = 1, p = 0.007 ; 6 months : = 9.08, df = 1, p = 0.003 ; 12 months : = 4.67, df = 1, p = 0.03). among the weight losers (n = 300), there were significantly more women in the wpi group (90.2%) than the web group at the 6-month assessment (79.1%, = 5.26, df = 1, p = 0.02). for the remaining variables, there were no significant differences between the groups (all p 's > 0.15). because of the difference in gender representation of the groups, gender was entered as a covariate in all analyses. study staff, blinded to treatment condition, collected objective weight measurements at the start of the program and at months 3, 6, and 12. participants were weighed with shoes off, wearing light street clothing using a digital scale (tanita bwb, 800). weight change was computed by subtracting the baseline weight from the weight at each follow - up assessment visit. motivation for participating in a weight loss program was measured using the treatment self - regulation questionnaire and was completed at the same time points as the weight measurements. this questionnaire assesses motivation for starting, or continuing, participation in a weight loss program via the participant 's endorsement of statements of autonomous and controlled motivation. an example item from the autonomous subscale is i have remained in this program because i feel like it is the best way to help myself. the controlled subscale included items such as i have remained in the program because others would have been angry at me if i did not. responses to these items were given on a scale of 1 (not at all true) to 7 (very true) and were averaged to indicate a summary assessment of autonomous and controlled motivation. at baseline, participants completed the full tsrq assessing motivation to begin a weight loss program ; a subset of items assessing motivation to continue in a weight loss program were used at later assessments to reduce participant burden. the internal consistency of this scale at the four time points ranged from 0.63 to 0.78 (cronbach 's coefficient alpha). the internal consistencies of these scales were also acceptable with values between 0.66 and 0.88. the primary aim of this analysis was to test whether the offer or receipt of an incentive would lead individuals to show differential changes in autonomous and controlled motivation for remaining in a weight loss program. for the first set of analyses, the motivation to remain in a weight loss program of individuals who were randomized to receive an offer of a financial incentive were compared to those in the same program but were randomized not to receive the offer of the incentive. it was hypothesized that among those who were offered a financial incentive (wpi), autonomous motivation would decrease at a greater rate than those who were not offered an incentive (web). conversely, controlled motivation was hypothesized to increase in wpi at a greater rate than in web. the second set of analyses compared the motivation of individuals who received an incentive for weight loss relative to individuals who also lost weight but were randomized to a condition that did not provide an incentive. again it was hypothesized that wpi would show greater decreases in autonomous motivation after the receipt of the incentive than web. controlled motivation was expected to increase in wpi compared to web. to test these hypotheses, the first level of the models included the individual growth curves and the time varying covariate weight loss (kilograms of weight loss). the second level included gender as a control variable, the dummy variable for intervention group, and the interaction term between month and the intervention group. a third level of the model was tested that would account for the nesting of employees within the worksite. however, this model was ultimately rejected because there was too little variance at the third level to estimate random intercepts. the final model tested used the following equations : (1)level 1 : yij=0j+1j(month)ij+2j(weight change)ij + rij, level 2 : oj=00+01(intervention)j+02(female)j + 0j,1j=10+11(intervention),2j=20. in this model, fixed effects were estimated for changes by time (month) and intervention group while controlling for the effect of weight loss and the dummy coded control variable for gender. the cross - level interaction term (indicated by 11) was tested to assess if the effect of the intervention group varied over time. the only random effect included in the model was for the intercept. if support were found for the hypotheses about autonomous motivation decreasing more in the wpi group, the coefficient for the intervention month interaction (11) would be significant and negative. if support for the controlled motivation hypotheses were found, the intervention month coefficients in those analyses would be positive. the analyses of changes in motivation after receiving an incentive included motivation measured at the current and the subsequent assessment (i.e., weight losers at 3 months were used to assess changes in motivation between 3 and 6 months). the scores on the autonomous and controlled motivation scales were first examined in a cross - sectional manner. there was a significant difference between the web and wpi groups at 3 months (t(df = 407) = 2.17, p = 0.03), where the wpi group reported higher levels of autonomous motivation (see table 2). at 6 months, the difference was marginally significant (t(df = 368) = 2.01, p = 0.05) but the difference was not significant at 12 months (t(df = 333) = 0.98, p = 0.33). there were no significant differences by intervention group on controlled motivation at any time (p 's > 0.29). although this comparison of means provides some evidence that the wpi group that was offered incentives did not have autonomous motivation for remaining in a weight loss program that was significantly lower nor controlled motivation that was higher from web, it does not account for individual changes in motivation over time. therefore, the effect of the offer of incentives was analyzed over time using longitudinal methods. between baseline and the end of the intervention, there was a significant decrease in autonomous motivation for participating in a weight loss program (see table 3, p < 0.001). there were no differences between the web and wpi groups on autonomous motivation throughout the study (p = 0.42). there were also no differences in changes between the groups over time (intervention month, p = 0.83). this indicates that any changes in autonomous motivation over time were not related to the intervention group assignment. as suggested by prior studies, weight loss was significantly associated with changes in autonomous motivation such that, all other things being equal, on the 7-point scale, a one - kilogram weight loss was associated with a 0.08 unit increase in autonomous motivation (p < 0.001). finally, women reported having autonomous motivation for participating in the weight loss program 0.34 units higher than men (p < 0.001). similar to autonomous motivation, controlled motivation for remaining in a weight loss program was associated with weight loss. for controlled motivation, a one - kilogram weight loss was associated with an increase in controlled motivation of 0.02 units (see table 4 ; p = 0.005). there were no changes in controlled motivation over time (p = 0.19). no differences between intervention groups were found (p = 0.96), and there were no differences in changes in controlled motivation between the groups (p = 0.30). next, the effect of receiving an incentive was tested using the subsample of weight losers. as shown in table 3, there was a significant increase in autonomous motivation between 3 and 6 months such that all other things being equal, there was an increase of 0.13 units of autonomous motivation for each additional month of the intervention (p = 0.003). among weight losers, there were no significant differences in the level of autonomous motivation between the web and wpi groups (p = 0.07). additionally, there was no significant group by time interaction (p = 0.94). weight loss was associated with autonomous motivation, where each additional kilogram of weight loss was associated an increase of 0.08 of reported autonomous motivation (p < 0.001), all other things being equal. in 3-month weight losers, autonomous motivation did not differ between men and women (p = 0.15). the same pattern of results was found for when weight losers at 6 months were used to assess changes in autonomous motivation between 6 and 12 months, although in this analysis, women reported higher autonomous motivation than men (p = 0.03). similar to the analyses run for autonomous motivation, the effect of receiving an incentive on controlled motivation was first assessed using weight losers at 3 months examining changes in controlled motivation between 3 and 6 months (see table 4). there was a trend for controlled motivation to increase over time (p = 0.09) ; however, this did not reach statistical significance. there were no significant differences by intervention treatment group (p = 0.24) nor were there any differences by intervention groups over time (p = 0.72). unlike autonomous motivation, changes in controlled motivation were not related to changes in weight (p = 0.72), and there were no differences by gender (p = 0.65). finally, data from weight losers at 6 months was used to assess changes between 6 and 12 months on controlled motivation for participation in a weight loss program. although research has been conducted using financial incentives to encourage weight loss, no study to date has looked at the effect of these incentives on motivation. this paper addresses this gap by testing whether the assertions of the cet extend to motivation for participating in a weight loss program within a program offering financial incentives. the results of this study compared a group randomized to receive a financial incentive for weight loss with a group randomized not to receive the incentives. comparisons were made based on the offer of the incentive, as well as comparisons within a subset of the groups who were eligible to receive the incentives. the results suggest that neither the offer nor the receipt of a small incentive for weight loss leads to decreases in autonomous motivation or increases in controlled motivation for participating in a weight loss program. additionally, this study found that weight loss was consistently associated with changes in autonomous motivation to continue participating in the weight loss program. these results support past research suggesting that autonomous motivation measured after the weight loss program begins is a predictor of overall weight losses in both short and longer weight loss interventions. in this analysis, motivation was assessed at months 3 and 6 of a one - year trial. moreover, this repeated finding suggests that focusing on improving autonomous motivation for weight loss during a weight loss attempt may be beneficial. similar results for controlled motivation for participation were only found when examining individuals who did and did not lose weight. williams and colleagues found controlled motivation measured by the tsrq was associated with bmi change at the end of a weight loss intervention, but it was not associated with weight loss maintenance. webber and colleagues found controlled motivation was not associated with overall weight losses in a shorter term study. clearly, more research is needed before conclusions about the relationship between controlled motivation for participating in a weight loss program and weight loss can be solidified. in this study, there were no statistically significant relationships found between receiving an offer of a financial incentive and changes in motivation nor were there relationships found for those who actually received an incentive. first, the incentives paid for weight losses in this study were small and perhaps inadequate to lead to changes in motivation for weight loss program participation. although some individuals received this maximum incentive, the actual mean payment across the three assessment visits was $ 18.90 (median = $ 15). for most participants, this is less than the attendance stipend (e.g., $ 15 at 6 months or $ 20 at 12 months). this may have diluted the effect of the incentive as a motivator for continuing efforts to lose weight. a second plausible explanation for the lack of changes in motivation related to the incentives is the delay between the behaviors required for weight loss and the payment of the incentive. in this study, incentive payments were made during the study assessment visits at months 3, 6, and 12 of the intervention. by comparison, in the study by volpp and colleagues, where mean payments were $ 273 during a 16-week program, the incentives were provided either weekly (in the lottery condition) or monthly (in the contract condition ;). the longer lag between the behavior change and the receipt of the incentive may have forced participants in this study to rely more on other sources of motivation rather than the incentives. further research into the perceived value, the amount, and the timing of cash incentives, as well as their impact on motivation for weight loss, will help clarify this relationship. another possibility is that changes in motivation may have occurred, but the measurement of motivation was too distal from when the incentive was received for the change to be detected. in other words, changes in motivation may have occurred immediately after the incentive was received but then dissipated between then and the next measurement. no research to date has investigated the duration of impact that financial incentives may have on motivation, but prior weight loss studies have found that the effect of incentives disappears during weight loss maintenance. future studies may want to include more observations of motivation to explore this relationship. finally, the lack of significant relationships between the incentives and motivation in this paper may be a result of insufficient sample size. the analyses presented in this paper have adequate power to detect effect sizes equal to or greater than d = 0.23 for the analyses investigating the offer of incentives and effect sizes greater than d = 0.54 for the analysis of receiving the incentives (estimated using the optimal design software). the effect sizes for changes in motivation between the intervention groups over time ranged from 0.004 to 0.08 (very small to small effects). this study is the first to look at the relationship between financial incentives for weight loss and motivation to participate in a weight loss program. this is an important area for exploration because there is strong support from both employees and employers for using incentives within worksites to promote weight loss among employees, and theory suggests that use of such incentives may decrease autonomous motivation. this study utilized data from a worksite weight loss program of a similar intensity to what may be offered in employer sponsored weight loss programs. with these similarities, additionally, the amount of the incentives offered was decided upon based on the results of a survey of employers. the incentives used in this study were similar in magnitude to what employers may be willing to pay as part of an independent worksite weight loss program. additionally, the demographics of this sample are similar to the demographics of participants in other worksite health promotion programs, namely, that the sample was predominately white, college - educated women. thus, results may be generalizable to the typical worksite - based weight loss program participants within some, but not all, worksites. this study was also large enough to allow for a secondary analysis restricted to individuals who lost weight. by focusing only on participants who lost weight within these two groups, finally, weight loss in this study was measured using standardized protocols with in - person weights and the assessment of motivation included reliable / valid measures of motivation. despite these strengths, there are limitations that need to be considered. first, because this study focused on changes in motivation for participating in a weight loss program over time, it only included participants who completed the study questionnaires and excluded those with incomplete data. this may have introduced bias into the analyses where only participants who were highly motivated to lose weight, or earn incentives, completed follow - up assessments. additionally, only one measure of motivation for participating in a weight loss program was used in this study (treatment self - regulation questionnaire). this reliable measure assesses overall motivation for participating in a weight loss program but does not specifically assess money as a motivator. additionally, not all items of this measure were included in order to reduce participant burden. future research may want to consider using additional measures of motivation for weight loss as well as motivation to participate in a weight loss program. this is the first study to investigate the relationship between financial incentives and motivation for participating in a weight loss program. in this sample, there was no relationship between either the offer or the receipt of an incentive for achieving weight losses and subsequent changes in either autonomous or controlled motivation for participating in a weight loss program. further research is needed to investigate this relationship using other measures of motivation (e.g., including directly assessing the motivation for money as a catalyst for changing behavior). additional research to identify how the amount of the incentive and the timing of the incentive payments influence motivation, and ultimately weight loss, will make an important contribution to the field of obesity research.	this analysis investigated if changes in autonomous or controlled motivation for participation in a weight loss program differed between individuals offered a financial incentive for weight loss compared to individuals not offered an incentive. additionally, the same relationships were tested among those who lost weight and either received or did not receive an incentive. this analysis used data from a year - long randomized worksite weight loss program that randomly assigned employees in each worksite to either a low - intensity weight loss program or the same program plus small financial incentives for weight loss ($ 5.00 per percentage of initial weight lost). there were no differences in changes between groups on motivation during the study, however, increases in autonomous motivation were consistently associated with greater weight losses. this suggests that the small incentives used in this program did not lead to increases in controlled motivation nor did they undermine autonomous motivation. future studies are needed to evaluate the magnitude and timing of incentives to more fully understand the relationship between incentives and motivation.
rheumatoid arthritis (ra) is a chronic, inflammatory, multisystemic autoimmune disorder that affects (mostly) diarthrodial joints1. it is associated with an increased morbidity and mortality due to cardiovascular disease (cvd) ; however, the reason for the increased risk of cvd is not explained by traditional risk factors2, 3. in several studies, positive correlations have been shown between the increased disease activity in ra and increased erythrocyte sedimentation rate (esr)4, severe extra - articular manifestations5, rheumatoid factor seropositivity6, elevated c - reactive protein levels (crp)6, 7, and cardiovascular mortality. the presence of inflammation leads to endothelial activation and dysfunction, and this is regarded as the primary finding of atherosclerosis8, 9. subclinical atherosclerosis and vascular structure are measured through a noninvasive method of carotid intima - media thickness (cimt) measurements in patients with ra10. in addition to predicting the presence of local atheroma, the findings of cimt can reveal possible damage to the arterial system11. recent studies have shown increased cimt scores in ra patients in contrast to healthy controls12, 13. in ra, radiographic assessment of joint damage is the most widely accepted standard for following the course of the disease14. ra is more severe, progressive, and destructive during the early stage15, and although inflammation has a negative impact on functional capacity in early ra, increased erosion and joint destruction become more pronounced at the further established stages16. this joint destruction increases the health assessment questionnaire (haq) scores of established ra patients16 ; however, it has been clearly indicated that the real marker of the increment in haq score in established ra patients is the disease activity score (das)17. in several studies, the joint destruction of ra patients has been measured using the van der heijde modification of the sharp method (mtts)18, 19. inflammation in ra patients leads to joint destruction and subclinical atherosclerosis, and the presence of elevated joint destruction can be a preclinical condition for cardiovascular risk detection. based on the relationships of inflammation and rheumatoid arthritis with the atherosclerosis and joint destruction mentioned above, no research study has been performed, as of yet, to clarify this issue. we, herein, aimed to investigate the potential relationship between joint destruction and carotid intima - media thickness. thirty - four ra patients (10m/24f) admitted to the physical medicine and rehabilitation outpatient clinic at harran university school of medicine, fulfilling the american college of rheumatology criteria20 for the diagnosis of ra, were consecutively enrolled in our cross - sectional study. thirty - one healthy controls matched for age, sex, and bmi were recruited from the staff of the same hospital. the research protocol was approved by the ethics committee of the harran university school of medicine, and written informed consent was obtained from all subjects. patients with any other inflammatory diseases, coronary arterial disease, liver disease, neoplastic disease, diabetes mellitus, hypertension, renal failure, history of stroke, or iron deficiency anemia were excluded from the study. each patient s disease activity was measured using the disease activity score for 28 joints with the erythrocyte sedimentation rate (das28esr)21, and a visual analog scale (vas010 cm) was used to measure pain. the disability of the ra patients was measured using the health assessment questionnaire (haq) index, which ranges from 0 to 3. the patients fasting total cholesterol, high - density lipoprotein (hdl), low - density lipoprotein (ldl), triglycerides, esr, and crp concentrations were measured in the morning. the patients x - ray images were scored according to the modified sharp / van der heijde method by consensus of the readers : one experienced musculoskeletal radiologist and two clinical researchers. the maximum erosion score is 160 for the hand and 120 for the feet ; the maximum score for joint space narrowing is 120 for the hands and 48 for the feet, resulting in a maximum total score of 44822. the images were obtained using a high - resolution doppler ultrasound (mylab twice ; esaote, genoa, italy) with a 12 mhz linear - array transducer, and the cimt was automatically measured with software. the patients were examined in a supine position at 3045 degrees opposite the direction of neck extension. the cca, ica, and eca carotid bulbs were morphologically evaluated with all visual segments at the axial level, and the cimt was measured at the posterior wall when they were clearly visible in the longitudinal plane at 11.5 cm proximal to the distal carotid bulbs. intima - media thickness (imt) was measured as the distance from the leading edge of the first echogenic line to that of the second echogenic line. the first line represents the lumen - intima interface, and the second line the collagen - containing upper layer of the tunica adventitia. all of the sonographic examinations were performed by the same examiner, who was unaware of the subjects clinical status throughout the study. statistical analyses were carried out using spss 18.0 for windows (pasw statistics for windows, spss inc., chicago, il, usa), and between group comparisons were made using independent sample t - tests. pearson correlation tests were used to determine the relationships between the variables, and multivariate linear regression analyses were performed to identify independent predictors of cimt. all demographic and quantitative data are expressed as the mean standard deviation (sd). differences with p values < 0.05 were considered to be statistically significant, and all results are expressed with a 95% confidence interval. demographic and clinical data of the patients and healthy controls are shown in table 1table 1. demographic and clinical data of patients and healthy controls (mean sd)patientsn=34controlsn=31age (years)40.558.0038.836.21male / female (number)10/248/23bmi (kg / m)24.841.8924.671.86disease duration (months)63.6169.13painvas (010 cm)6.792.25sjc281.141.70tjc287.764.43das28esr4.891.22haq1.890.77esr (mm)33.9422.028.933.45crp (mg / dl)1.502.560.330.13rf (iu / ml)47.7623.23total cholesterol (mg / dl)167.4934.68152.8033.28ldl cholesterol (mg / dl)109.6931.7097.4123.04hdl cholesterol (mg / dl)46.829.5041.775.21triglyceride (mg / dl)120.6150.75123.1943.91tc / hdl3.691.003.680.80 p<0.001 ; p<0.05. bmi, body mass index ; crp, c reactive protein ; das, disease activity score ; esr, erythrocyte sedimentation rate ; haq, health assessment questionnaire ; hdl, high density lipoprotein ; ldl, low - density lipoprotein ; rf, rheumatoid factor ; sjc, swollen joints count ; tc, total cholesterol ; tjc, tender joints count ; vas, visual analog scale ; tmss, total modified sharp score. there was no significant difference in terms of age, gender, and bmi between the patients and the controls. the hdl levels of the patients were significantly higher than in the controls, and the esr and crp levels of the ra patients, right cimt, left cimt, and mean cimt scores were significantly elevated. the cimt of the patients and controls and the mtts of the patients are shown in table 2table 2. common carotid artery imts and total modified sharp score (mean sd)patientsn=34controlsn=31right cimt0.590.130.500.06left cimt0.610.150.490.06mean cimt0.600.140.490.06tmss66.1730.79 p<0.001 ; p<0.05. cimt, common carotid artery intima - media thickness ; tmss, total modified sharp score. bmi, body mass index ; crp, c reactive protein ; das, disease activity score ; esr, erythrocyte sedimentation rate ; haq, health assessment questionnaire ; hdl, high density lipoprotein ; ldl, low - density lipoprotein ; rf, rheumatoid factor ; sjc, swollen joints count ; tc, total cholesterol ; tjc, tender joints count ; vas, visual analog scale ; tmss, total modified sharp score p<0.001 ; p<0.05. cimt, common carotid artery intima - media thickness ; tmss, total modified sharp score positive correlation was detected between the mean cimt score and age, and the crp levels, ldl concentration and triglycerides level were positively correlated. in the regression model in which the mean cimt was the independent variable and age, crp, ldl, and tg were the dependent variables, age was found to be an independent predictor of cimt. to the best of our knowledge, this is the first study to evaluate the relationship of cimt and joint destruction with rheumatoid arthritis, and the main findings of the present study were that : (i) that contrary to our hypothesis, mtss and cimt were not significantly associated and (ii) that the cimt scores were positively correlated with age, crp levels, ldl concentration, and triglycerides level. radiographic follow - up is one of the most widely accepted methods of monitoring disease progression in ra patients. additionally, mtss as a radiographic measure evidently shows the effectiveness of medical options18, 19. it has also been proven that mtss is associated with impaired hand function and elevated haq scores in ra patients23. these findings presumably differ from the cvd risk in the radiographic progression. in our study, similarly, veselinovic. determined that their ra patients cimt scores were significantly higher than in the controls, and the brachial artery flow - mediated vasodilatation values were lower, which was attributed to the development of endothelial dysfunction and accelerated atherosclerosis13. adhikari. also studied early ra patients, and they reported the same findings as previous researchers12. according to the eular guidelines for cardiovascular risk management, low hdl levels with increased ldl and tg levels are a predictor of cvd in the general population24. furthermore, if the score model is used, increased tc / hdl levels could be an additional prognostic indicator in terms of cv risks25. in our study, the hdl levels of the patients were significantly higher than in the controls ; however, the ldl, tg, and tc / hdl levels were not significantly different between the groups. these findings show that endothelial dysfunction and atherosclerosis in ra patients are seen at the early stages of ra and that existing data about the general population are not sufficient to evaluate ra patients. previously, the relationships between cimt scores and cardiac events (myocardial infarction, angina pectoris, and coronary revascularization), cerebrovascular events (stroke or transient ischemic attacks), and hypertension were evidently shown26, 27. moreover, the cimt findings were worse, despite treatment with disease modifying drugs and biological agents like anti - tnf inhibitors28, 29. in order to determine the potential factors affecting cimt in patients with ra, for that reason, age was found to be the only independent predictor of cimt in multivariate regression analysis. in parallel with our findings, one study has revealed increased cimt scores with older ages30. additionally, we detected a positive correlation between crp and cimt among the controls, but the regression analysis excluded crp as an independent predictor. unlike our results, gonzalez - gay. evidently proved that there is a positive association between the maximum crp and cimt scores31 (table 3table 3. the relationship between patients mean cimt score and demographic, clinical, and laboratory characteristicspearsonscoefficientb regression coefficientage (years)0.628 0.499disease duration (months)1.000painvas0.201sjc280.307tjc280.021das28esr0.249haq0.246esr0.261crp0.4290.277rf0.115total cholesterol0.083ldl cholesterol0.3870.102hdl cholesterol0.265triglyceride0.4100.061tc / hdl0.252tmss0.164 p<0.001 ; p<0.05. from multiple lineer regression analysis ; bmi, body mass index ; crp, c - reactive protein ; das, disease activity score ; esr, erythrocyte sedimentation rate ; haq, health assessment questionnaire ; hdl, high - density lipoprotein ; ldl, low - density lipoprotein ; rf, rheumatoid factor ; sjc, swollen joints count ; tc, total cholesterol ; tjc, tender joints count ; vas, visual analog scale ; tmss, total modified sharp score). p<0.001 ; p<0.05. from multiple lineer regression analysis ; bmi, body mass index ; crp, c - reactive protein ; das, disease activity score ; esr, erythrocyte sedimentation rate ; haq, health assessment questionnaire ; hdl, high - density lipoprotein ; ldl, low - density lipoprotein ; rf, rheumatoid factor ; sjc, swollen joints count ; tc, total cholesterol ; tjc, tender joints count ; vas, visual analog scale ; tmss, total modified sharp score the limitations of our study include a cross - sectional design and a small sample size. despite the fact that our patients were not classified in terms of disease duration, we have tried to minimize the potential negative factors that may affect cimt measurements. in conclusion, patients suffering from ra require close monitoring for cardiovascular risks, and the comorbidity of age - related cardiovascular disease should not be overlooked. although we could not detect a positive relationship between cimt and mtss, further large - scale, multicenter, prospective studies are needed to clarify this issue.	[purpose ] the purpose of this study was to investigate the possible relationship between joint destruction and carotid intima - media thickness in patients with rheumatoid arthritis. [subjects and methods ] thirty - four ra patients and 31 healthy controls were enrolled in this study. the disease activity for 28 joints was recorded for each patient using the erythrocyte sedimentation rate (das28esr), a visual analog scale (vas010 cm), and a disability index, the health assessment questionnaire (haq). x - ray imagesof the patients were scored according to the modified sharp / van der heijde method, and the common carotid intimal medial thickness (cimt) was automatically measured with software using high - resolution doppler ultrasound. [results ] contrary to our hypothesis, the modified total sharp score (mtss) and cimt were not significantly associated. the erythrocyte sedimentation rate (esr) and c - reactive protein (crp) levels of the ra patients and the right cimt, left cimt, and mean cimt scores were significantly elevated. positive correlation was detected between the mean cimt score and age, crp levels, ldl concentration and triglycerides (tg) level. in the regression model, where the mean cimt was the independent variable and age, crp, ldl, and tg were dependent variables, age was found to be an independent predictor of cimt. [conclusions ] patients suffering from ra require close monitoring for cardiovascular risks, and the comorbidity of age - related cardiovascular disease should not be overlooked.
anti - neutrophil cytoplasmic antibody (anca)-associated vasculitis (aav) is characterized by pauci - immune necrotizing vasculitis of the small blood vessels,1 which comprise three different clinical syndromes : eosinophilic granulomatosis with polyangiitis (egpa, previously called churg - strauss syndrome), granulomatosis with polyangiitis (gpa, previously called wegener s granulomatosis), and microscopic polyangiitis (mpa). patients with aav are usually defined and classified according to the european medicines agency (emea) algorithm.2 this uses an algorithm to classify vasculitis and utilizes the american college of rheumatology (acr) criteria (1990) and the chapel hill consensus conference definitions.1 despite the common association with anca, however, these disease entities differ significantly in their clinical features and possibly in underlying pathophysiology. the association between interstitial lung disease (ild) and aav, particularly mpa, has been described in a number of case reports and case series reports in the last 2 decades. in addition, patients with pulmonary fibrosis and anca positivity but without other manifestation of systemic vasculitis have also been reported. the association between ild and anca / aav has received increasing attention both clinically and pathophysiologically. in this article, the association between ild and anca / aav is thoroughly reviewed. nada reported 2 patients with pulmonary fibrosis and p - anca - positive mpa in 1990.3 an association between aav or anca and ild has since been demonstrated. the association between ild and aav or anca in many patients was first reported in a japanese study by arinuma in 1994.4 in this retrospective study of 46 myeloperoxidase (mpo)-anca - positive patients with collagen - vascular disease or glomerulonephritis, 20 (43%) of the mpo - anca - positive patients had ild. of the 20 patients with ild and mpo - anca, 9 had mpa and 4 had crescentic glomerulonephritis. ild was diagnosed before the onset of renal diseases in 7 patients, whereas ild never developed after the diagnoses of renal diseases in the study population. since then, several studies have reported similar findings from japan59 and other countries1015 ; however, it is still recognized that ild is more frequently associated with anca - positive japanese patients than western patients.16 in a japanese nationwide rapidly progressive glomerulonephritis (rpgn) survey, 301/1147 (26.2%) patients with aav had ild.5 in another japanese nationwide, prospective, inception cohort study of aav (remission induction therapy in japanese patients with anca - associated vasculitides ; remit - jav), 61/156 (39.1%) patients had ild.6 in contrast, ild was observed in only 14/510 (2.7%) aav patients at a renal vasculitis clinic in london.10 this discrepancy between japan and other countries has been usually explained by the ethnicity and the predominance of mpo - anca (which will be discussed in later section of this review). in addition, the varied accessibility to computed tomography (ct) could somewhat affect the diagnostic rates of ild, especially subclinical or very mild cases, and cases with pulmonary limited vasculitis. according to the organization for economic co - operation and development (oecd) health statistics 2014 (http://stats.oecd.org/index.aspx?datasetcode = health_stat), the numbers of ct scanners per million population were 92.62, 29.26, 7.29, and 7.62 in 2002, and 101.28, 43.44, 8.95, and 13.49 in 2013 in japan, united states, united kingdom, and france, respectively. however, the prevalence of ild among patients with mpa (not entire aav) may actually be similar throughout the world. in a study of 33 consecutive mpa patients with rpgn and/or alveolar hemorrhage from greece, pulmonary fibrosis was found in 13 (39%) patients.11 in a study of mpa patients form argentina, 9 of the 28 (32%) patients had pulmonary fibrosis.12 most of the studies have indicated that ild developed more frequently in patients with mpo - anca - positive aav, mainly in those with a diagnosis of mpa, compared to patients with proteinase-3 (pr3)-anca - positive aav. in a japanese nationwide rpgn survey, patients with mpo - anca (291/1088, 26.7%) were more frequently associated with ild than patients with pr3-anca (20/114, 17.5%, p = 0.03).5 in another japanese nationwide, prospective, inception cohort study of aav,6 mpo - anca was more frequently associated with ild than pr3-anca (60/130 vs. 3/18, p = 0.02), and the prevalence of ild in patients with mpa / renal - limited vasculitis (37/78) was higher than that in patients with gpa (3/33) or egpa (2/14). it is well recognized that patients with mpa and with positivity for mpo - anca are predominant in japanese patients with aav.16 in fact, fujimoto confirmed that there was no major difference in aav incidence between japan and the united kingdom, whereas mpa and mpo - anca was more common in japan and gpa and pr3-anca was more common in the united kingdom.17 thus, this may contribute to the high prevalence of ild in japanese patients with aav as described in the previous section. a renal vasculitis clinic in london reported that all 14 patients with aav and ild had mpo - anca and a clinical diagnosis of mpa.10 the french vasculitis study group reported mpo - anca in all of the 12 patients who had systemic vasculitis related to anca (10 mpa and 2 gpa) and pulmonary fibrosis.13 six university pulmonology french departments with an expertise in the field of ild reported that they retrospectively detected 17 patients with pulmonary fibrosis and positive anca testing and that anca exhibited perinuclear fluorescence in 14 patients (6 with mpo - anca).14 mpa was diagnosed in 7/17 patients. in a retrospective multicenter study by 16 french medical centers, 49 patients with pulmonary fibrosis associated with aav were identified, and 43 patients had mpo - anca while 2 had pr3-anca, and 40 patients had mpa while 9 had gpa.17 thus, there is a significant predominance of mpo - anca and mpa in patients with aav and ild throughout studies from different countries. pulmonary fibrosis was clinically manifested at the time of diagnosis in the majority of aav patients that developed it. in a study of 33 consecutive mpa patients from greece,11 pulmonary fibrosis was present in 12 patients at the time of diagnosis, whereas pulmonary fibrosis developed in only one patient while on therapy 10 years after disease diagnosis. in most of the cases in a retrospective study of 17 patients presenting with pulmonary fibrosis and a positive anca testing by the french pulmonology group, lung fibrosis preceded the development of mpa by 110 years (if ever occurred), or the 2 diseases were diagnosed concomitantly.14 in a french retrospective multicenter study including 49 patients with pulmonary fibrosis associated with aav, the diagnosis of pulmonary fibrosis preceded the onset of vasculitis in 22 (45%) patients.15 in a retrospective study of 61 consecutive japanese patients with an initial diagnosis of idiopathic pulmonary fibrosis (ipf) at hospital presentation, mpo - anca was positive in 3 patients (5%) and mpo - anca positive conversion occurred in 6 patients (10%), of whom 2 were complicated by mpa.7 the median duration between initial ipf diagnosis and conversion to mpo - anca positivity was 23 months (range, 0 to 71 months). in a retrospective study of 966 patients with ipf from japan, anca was initially negative and measured repeatedly thereafter in 264 patients.8 in these patients, mpo - anca and pr3-anca seroconversion occurred in 15 (5.7%) and 14 (5.3%) patients, respectively, and mpa developed in the 6 patients with seroconversion to mpo - anca. collectively, the development of ild after a diagnosis of aav is very rare, whereas mpa developed in some patients with ipf with mpo - anca positivity at ipf diagnosis or with mpo - anca - positive conversion during follow - up ; however, there is no consensus on whether patients with ild and mpo - anca positivity but without other manifestations of systemic vasculitis should be called pulmonary limited vasculitis as a phenotypic variant of mpa. the french vasculitis study group reported that there were signs of usual interstitial pneumonia (uip) in 6 cases and non - specific interstitial pneumonia in one case, whereas the type of interstitial diffuse pneumonia was unspecified in 5 cases among the 12 patients with aav and pulmonary fibrosis by high - resolution computed tomography (hrct).13 in a retrospective study of 17 patients presenting with pulmonary fibrosis and a positive anca testing by the french pulmonology group, hrct analysis showed honeycombing, reticular intralobular opacities and traction bronchiectasis in all the patients with some degree of ground - glass attenuation (usually limited), whereas air - space consolidation was rare.14 in a french retrospective multicenter study including 49 patients with pulmonary fibrosis associated with aav, 42/49 patients were retrospectively reviewed, and typical uip was the main hrct pattern (n = 18, 43%).15 in a japanese retrospective study of 31 patients with pulmonary fibrosis and mpo - anca, chest hrct scan images showed reticulonodular shadows, honeycombing, and decreased lung volume, all of which were found predominantly in the lower and outer regions of the lung, and the histopathological pattern of pulmonary fibrosis could be classified as a uip pattern in all 11 autopsied cases.9 in another japanese retrospective study of 61 patients with ipf, the initial hrct scans of the 9 mpo - anca - positive patients showed subpleural reticular opacities, traction bronchiectasis, and honeycombing7 ; however, because these findings were also frequently observed in mpo - anca - negative cases, no differences were found between the 2 groups. in a japanese retrospective study of 31 patients with pulmonary fibrosis and mpo - anca, the histopathological features were analyzed in 15 patients : autopsy in 11, video - assisted thoracoscopic surgery (vats) in 2, and trans - bronchial lung biopsy (tblb) in 2.9 in 13 patients, interstitial fibrosis was not confined to the alveolar walls but extended to the proximal interstitial tissues and interlobular septa. the vasculitides were observed in pulmonary arterioles in 3 cases, in bronchial arteries in 3, and in capillaries presenting as alveolar hemorrhages in 2 ; however, vasculitis has almost never been pathologically proven in ild / pulmonary fibrosis from other studies. for example, in the other japanese retrospective study of 9 patients with ipf and mpo - anca,7 the histologic pattern identified in the surgical lung biopsies in 6 patients was consistent with that of uip, whereas alveolar hemorrhage or small vessel vasculitis was not observed. in addition, postmortem examination of the lung for 2 cases showed alveolar hemorrhage but no evidence of small vessel vasculitis. in most studies, aav patients with ild have a worse prognosis than those without it, and patients with pulmonary fibrosis and anca had as low a prognosis as patients with ipf without anca. the french vasculitis study group reported that the respiratory status of 5 of the 12 patients with aav and pulmonary fibrosis worsened, and 3 of them died from exacerbation of end - stage respiratory failure.13 in a study of 33 consecutive mpa patients from greece,11 the presence of pulmonary fibrosis was associated with increased mortality (p = 0.02), with 6 deaths occurring in the fibrotic group (n = 13) and one occurring in the nonfibrotic group (n = 20). in the fibrotic group, most deaths were related to pulmonary fibrosis. in a japanese retrospective study of 31 patients with pulmonary fibrosis and mpo - anca, a comparison of their institution 's survival rates in mpo - anca - negative pulmonary fibrosis with collagen vascular diseases, cryptogenic fibrosing alveolitis, and mpo - anca - positive pulmonary fibrosis revealed that the 5-year survival rate of mpo - anca - positive pulmonary fibrosis was worse than in mpo - anca - negative pulmonary fibrosis with collagen vascular diseases and was the same for cryptogenic fibrosing alveolitis.9 in a japanese nationwide, prospective, inception cohort study of aav, patients with ild had significantly lower birmingham vasculitis activity scores (bvas) than those without ild (p = 0.019)6 ; however, it does not necessarily mean that aav patients with ild have a better prognosis because ild is not included in these definitions. actually, in another japanese nationwide rpgn survey, the 5-year survival rate was 50.2% in the patients with ild and 73.3% in those without pulmonary involvement ; ild was added as one of the predictors of 5-year mortality.5 in contrast, in a retrospective study by the renal vasculitis clinic in london,10 there was no difference in mortality between patients with mpa and ild (n = 14) and those with mpa without ild (n = 180) over the same time period (p = 0.07), which should be treated with some caution due to the small cohort size. currently, there is no specific treatment for patients with ild and aav / anca - positivity.10 corticosteroids and/or cyclophosphamide, cyclosporine, or n - acetylcysteine were administered to these patients18 ; however, their clinical benefit and indication are not well established. in a japanese retrospective study of 61 patients with ipf,7 corticosteroids were administered more frequently in patients with mpo - anca than those without it (8/9 = 89% vs. 26/52 = 50%, p = 0.036). immunosuppressants, including azathioprine, cyclophosphamide and mizorbine, were administered to 4 (44%) patients with mpo - anca - positive fibrosis and only one (2%) patient with anca - negative ipf. as for mpo - anca - positive fibrosis, 6 patients died during the follow - up period. four patients died from the acute exacerbation of pulmonary fibrosis, one patient died of intractable pneumothorax, and one patient died from a cytomegalovirus infection. as for ipf, therapy was started for progressive respiratory failure in all patients. eight patients died of an acute exacerbation of pulmonary fibrosis, 6 patients died of progressive respiratory failure, 6 patients died of pneumonia and 3 patients died of lung cancer. the median survival of patients with mpo - anca - positive fibrosis and ipf was 62 months (95% ci 29.1394.87) and 63 months (95% ci 44.5581.42), respectively. however, induction therapy with cyclophosphamide might improve the outcome.15 in a french retrospective multicenter study including 49 patients with pulmonary fibrosis associated with aav, the 3-year survival rate in patients treated with an immunosuppressant (cyclophosphamide or rituximab) combined with glucocorticoids as induction therapy was better than that in patients treated with glucocorticoids alone (94% vs. 64%, p = 0.03). the association of ild and anca or aav does not seem to be fortuitous, considering its higher prevalence than in the average population. although the pathogenesis of ild in aav remains poorly understood, 3 major hypotheses have been proposed by several groups on the development of ild in patients with anca or aav as summarized elsewhere by kagiyama.8 first, repeated episodes of alveolar hemorrhage due to pulmonary capillaritis could be the pathogenesis of pulmonary fibrosis.13 schnabel reported that subclinical alveolar bleeding was, indeed, a common finding in aav.19 second, mpo - anca may play a direct role in the pathogenesis of pulmonary fibrosis. guilpain suggested that oxidative stress, in particular the production of hypochlorous acid (hocl) through the interaction of mpo with anti - mpo antibodies, could trigger the fibrotic process observed in mpa.20 foucher observed patchy inflammatory cell infiltrates throughout the parenchyma of the lung in their mpo - induced rat model of aav and suggested that the presence of an anti - mpo directed autoimmune response contributes to generalized pulmonary tissue injury.21 third, to the extent that pulmonary fibrosis is clinically manifested at the time of diagnosis in the majority of patients, ipf may induce anca and aav.11 namely, anca might be produced as a result of neutrophil destruction during the chronic inflammation process. in addition, tobacco smoke exposure may play an initiating role in the pathophysiology of the disease by activating epithelial cells and stimulating their mpo expression. in a japanese retrospective study of 9 patients with ipf and mpo - anca, they were all smokers and frequently demonstrated low attenuation areas on their hrct scans.7 similarly, in a retrospective study of 17 patients presenting with pulmonary fibrosis and a positive anca testing, 11 patients were either current or past smokers.14 furthermore, churg suggested that acute exposure to cigarette smoke leads to macrophage activation and neutrophil recruitment, with consequent elastin and collagen degradation, resulting in accelerated matrix destruction and emphysema.22	the association between interstitial lung disease (ild) and anti - neutrophil cytoplasmic antibody (anca)-associated vasculitis (aav), particularly microscopic polyangiitis (mpa), has been described in a number of case reports and case series reports in the last 2 decades. in addition, patients with pulmonary fibrosis and anca positivity but without other manifestations of systemic vasculitis have also been reported. pulmonary fibrosis was clinically manifested at the time of diagnosis in the majority of aav patients that developed this condition. moreover, anca - positive conversion occurs in patients initially diagnosed with idiopathic pulmonary fibrosis, and as a result, other manifestations of systemic vasculitis develop in some of these patients. there is significant predominance of myeloperoxidase (mpo)-anca and mpa in patients with aav and ild. radiological and pathological findings generally demonstrate usual interstitial pneumonia (pattern) in the lungs of these patients. in most studies, aav patients with ild have a worse prognosis than those without it.
acquired amegakaryocytic thrombocytopenia (amt) is a rare hematologic disorder characterized by thrombocytopenia and is association with a markedly diminished number of bone marrow megakaryocytes. this condition has been observed in patients with lupus erythematosus, t - cell large granular lymphocyte leukemia and eosinophilic fasciitis. amt is associated with a marked increase of t - activated suppressor cells (cd8 + /dr+) and a high level of autoantibodies against the thrombopoietin receptor (c - mpl). no cytogenetic abnormality has been shown to be consistently present in amt. in most patients we describe a rare case of amt syndrome which did not respond to any of the previous therapies except rituximab (ant - cd20 antibody). in september 2008 ; a 50-year - old man with petechial rash, large ecchymosed, gross hematuria and severe shoulder and periumbilical pain was admitted to our center. in the past medical history : he had symptoms of bleeding for 15 months ago and laboratory studies revealed a severe thrombocytopenia with platelet count of 12000/l, a leukocytosis with white blood cell (wbc) count of 25000/l and hemoglobin (hb) of 15 gr / dl. there was an increased level of myeloid / erythroid series and a severe decrease of megakaryocytes series, in the bone marrow examinations. patient was treated with intravenous immunoglobulin (ivig) and transient clinical response was taken. after six months, he was referred to our center for the complaint of severe bleeding. he had a wbc count of 12100/l, hgb of 13 gm / dl, hematocrit (hct) of 31.3%, a mean corpuscular volume (mcv) of 93fl, and a platelet count of 7000/l. the patient undergone bone marrow examination again, cellularity was 75%, myeloid and erythroid series were mildly increased and megakaryocytes severely decreased to absented. additional studies including antinuclear antibodies (ana), rheumatoid factor (rf), and igm / igg antiplatelet antibody tests were normal. the patient with diagnosis of amegakaryocytic thrombocytopenia was treated with ivig again, but clinical and laboratory response were not taken. there was not any improvement in patient 's signs and symptoms (figure 1). platelet count was lower than 10000/l and he was experiencing diffuse petechial rash, easy bruising, gingival bleeding and hematuria. bleeding symptoms were controlled by platelets transfusions, however it did not cause into a dramatic increase in the platelets count. we explained the treatment options, including anti - thymocyte globulin (atg) and rituximab to the patient. hence rituximab (anti - cd20 antibody) with dose of 375 mg / m, with three weeks interval, for three consequent doses was started. the platelet count rose dramatically to 20000/l on the 6th day, to 30000/l on the 29th day and to 200 000/l on the 42th day. in 25 months follow up ; the patient had normal blood counts without any medications, except that wbc was mildly increased (figure 1). hemoglobin the patient had a hypercellular marrow with adequate to increased megakaryocyte in the 63th day after treatment. we diagnosed myeloproliferative disease according to the morphological changes observed in the bone marrow examinations. the search for bcr / abl, philadelphia chromosome, and janus kinase2 (jak2) v617f by pcr test was also negative, and the diagnosis of myeloproliferative disease was not approved in our patient. here we presented a case of refractory amt which responded to anti cd-20 antibody therapy. the differential diagnosis of patients suspected to have amt are idiopatic (immune) thrombocytopenic purpura, with misinterpretation of morphologic findings, hereditary and acquired aplastic anemia, preleukemia and systemic lupus erythematosis. the clinical course of the disease is variable, and suggested treatment have shown variable efficacy in the management of disease. immunosuppressive therapies including administration of steroids, cyclophosphamide, cyclosporine, androgens, atg have been used with varying degrees of success. ivig, prednisone, cyclophosphamide, and vincristine have not been efficacious in amt, unlike the response to these agents in immune - mediated thrombocytopenia, although there are isolated reports of prednisone, ivig, and cyclophosphamide being transiently effective in occasional patients with amt. myeloablative chemotherapy (busulfan and cyclophosphamide) followed by allogeneic bone marrow transplant from a fully hla - matched sibling has been reported to be effective. atg was also reportedly effective in a case of amt associated with a marked increase of t - activated suppressor cells (cd8+/dr+). cyclosporine alone or in combination with atg has been shown to be very effective in treatment of amt. in one of the reports of cyclosporine use, longitudinal follow - up revealed a relapsing and remitting disease course of amt, which correlated with the dosing of cyclosporine. it has been shown to be effective in patients with bone marrow failure due to myelodysplastic syndromes (mds), graft failure, chemotherapy, or aplastic anemia. there are a limited number of studies reporting hematopoietic cell transplantation in patient with the acquired amt.17 in 2008, fukushima., firstly reported a case of amegakaryocytic thrombocytopenia due to systemic lupus erythematosis. rituximab is a chimeric monoclonal antibody against the protein cd20, which is primarily found on the surface of b cells. cd20 is expressed on most stages of b cells, such as immature b cells, naive b cells, memory b cells, and germinal center b cells, but not early pro - b cells or plasma cells. it has been described that rituximab may suppress pathogenic b - cell clones which produce anti - tpo(thrombopoietin) receptor antibodies that are resistant to conventional immunosuppressive therapy, including cyclosporine and prednisone. anti - cd20 antibody acting against cd20 receptors over membrane of b - cell type lymphocytes, and suppressed the humoral immunity. anti - cd20 antibody was taken and this refractory amt patient that was depended to platelet transfusions, responded to this drug, and after first dose, the dependency of patient to platelet transfusions does not continued, and at the beginning of 3th dose, the platelet count became normal. rituximab was effective in this refractory case of amt, the platelet count is normal after 25 months of last dose without any medication, but mild leukocytosis (wbc 11000 to 25200/l) remained sustained. moreover we did not have any documentation for diagnosis of chronic myeloproliferative disease except morphology of bone marrow examination.	amegakaryocytic thrombocytopenia (amt) is a rare cause of acquired thrombocytopenia. the pathogenesis and treatment of amt is not clearly known. here we demonstrate a 50-year - old man presented with the clinical manifestations of severe thrombocytopenia (7000 platelets/l) with a marked decrease to absent of megakaryocytes in the bone marrow. the patient did not respond to intravenous immunoglobulin, cyclosporine or high dose prednisone. after the treatment with anti - cd20 antibody (rituximab), the patient 's clinical symptoms and platelet counts improved.
salivary gland tumors are known for their varied histomorphological appearance, which sometimes poses a diagnostic challenge for pathologists. one of such appearances is the oncocytic change in tumor cells of benign and malignant salivary gland tumors. morphologically, oncocytes are characterized by abundant eosinophilic granular cytoplasm with pyknotic nuclei, which is usually centrally placed or shifted towards one side. oncocytic nature of tumor has been associated with warthin s tumor, oncocytoma, and oncocytic carcinoma. pleomorphic adenoma (pa) is the most common salivary gland tumor and accounts for 70% of all the tumors of major salivary glands. as the name suggests, pa is characterized by plethora of morphological appearances including myxoid, hyalinized, chondroid, osseous, squamous etc. however, predominantly oncocytic cells in pa are extremely rare to occur. literature search indicates only 18 cases reported till date with largest case series (9 cases) reported by skalova. in 1999 (table 1). in the present paper, we report a case of pa showing predominantly oncocytic cells with cholesterol clefts and macrophages. a 45-year - old male patient reported with a slow growing asymptomatic swelling on the hard palate region since 5 months. physical examination revealed 4 cm 4.5 cm, diffuse, non - tender, smooth - surfaced, and soft to firm non - fluctuant swelling involving the posterior region of the palate (figure 1). based on the clinical and radiographic findings, a presumptive diagnosis of pa of palatal salivary glands was made. microscopic examination showed homogenous eosinophilic cellular mass composed of epithelial components in the form of tubular and solid pattern. the abluminal polygonal and oval cells showed abundant dark eosinophilic granular cytoplasm (figure 2). cholesterol crystals were evident at few places associated with large oval macrophages showing faint granular eosinophilic cytoplasm along with giant cells (figure 4). the tumor was very cellular and devoid of chondromyxoid stroma. based on these findings, in 1931, hamperl coined the term oncocyte (onkocyten) to describe a cell having abundant eosinophilic granular cytoplasm and a centrally located hyperchromatic nucleus. oncocytic metaplasia of ductal and acinar cells is commonly encountered in normal salivary glands in persons over 50 years of age, but primary oncocytic neoplasms and tumour - like lesions are comparatively rare, accounting for less than 1% of salivary tumours. histomorphological appearance of oncocytes is attributed to abundant mitochondria present in the cytoplasm of cell. presence of oncocytes in salivary gland tumors is not a rare phenomenon as they are considered as a prominent feature of warthin s tumor, oncocytoma, and oncocytic carcinoma. even some non - oncocytic tumors like myoepithelioma, acinic cell carcinoma, mucoepidermoid carcinoma, basal cell adenoma, polymorphous low - grade adenocarcinoma, and sebaceous adenoma can show predominantly oncocytic histomorphologic feature. however, their presence in pa is regarded as an extremely rare phenomenon and can cause diagnostic pitfall in the differential diagnosis. to our knowledge, only 18 cases of oncocytic pa were reported till date including present case (table 1). most common site was parotid gland (14 cases) followed by submandibular (2 cases), para - pharyngeal area and palate. recurrence and metastasis has not yet been reported in the literature. in the present case, we have reported cholesterol cleft formation at few places associated with prominent macrophages and giant cells (figure 4). macrophages were large oval cells showing faint granular eosinophilic cytoplasm with centrally or peripherally placed pyknotic nuclei. since cholesterol clefts act as foreign material, they are usually surrounded by macrophages and giant cells. only one case of pa (non - oncocytic) associated with cholesterol crystals and macrophages have been reported in the literature till date. in the present case, we observed clear change in the cytoplasm of oncocytic cells, which again can be regared as a rare phenomenon in oncocytic pa. oncocytic metaplasia has long been regarded as affecting both secretory and ductal epithelial cells in salivary glands, although askew. provided ultrastructural and light microscopic evidence of both epithelial and myoepithelial oncocytes. in agreement with this, feiner described a case of an oncocytic adenoma of the parotid gland revealing immunohistochemical co - expression of s-100 protein and cytokeratins (cks), which was interpreted as an indication of myoepithelial differentiation of oncocytes. oncocytic pa showed positivity for ck5/6, ck8/18, ck14, vimentin, alpha - smooth muscle actin, s100 protein, p63, epidermal growth factor receptor and b - catenin. however, oncocytic cells in pa usually show a luminal phenotype, expression of anti - mitochondria antibody and reduced b - catenin staining. in normal age related oncocytic metaplasia, both luminal and abluminal cells show similar oncocytic morphology indicating similarity with the molecular pathogenesis of oncocyte formation in pa. in oncocytic pa, mariano. reported positivity for high molecular weight ck in luminal cells and myoepithelial cells. this suggests that mitochondrial phenotype is present in both the luminal and myoepithelial cells and mutation occurs in more differentiated cells during tumor formation. the prognosis is good and no recurrent cases have been reported till date. in conclusion, we have reported a rare case of oncocytic pa with histopathological evidence of cholesterol crystals, macrophages and giant cells. presence of clear cell change in oncocytic cells can also be regarded as a rare event in oncocytic pa as it has not yet been reported in the literature.	pleomorphic adenoma (pa) is the most common salivary gland tumor characterized by histo - morphological diversity in the form of myxoid, hyalinized, chondroid, osseous, and squamous areas. in this paper, we report a rare case of predominantly oncocytic variant of pa in a 45-year - old male patient on the posterior palatal region. microscopic examination showed homogenous eosinophilic cellular mass composed of epithelial components arranged in the form of tubular and solid patterns. the polygonal and oval cells showed abundant dark eosinophilic granular cytoplasm. the cell borders were distinct with a central nucleus showing prominent nucleoli. interestingly at few places, cholesterol clefts were seen surrounded by macrophages and giant cells. the tumor was surgically excised with no evidence of recurrence after 2 years.
according to the brazilian institute of geography and demography (instituto brasileiro de geografia e estatstica or ibge),1 brazil presents an aged population that corresponds to 9.7% of its total population. the aging index grew from 0.11 in the 1980 s to 0.25 in 2004.1 these values show that brazilian society is aging, but it can still be considered young when compared to other countries, such as italy, japan and germany, which present larger elderly populations.1 the aging process has been widely studied in the last decade, enhancing the understanding of diseases that affect elderly people and, therefore, promoting a shift towards a healthier and more independent aging process. amongst the physiological modifications observed during aging, the loss of functional capabilities and the modifications of metabolic functions are the most important.2,3 studies show that regular physical activity is a key feature for more healthful aging.3,4 the risk of disease and health problems can decrease with exercise,3,4 which may also help the immune system recover in the elderly, reducing the prevalence of infections and neoplasias among exercisers.3 regular exercise can also decrease fat mass and increase lean mass and aerobic performance.5 in general, physical exercise has proven to be beneficial for diseases associated with aging, and these benefits may result from any type of physical activity. however, resistance exercises, as the most effective method for developing muscle strength, have shown specific benefits and are currently prescribed by many major health organizations to improve health and fitness.3 when sedentary for a long time, elderly people can present significant alterations in body composition and physical fitness, compromising their quality of life in many important ways. some chronic diseases, for instance aids, can even worsen this situation because such diseases exacerbate the effects of aging on body composition and muscle strength. recovering strength and physical fitness is the major goal of exercise in patients with aids wasting syndrome. therefore, a resistance training program could form the basis of an exercise prescription for this group of patients. it should be progressive, so resistance increases as the patient becomes stronger.6 some studies involving hiv - positive adults and resistance training have been carried out, but no studies focusing on elderly hiv - positive people have been conducted yet.79 the present study aims to describe a case series of hiv - positive elderly people who participated in a progressive resistance training program and to evaluate their body composition, muscular strength, physical fitness and evolution of cd4 and cd8 cell counts. this is a case series study10 describing the effects of a one - year resistance training program among elderly hiv - positive patients. all hiv - positive patients, 60 or more years old and who were being treated for hiv / aids at the hospital das clnicas da faculdade de medicina da universidade de so paulo by the end of 2003 were invited to participate in this study. the first invitation was made either by telephone or directly at the medical consultation, followed by written correspondence to their homes. after obtaining their written agreement to participate in the research, orientation to the program the institutional research ethical committee approved the study after review, and all participants signed a formal written, informed consent form. inclusion criteria were the following : hiv - positive, older than 60 years by july 2003, lack of regular physical activity practice and agreement to participate. exclusion criteria were the following : medical contraindication to perform exercises, any physical condition that limited resistance of training, use of corticosteroids or other anabolic steroids and non - adhesion to the training program (defined as an absence of at least three consecutive months from the training program). after the initial interview, patients were questioned about the time since their hiv diagnosis, in addition to their health and life conditions. patients were clinically followed by their own infectious disease - specialized physicians throughout the study. both immunological evaluations, by means of t - cd4 and t - cd8 lymphocytes counts, and virological evaluations, by means of quantitative hiv - rna pcr, were standard practice and performed every four months for those who attended the clinic. the anthropometric measurements collected in this study were body mass, circumferences and skinfolds, assessed according to protocols previously described.11,12 body composition assessments by dual - energy x - ray absorptiometry (dexa) were carried out before and after the training period, following the methods described by ellis.13 the supervised progressive resistance training program consisted of four different exercises (muscles trained) : 1) leg press (quadriceps), 2) seated row (latissimus dorsi), 3) lumbar extension (paravertebral muscles), and 4) chest press (pectoralis major). exercises were performed using free weight machines (maxiflex biodelta, joinville, sc, brazil). exercises were performed in three sets of 812 repetitions at light, moderate and heavy resistance, respectively, with 12 minutes of rest between the series, twice a week for one year, from march 2004 to september 2005. according to our protocol, adapted from the american geriatric society recommendations,14 all subjects were submitted to five training sections, prior to the beginning of the training program, in order to assure an adequate and safe evaluation of their initial working load.15 sub - maximum weight supported (in the heavy resistance series) was defined as the maximum weight lifted smoothly without valsalva maneuver, apnea or isometry. for the purposes of this work, it was a surrogate of muscular strength. patients were also submitted to two functional tests every four months in order to better evaluate their physical performance evolution along the training period. the tests included an assessment of a timed 2.4-meter walk (walking 2.4 m) at a normal pace and a timed test of five repetitions of rising from a chair and sitting down (sit - standing), according to the protocol described by guralnik.16 data were recorded and stored in an excel sheet and analyzed with statistica for windows, version 7.5. the differences seen in strength, anthropometric variables and functional tests before and after the one - year training period were compared with wilcoxon matched pair test, considering the entire sample and controlling for age and gender effects as well as for baseline hiv infection stage and co - morbidity presented by them. one hundred and eight hiv - positive patients, more than 60 years old by the end of 2003, were invited to participate in the study. of those, two refused, two died, and two did not have the physical condition necessary to participate in the training program. in addition, 88 patients did not show interest or were unable to participate in the study because they lived outside so paulo city and/or presented poor clinical conditions. thus, only 14 patients, aged 6271 years (mean sd : 65.6 2.9), of both genders and with average of nine years duration of hiv infection, agreed to participate. in addition, three of these patients abandoned the training program for more than three months and were excluded from the study protocol. therefore, only 11 completed the period of training and remained in the study for the final analysis. among those 11 participants, only one did not report previous use of highly active antiretroviral therapy (haart), and four presented previous histories of hiv - associated diseases. on average table 2 shows the weight load variation after one year of resistance training program with four different exercises. the average load of each muscular group trained increased significantly : leg press 97%, p = 0.004 ; seated row 78%, p = 0.021 ; lumbar extension 122%, p = 0.003 ; and chest press 74%, p = 0.003. the increased muscular strength is reflected in the results of the two functional tests performed. there was a significant reduction in both the sit - standing (2.00 1.57s, p=0.003) and the walking 2.4 m (9.25 6.58s, p=0.003) times when comparing before and after the year of resistance training, as shown in figures 1 and 2. weight did not change significantly, nor did most of the body composition measures analyzed (see table 3), with the exception of triceps and thigh skinfolds, which both showed significant reduction (11.4 9.2 mm, p = 0.037 ; and 13.9 12.1 mm, p = 0.011, respectively), as shown in figure 3. the differences seen in all variables described above were not affected by age, gender, hiv infection stage or co - morbidity with the exception of strength variation in seated row, which increased more among men (p=0.02). in addition, no significant adverse effect related to the exercise program or any new (or worsening of previously existent) aids - related condition developed during the training period. finally, with respect to the evolution of virological and immunological markers of hiv infection, all but two patients (patients aap and afs of table 1) had persistently undetectable viral loads. also, there were significant increases in the cd4 cell counts (388 163 before vs. 539 225 after, = 151 cells, p=0.008) and in the cd4/cd8 ratio (0.63 to 0.81, p=0.009) and a non - significant increase in the cd8 cell counts (762 423 before vs. 816 376 after, = 54 cells ; p=0.464). to the best of our knowledge, this is the first study to investigate the influence of progressive resistance training in hiv - infected people older than 60 years. the training protocol followed the recommendations of american college of sports and medicine3, which suggest a progression model for resistance exercises in healthy, older adults. for this population, the studies show favorable changes regarding the risk factors associated with osteoporosis, heart disease, cancer and diabetes and also show a reduction in fat mass and an increase in lean mass and muscular strength.4,18 as mentioned in the results, only 11 out of 14 hiv - infected people, aged 60 years or more, completed the one - year training period. among them, those who experienced some intercurrence during the training period did not suffer deconditioning. when they restarted training, it was easy for them to continue with the same loads they used before. a substantial strength increase was seen for all exercises in every patient who completed the training program regardless of their age, gender, baseline hiv infection stage or the presence of any hiv / aids - associated morbidity, and this was not different for the only patient who did not use antiretrovirals. the average increase in the load supported after 12 months of resistance training varied from 74 to 122% (p0.0030.021), depending on the muscular group considered. in addition, the functional tests results, which showed significant improvement in the sit - standing and walking 2.4 m times (p= 0.003), reflected this increase. before starting the training program, the patients were all sedentary and, in spite of having different baseline hiv infection stages and co - morbidity histories, they had stable clinical conditions. thus, the strength increase observed could hardly be attributed to any other cause but the training program. the strength increment is important for the quality of life of the aged population because it improves biomechanics and cardiovascular responses, thus facilitating daily life activities. our sample was comprised of elderly hiv - positive individuals who were experiencing a stable clinical evolution, almost all of them under haart treatment for more than one year prior to the beginning of the study. however, despite the significant benefits associated with haart, hiv infection and its therapy have been associated with the development of several metabolic complications : increased central adiposity, peripheral lipoatrophy, peripheral insulin resistance, diabetes, dyslipidemia and hypertriglyceridemia, osteoporosis and osteopenia. these complications may predispose patients to a premature risk of metabolic and cardiovascular diseases.19 in addition, aging predisposes them to the same biological effects,20 and one could expect that aging could act as a potentiator of those hiv infection- and haart - related alterations. on the other hand, resistance training improves many of those alterations in our study, no changes were seen either in body composition, assessed by dexa after 12 months of resistance training, or in anthropometric measures with the exceptions of triceps (p=0.037) and thigh (p=0.011) skinfolds. weight did not change significantly (p=0.84) either. we are well aware that this could be simply an effect of the lack of power of the study due to the small size. however, the low intensity of the alterations seen in table 3 and the fact that some of them are opposite to what is the expected effect of resistance training could suggest that the absence of statistical significance may be true. finally, the effects of exercises on immune function have been studied in both adult and elderly healthy populations, showing that moderate levels of training are helpful for both populations.21,22 in our study, assessment of immune response, which is usually performed for hiv patients, showed a significant increase in both cd4 counts (=151 cells, p=0.008) and in the cd4/cd8 ratio (0.63 to 0.81, p=0.009) in addition to a non - significant increase in the cd8 counts (= 54 cells ; p=0.464) after one year of resistance training. those are important variations in the number of cells, considering that there were no significant changes in viral load or haart use among them during the training period. almost all patients had undetectable viral load and were on haart therapy before, during and after the training period. therefore, the observed changes in the numbers of cd4 and cd8 cells, together with the absence of new hiv - related morbidity, should most probably be attributed to their stable hiv infection conditions. in conclusion, despite the relatively small sample, our results indicate that a progressive resistance training program can benefit elderly people living with hiv without any major adverse effects or worsening of hiv / aids related conditions, which favors its recommendation for such a population.	backgroundelderly people present alterations in body composition and physical fitness, compromising their quality of life. chronic diseases, including hiv / aids, worsen this situation. resistance exercises are prescribed to improve fitness and promote healthier and independent aging. recovery of strength and physical fitness is the goal of exercise in aids wasting syndrome.objectivethis study describes a case series of hiv - positive elderly patients who participated in a progressive resistance training program and evaluates their body composition, muscular strength, physical fitness and the evolution of cd4 + and cd8 + cell counts.methodssubjects were prospectively recruited for nine months. the training program consisted of three sets of 812 repetitions of leg press, seated row, lumbar extension and chest press, performed with free weight machines hts, twice / week for one year. infectious disease physicians followed patients and reported all relevant clinical data. body composition was assessed by anthropometric measures and dual - energy x - ray absorptiometry before and after the training program.resultsfourteen patients, aged 6271 years old, of both genders, without regular physical activity who had an average of nine years of hiv / aids history were enrolled. the strengths of major muscle groups increased (74%122%, p=0.0030.021) with a corresponding improvement in sit - standing and walking 2.4 m tests (p=0.003). there were no changes in clinical conditions and body composition measures, but triceps and thigh skinfolds were significantly reduced (p=0.037). in addition, there were significant increases in the cd4 + counts (n=151 cells ; p=0.008) and the cd4+/cd8 + ratio (0.63 to 0.81, p=0.009).conclusionresistance training increased strength, improved physical fitness, reduced upper and lower limb skinfolds, and were associated with an improvement in the cd4 + and cd4+/cd8 + counts in hiv positive elderly patients without significant side effects.
a total of 85 (30 women 55 men) consecutive patients routinely referred to coronary angiography for stable angina pectoris were included in the study after the following exclusions : any kind of rhythm abnormailities that could have interfered with p- wave analysis (af, freguent atrial and ventriculer beats, pacemaker rhythm), acute coronary syndromes, valvular heart disease, serum electrolyte disturbances, abnormal thyroid function, pulmonary hypertension, cardiomyopathies, use of any antiarrhythmic drug, history of myocardial infarction, percutaneus coronary intervention, and cardiac surgery. entry criteria included chest pain or other symptoms suggestive of myocardial ischemia whc clinically indicated coronary angiography. the clinical risk factors for the patients such as age, gender, hypertension (ht), diabetes mellitus (dm), history of hyperlipidemia, and smoking status were noted. patients were divided into 4 groups based on their extent of angiographic coronary artery disease. patients with normal coronary arteries were labeled as normal group (25 patients), 22 patients with signficant obstruction in 1 major epicardial artery were considered as having 1 vessel disease, 26 patients with significant obstruction in 2 major epicardial arteries were included in the 2 vessel disease group, finally 12 patients with significant obstruction in 3 major epicardial arteries were enrolled in the 3 vessel disease group. hypertension was defined based on blood pressure 140/90 mm hg or greater, and a history of antihypertensive drug use. dm was defined as fasting blood glucose126 mg / dl on two occasions or being on treatment. all demographic and clinical data were collected prospectively. a 12- lead surface electrocardiogram (ecg) recordings were acquired at a paper speed of 50 mm / s, with 1 mv / cm standardization. two in - vestigators without knowledge of the clinical status of the patients manually measured the maximum and minimum p - wave duration and pd. to improve accuracy we used calipers and magnifying lenses. the onset of p - wave was defined as the junction between the end of the p - wave deflection and the ofset of the p - wave as the junction between the end of the p - wave deflection and the isoelectric line. we calculated p maximum (p max) and p minimum (p min) and their diferences were defined as pd. echocardiographic measurements were performed by using a 2.5 mhz probe with acuson sequa echocardiographic device (siemens, usa). lv dimensions were generally measured with 2d - guided m - mode from the parasternal projections, using a leading edge to leading edge convention. the left atrium and the left ventricle diameters, left ventricular ejection fraction (lvef), and the presence of mitral insufficiency were evaluated. the angiographic characteristics, which included lesion location and percentage stenosis, of all coronary lesions in the index coronary angiogram were obtained by throughly reviewing the angiogram. angiographic analysis was carried out by two experienced cardiologists who were blinded to the study protocol. vessel score was the number of vessels with a significant stenosis (> % 50). we also used gensini scoring system. according to this method we defined narrowing of the lumen of coronary arteries as 1 for 1 - 25% stenosis, 2 for 26 - 50% stenosis, 4 for 51 - 75% stenosis, 8 for 76 - 90% stenosis, 16 for 91 - 99% stenosis and 32 for total occlusion. then the score is multiplied by a factor that shows the significance of the lesion s location. it is 2.5 for proximal left anterior descending artery (lad) and proximal circumflex artery (cx) lesions, 1.5 for a mid - lad lesion, and 1 for distal lad, mid / distal cx and right coronary artery lesions. baseline demographic data are presented as mean sd for continuous variables and frequancies for discrete variables. comparison of parametric values between the 2 groups was performed by means of an independent samples t - test. correlation between p wave measurements and angiographic, clinical, and echocardiographic variables were assessed by pearson correlation coefficient. to ascertain the independent contribution to pd multiple linear regression analysis a total of 85 (30 women 55 men) consecutive patients routinely referred to coronary angiography for stable angina pectoris were included in the study after the following exclusions : any kind of rhythm abnormailities that could have interfered with p- wave analysis (af, freguent atrial and ventriculer beats, pacemaker rhythm), acute coronary syndromes, valvular heart disease, serum electrolyte disturbances, abnormal thyroid function, pulmonary hypertension, cardiomyopathies, use of any antiarrhythmic drug, history of myocardial infarction, percutaneus coronary intervention, and cardiac surgery. entry criteria included chest pain or other symptoms suggestive of myocardial ischemia whc clinically indicated coronary angiography. the clinical risk factors for the patients such as age, gender, hypertension (ht), diabetes mellitus (dm), history of hyperlipidemia, and smoking status were noted. patients were divided into 4 groups based on their extent of angiographic coronary artery disease. patients with normal coronary arteries were labeled as normal group (25 patients), 22 patients with signficant obstruction in 1 major epicardial artery were considered as having 1 vessel disease, 26 patients with significant obstruction in 2 major epicardial arteries were included in the 2 vessel disease group, finally 12 patients with significant obstruction in 3 major epicardial arteries were enrolled in the 3 vessel disease group. hypertension was defined based on blood pressure 140/90 mm hg or greater, and a history of antihypertensive drug use. dm was defined as fasting blood glucose126 mg / dl on two occasions or being on treatment. a 12- lead surface electrocardiogram (ecg) was obtained from each patient while in supine position. recordings were acquired at a paper speed of 50 mm / s, with 1 mv / cm standardization. two in - vestigators without knowledge of the clinical status of the patients manually measured the maximum and minimum p - wave duration and pd. to improve accuracy we used calipers and magnifying lenses. the onset of p - wave was defined as the junction between the end of the p - wave deflection and the ofset of the p - wave as the junction between the end of the p - wave deflection and the isoelectric line. we calculated p maximum (p max) and p minimum (p min) and their diferences were defined as pd. echocardiographic measurements were performed by using a 2.5 mhz probe with acuson sequa echocardiographic device (siemens, usa). lv dimensions were generally measured with 2d - guided m - mode from the parasternal projections, using a leading edge to leading edge convention. the left atrium and the left ventricle diameters, left ventricular ejection fraction (lvef), and the presence of mitral insufficiency were evaluated. the angiographic characteristics, which included lesion location and percentage stenosis, of all coronary lesions in the index coronary angiogram were obtained by throughly reviewing the angiogram. angiographic analysis was carried out by two experienced cardiologists who were blinded to the study protocol. vessel score was the number of vessels with a significant stenosis (> % 50). we also used gensini scoring system. according to this method we defined narrowing of the lumen of coronary arteries as 1 for 1 - 25% stenosis, 2 for 26 - 50% stenosis, 4 for 51 - 75% stenosis, 8 for 76 - 90% stenosis, 16 for 91 - 99% stenosis and 32 for total occlusion. then the score is multiplied by a factor that shows the significance of the lesion s location. it is 2.5 for proximal left anterior descending artery (lad) and proximal circumflex artery (cx) lesions, 1.5 for a mid - lad lesion, and 1 for distal lad, mid / distal cx and right coronary artery lesions. baseline demographic data are presented as mean sd for continuous variables and frequancies for discrete variables. comparison of parametric values between the 2 groups was performed by means of an independent samples t - test. correlation between p wave measurements and angiographic, clinical, and echocardiographic variables were assessed by pearson correlation coefficient. to ascertain the independent contribution to pd multiple linear regression analysis the clinical, echocardiographical and electrocardiographic characteristics of the cases in group 1 and group 2 are shown in table 1. there was no difference in comparison of groups with regard to age, hypertension, diabetes and smoking. although p max was significantly higher in groups 2, 3 and 4, no difference was determined between groups 1 and 2. the relationship between pd, and clinical, and echocardiographic characteristics in patients with cad is shown in table 3. in cad group, pd was related to diabetes, smoking and ef (p=0.014, p=0.006, p=0.003) but not related to other clinical and echocardiographic characteristics (table 3). pmax and pd were related to vessel and gensini scores in patients with cad (table 4). in multivariate logistic regression analysis, increased pd was found to be independently associated with vessel (=4.139, p=0.002) and gensini score (=0.132, p=0.007). baseline clinical, echocardiographical and electrocardiographic characteristics of the study population cad : coronary artery disease ; lv : left ventricle ; edd : end - diastolic dimension ; esd : end - systolic dimension ; ef : ejection fraction ; lad : left atrial diameter ; p max : p maximum ; p min : p minimum ; pd : p dispersion ; ns : non - significant. comparison of p wave measurements of the groups according to vessel score p1 : comparision of variables between group 1 and 2 ; p2 : comparision of variables between group 2 and 3 ; p3 : comparision of variables between group 2 and 4 ; p max : p maximum ; p min : p minimum ; pd : p dispersion. the relationship between p wave dispersion, and clinical and echocardiographic characteristics in patients with coronary artery disease lv : left ventricle ; edd : end - diastolic dimension ; esd : end - systolic dimension ; ef : ejection fraction ; lad : left atrial diameter ; mr : mitral regurgitation the relationship between p wave measurements and gensini and vessel scores in patients with coronary artery disease p max : p maximum ; p min : p minimum ; pd : p wave dispersion our study showed, increased p wave duration and pd was related to the extent and severity of cad in stable coronary artery disease patients. similarly, increased pd has been observed to be associated with coronary artery disease severity. af is the most common cardiac rhythm abnormality and its incidence was 0.6% in the coronary artery surgery study (cass) registry. interatrial conduction delays have been shown to be implicated in initiating and maintaining af [13 - 15 ]. another mechanism for increased pd may be the increase in collagen fiber deposition in the cardiac interstitium. it was reported that pd was associated with inhomogeneus and discontinuous propogation of sinus impulses. electrocardiographic markers of abnormal atrial conduction, such as pd, p maximum, and p minumum, may be influenced by myocardial ischemia. previous studies have demonstrated that atrial ischemia is implicated in the pathogenesis of af [21, 22 ]. dilaveris. reported that myocardial ischemia prolongs pd in 95 patients with documented cad and zmen. in addition, it has been shown that p - wave dispersion is increased in coronary slow- flow phenomenon. ischemia- induced inhomogeneous and discontinuous atrial conduction may be related to increased p maximum and pd. reduced blood flow due to coronary atherosclerosis may contribute to the development of tissue injury and fibrosis. another explanation for this is that ischemia causes renin angiotensin system activation [28, 29 ]. the regional fibrosis in the atrial wall, due to chronic ischemia could cause different atrial conductions leading to increased pd in surface ecgs. another pathophysiological explanation for increased p - wave duration and dispersion in cad may be autonomic tone associated with cad. ischemic left ventriculer dysfunction may increase left atrial pressure, and might another fundamental causes of increased p wave duration and pd in patients with cad compared to control subjects. atrial strain, which is a sugnificant factor in the pathophysiology of af together with ischemia- induced hetergeneous atrial conduction, may results an increase in p wave duration and pd. similarly, in our study there was no significant association between p min and gensini and vessel scores. the major limitation of our study is the small number of patients included in the study. for evaluation of ecg results we did not use the high - resolution computer software program. previous studies have found a low error of the measurement of pd on paper printed ecgs, contrarily other studies reported that manual pd measurement on paper printed ecgs obtained at a standard signal size may effect the accuracy and reproducibility of the results. in conclusion, our results suggest that there is a considerable association between increased pd and the severity of cad.	objective : p- wave dispersion (pd) is an indicator of inhomogeneous and discontinuous propagation of sinus impulses. in the present study we aimed to investigate the pd and its association with the severity of the disease. in patients with stable coronary artery disease.methods:we prospectively analyzed 60 subjects with coronary artery disease (cad) and 25 subjects with nor - mal coronary angiograms (control group). the maximum and minimum p - wave duration and pd were measured from the 12-lead surface electrocardiograms. the cad severity was assessed by the severity score (gensini score) and the number of vessels involved (vessel score).results : p max was longer in cad group compared with the control group (p<0.001). pd was greater in the cad group, compared with the control group (p<0.001). however, p min did not differ between the two groups. in bi - variate correlation, increased pd was correlated with presence of diabetes mellitus (r=0.316, p=0.014), smoking (r=0.348, p=0.006), left ventricular ejection fraction (r=-0.372, p=0.003), vessel score (r=0.848, p=0.001), and gensini score (r=0.825, p=0.001). multiple linear regression analysis showed that pd was independently associated with vessel score ((3=0.139, p=0.002) and gensini score ((3=0.132, p=0.007).conclusion : pd was greater in patients with cad than in controls and it was associated with cad severity.
otomycosis or fungal otitis externa has typically been described as fungal infection of the external auditory canal with infrequent complications involving the middle ear. although rarely life threatening, the disease is a challenging and frustrating entity for both patients and otolaryngologists as it frequently requires long - term treatment and follow - up. despite this otomycosis is one of the common conditions encountered in a general otolaryngology clinic setting and its prevalence has been quoted to range from 9% to 27.2% [2, 3 ] among patients who present with signs and symptoms of otitis externa and up to 30% [46 ] in patients with discharging ears. it is worldwide in distribution with a higher prevalence in the hot, humid, and dusty areas of the tropics and subtropics [2, 3, 5, 6 ]. overview of the literature reveals otomycosis to be a common medical problem in india [7, 8 ]. most patients suffering from early otomycosis complain of severe itching which often progress to pain, hearing loss, and often leading to tympanic membrane perforations [810 ]. although aspergillus niger and candida albicans are by far the most common offenders [2, 58, 10 ], a wide spectrum of other fungi can cause otomycosis. various factors have been proposed as predisposing factors for otomycosis, including a humid climate, presence of cerumen, instrumentation of the ear, immunocompromised host, and, more recently, increased use of topical antibiotic / steroid preparations. in this study conducted in a rainy and humid coastal city in south india, we identify the organisms isolated in otomycosis, including fungi and bacteria and compare them with normal ears. a total of 150 immunocompetent individuals attending the outpatient department of otolaryngology head and neck surgery at kasturba medical college in mangalore, a coastal city in the west coast of india, were chosen for the study. this was performed over a two - year period between january 2008 and january 2010. inclusion criteria. 100 consecutive patients with clinically diagnosed otomycosis in the absence of other ear conditions like chronic otitis media further 50 consecutive outpatients without clinical otomycosis or any aural symptoms, seeking treatment for minor nonotological ailments with no bearing on otomycosis, were included in the control group. the criteria used for establishing a diagnosis of otomycosis were based on any two or more of the following findings : presence of symptoms like itching, pain, feeling of blocked ear, tinnitus, deafness, and otoscopy revealing masses of hyphae / spores or a curd - like grey / white discharge, the demonstration of fungal elements in 10% potassium hydroxide - methylene blue preparation, culture of clinical material (debris, discharge, and moulds) on sabouraud 's dextrose agar slants. presence of symptoms like itching, pain, feeling of blocked ear, tinnitus, deafness, and otoscopy revealing masses of hyphae / spores or a curd - like grey / white discharge, the demonstration of fungal elements in 10% potassium hydroxide - methylene blue preparation, culture of clinical material (debris, discharge, and moulds) on sabouraud 's dextrose agar slants. the following conditions were classified as secondary otomycosis and were excluded from the study:(1) all patients with otomycosis alongside history of chronic otitis media, active otitis, tympanic membrane perforations, prior ear surgery or aural procedures, and diabetics,(2) patients with any serious debilitating diseases like malignancies, tuberculosis, and other chronic granulomatous diseases,(3) patients with immunocompromised conditions and history suggestive of fungal diseases elsewhere in the body. all patients with otomycosis alongside history of chronic otitis media, active otitis, tympanic membrane perforations, prior ear surgery or aural procedures, and diabetics, patients with any serious debilitating diseases like malignancies, tuberculosis, and other chronic granulomatous diseases, patients with immunocompromised conditions and history suggestive of fungal diseases elsewhere in the body. clinical presentations of cases such as itching, pain, feeling of ear blockage, and ear discharge were recorded. any history of trauma, use of wooden sticks, metal wax pickers or any other objects in an attempt to remove ears wax from ear, use of oils, topical antibiotic ear drops, and/or other aural preparations were noted. age, sex, socioeconomic status, and occupation of the patient were also recorded. a history indicative of fungal infections elsewhere in the body like onychomycosis and recurrent vaginal infections (in females) patients ' nails were checked for any evidence of onychomycosis. in patients with otomycosis, scrapped material was collected from the external auditory canal with the help of a sterile jobson horne probe with ring curette and cotton carrier. in all other cases, specimens were collected using three sterile cotton tipped swabs and transported to the laboratory within half an hour for mycological and bacteriological examination. routine blood and urine analysis, elisa for hiv, and blood sugar tests for diabetes (diabetes was determined by fasting blood sugar result above 100 milligrams of glucose per deciliter of blood) were done in all cases to rule out immunocompromised states among the study and control populations. a portion of the scrapped material or one of the swabs was cultured on blood and macconkey 's agar at 37c for 24 h and 48 h and examined for bacterial growth. second swab / scrapped material was digested on a microscopic slide with 10% potassium hydroxide - methylene blue (2 : 1). a 22 mm square number 1 cover slip was placed on the digested material and examined microscopically using 10 x and 40 x objectives for fungal elements. the third swab / scrapped material was inoculated on two sabouraud 's dextrose agar with chloramphenicol. one of the agar slants was incubated at room temperature (25c) and the other was incubated at 37c for 2 to 3 weeks. all patients diagnosed with otomycosis were subjected to a thorough aural toilet by suctioning and removal of the fungal debris. following this patients were prescribed clotrimazole antifungal preparations, 4 - 5 drops to be instilled 3 times a day for a minimum of 7 days and a maximum of 14 days. in case of associated coexisting otitis externa, ear swabs collected from the control group were also processed by similar methods as for otomycosis patients. data was analysed with a statistical software program (spss statistics for windows version 20, chicago, il). the association between the predisposing factors (self - cleaning, eardrops, and oil instillation) and the prevalence of otomycosis was analyzed with the chi - square test. 63 (63%) of the patients with otomycosis (study group) were males and 37 (37%) were females (table 1). in the control group 30 (60%) were males and 20 (40%) were females. the highest incidence of otomycosis was in the age group of 2130 years and the lowest was noted in the age group of less than 10 years and over 60 years of age (figure 1). the highest incidence of isolation of fungi in the control group was in the age groups of 21 to 40 years. 60 (40%) of the males and 45 (30%) of the females were engaged in agriculture related jobs in a day - to - day basis. 94 (63%) of the patients had an average family income of inr (indian national rupees) 1050 to 5000 per month and 56 (37%) had an average family income of inr 20,000 per month and above. the right ear was most commonly involved (23/37 cases, 62%) whereas in males the distribution was almost equal in both ears (right ear 49% and left ear 46%). the incidence of otomycosis was high in patients with history of instilling coconut oil (42%) into the ear. in the otomycosis group, history of habitual cleaning of the ear by the patient with unsterile sticks, metal wax picks, or rolled bus tickets was recorded in 32% of the cases ; the use of topical antibiotics eardrops in 20% and the habit of instilling oil in the ear was recorded in 42% of patients. in the control group, history of habitual cleaning of the ear, use of ear drops, or instillation of oil was also observed in 20%, 9%, and 29% of the subjects, respectively (figure 2). the prevalence of history of self - cleaning, use of eardrops, or oil instillation (32%, 20%, and 42%, resp.) was significantly higher in the otomycosis group when compared to the control group (20%, 9%, and 29%). these three factors were statistically associated to a higher incidence of otomycosis (p = 0.0265, p = 0.0136, and p = 0.0274, resp.). itching was the predominant symptom seen in 73% of the otomycotic patients followed by a blocked sensation in the ears (38%). other symptoms were ear discharge (38%), ear pain (35%), and tinnitus (8%). fungi were isolated from all 100 patients constituting the study group who were diagnosed clinically to have otomycosis (table 2). a. niger complex was the commonest (38%) followed by a. fumigatus complex (27%) and a. flavus complex (15%). three had coexisting a. niger complex and a. fumigatus complex and three had coexisting a. niger complex and a. flavus complex. a. niger complex was the commonest (22%) followed by a. fumigatus complex(4%) and a. flavus complex (4%). 63% of patients with otomycosis had associated bacterial infections (table 2). in the study group, the commonest bacterial association observed was coagulase positive staphylococci (31%) followed by pseudomonas aeruginosa (20%), proteus spp. s. aureus 1 (9%) and s. epidermidis (37%) were the most common organisms isolated. 77% were treated with combined antifungal and antibiotic preparations and were cured completely of the disease. andrall and gaverret were the first to describe fungal infections of the ear [7, 13 ].. the infection may be either subacute or acute and is characterized by inflammation, pruritus, scaling, and severe discomfort. the mycosis results in inflammation, superficial epithelial exfoliation, masses of debris containing hyphae, suppuration, and pain. the incidence of otomycosis is reported to be high in tropical countries [2, 3, 5, 6, 14 ]. mangalore, the city where the present study has been done, is a coastal city in south india where the ambient temperature varies from a minimum 17c to a maximum 38c. there is a heavy rainfall of about 4000 mm per annum of which about 90% was received in the monsoon months between june and september. the incidence of otomycosis was high (78%) in the rainy months of july, august, and september compared to the months between january and march (22%). while some studies have reported otomycosis to be more common among young men [1, 5, 7, 8, 15 ] as in our series, many others have reported it to be so among young females [2, 3, 14, 16, 17 ]. in our series the male : female ratio was 1.7 : 1. in our series, patients with otomycosis mainly came from an agricultural background. in a study by wadhwani and srivastava, 24 fungi were isolated from earwax or otitis media of agricultural field workers, of which 18 were being reported for the first time from india. previous reports have stated an increased incidence in people of lower socioeconomic status [7, 14, 16 ] and this was also seen in our series. the skin covering the external auditory canal is similar to the other parts of the body, but it is exposed to the atmosphere by a small meatal inlet. in other words, the anatomical disposition of external auditory canal simulates a culture tube lined with skin that provides ideal condition for fungal and bacterial growth. the presence of excessive cerumen in patients with poor personal hygiene favors the germination of spores and conidia of the prevalent fungi. obsessive manipulation of the external ear with any hard objects such as wooden sticks or metal wax picks to clean the ear of wax and vigorous rubbing of the ears for relief from itching (in case of otalgia due to eustachian catarrh, serous otitis, or acute otitis media) is a common practice. these practices may cause trauma (usually minor and hence unnoticed) to the skin of external auditory canal and deposition of fungal conidia in the wound leading to fungal infection. the moisture, warmth, and acidic ph of the external auditory canal provide ideal growth requirements for the fungi. aspergilli have an optimum ph range of 5.7 and a maximum growth rate at a temperature of 37c and this is conducive for all species of aspergillus isolated in the present study. this is supported by the predilection of fungi to grow in the inner one - third of external auditory canal. swimming is also indicated as a predisposing factor for otomycosis [2, 5, 16 ]. onychomycosis and other forms of dermatomycoses are a potential source of repeated autoinoculation [18, 19 ]. if the nails are thickened, white, and crumbling, they should be trimmed and sent for culture and systemic therapy should be considered. the flare - ups in these cases are hormonally related and also require systemic therapy. lack of formal education in people in many parts of india has led them to believe myths that coconut oil application for ears is beneficial for a variety of ear ailments. our study revealed high association (42%) of otomycosis with instillation of coconut oil into the external ear. coconut oil has been reported to be sporostatic and therefore may help preserve the viability of fungal conidia deposited in the external ear long and indirectly contribute to occurrence of otomycosis. similarly the use of mustard oil is associated with high incidence of otomycosis. in a series by oliveri and coworkers lack of cerumen, chronic otitis, previous antibiotic therapy, and eczema were common predisposing factors. they also mention that working in gardens or using mechanical removing devices was significant predisposing factors. cerumen, whose role is protective in the external auditory canal, was absent in majority (93%) of the patients in our series. prior treatment of a bacterial infection with long - term topical antibiotic therapy is indicated as a predisposing factor presumably due to the protective cerumen layer [6, 23, 24 ]. 20% of the patients in our series used over - the - counter antibiotic eardrops for itching and pain in the ear without medical consultation. the lumen is filled with large shed sheets of keratin that have a wet tissue - paper appearance. otomycosis can also lead to tympanic membrane perforations [5, 9, 17, 25 ] and spread to the middle ear. for documentation purposes and standardization, it is essential for the treating otolaryngologist to define otomycosis as primary or secondary. otomycosis present in the external auditory canal in an immunocompetent individual and in the presence of an intact tympanic membrane and absence of any other external or middle ear pathology.without otitis externa. clear absence of clinical signs of otitis externa at the time of presentation like inflamed canal wall or stenosis of the auditory canal.with otitis externa. presence of clinical signs of otitis externa, when no clinical manifestation of otitis externa was present prior to the time of presentation. otomycosis present in the eac or middle ear alongside history of and/or existing otitis media or externa, trauma, or postoperative ears or with history of and/or existing fungal infections in other parts of the body.without immunocompromise. no hiv, diabetes mellitus, or granulomatous diseases or other immunocompromised conditions detected by lab studies.with immunocompromise. otomycosis present in the external auditory canal in an immunocompetent individual and in the presence of an intact tympanic membrane and absence of any other external or middle ear pathology. without otitis externa. clear absence of clinical signs of otitis externa at the time of presentation like inflamed canal wall or stenosis of the auditory canal.with otitis externa. presence of clinical signs of otitis externa, when no clinical manifestation of otitis externa was present prior to the time of presentation. that is, otitis externa was due to otomycosis and not vice versa. without otitis externa. clear absence of clinical signs of otitis externa at the time of presentation like inflamed canal wall or stenosis of the auditory canal. with otitis externa. presence of clinical signs of otitis externa, when no clinical manifestation of otitis externa was present prior to the time of presentation. otomycosis present in the eac or middle ear alongside history of and/or existing otitis media or externa, trauma, or postoperative ears or with history of and/or existing fungal infections in other parts of the body. without immunocompromise. no hiv, diabetes mellitus, or granulomatous diseases or other immunocompromised conditions detected by lab studies.with immunocompromise. no hiv, diabetes mellitus, or granulomatous diseases or other immunocompromised conditions detected by lab studies. with immunocompromise. presence of any immunocompromised condition detected by lab studies. simple culture and microscopic examinations under sterile conditions are sufficient to confirm otomycosis. aspergillus is the most commonly reported isolate in otomycosis across the world, followed by candida [2, 57, 10, 1315, 22, 26 ]. this is not different from reports from the indian subcontinent, including our study [5, 7, 8, 16, 2730 ]. in a few series the incidence of candida has been reported to be higher than aspergillus [2, 4, 31 ]. however, in our study, penicillium (8%) was the second most common isolate after aspergillus with candida albicans constituting only 4%. was also found to be higher compared to earlier reports from india and other parts of the world [7, 8, 16, 3234 ]. the isolation of chrysosporium spp. in our series is the first of its kind and its pathogenic role in otomycosis yet to be elucidated. have occasionally been isolated from systemic infections in bone marrow transplant recipients and in patients with chronic granulomatous diseases. apart from otomycosis, aspergilli cause a wide spectrum of infections including cutaneous manifestations and invasive infections such as pulmonary aspergillosis and endocarditis. clinical microbiology laboratories rely heavily on morphology - based identification methods for aspergillus species wherein diagnostic criteria include the recognition of asexual or sexual structures and their characteristics such as shape, size, color, ornamentation, and/or mode of attachment. unfortunately, such a phenotype - based scheme is not effective in identifying the species because largely these characteristics are unstable, and clinical aspergilli sometimes manifest atypically with slow sporulation and aberrant conidiophore formation. additionally, members of the section a. fumigatus have overlapping morphological characteristics, with several genetically distinct species existing within a single morphospecies. clinically, it is important to identify aspergillus species because different species have variable susceptibilities to multiple antifungal drugs thereby influencing the choice of appropriate antifungal therapy. for example, studies have demonstrated that a. terreus isolates are largely resistant to the antifungal drug amphotericin b. comparative dna sequence - based identification formats appear to be promising in terms of speed, ease, objectivity, and economy for species identification. not using molecular methods to ascertain the species could be a drawback in our study. however, in spite of the shift of fungal identification formats into the molecular arena there is no consensus on the gene / genes that can be used for species identification in the genus aspergillus. also, this methodology involves significant cost and phylogenetic expertise that are limiting factors in most clinical microbiology laboratories. additionally, consideration should also be given to the fact that most of these isolates may not be true causative agents of disease and therefore may not warrant species level identification in a diagnostic laboratory. taken together, a universal single marker that would rapidly and accurately identify aspergillus isolates to the species level would help support diagnostic microbiology laboratories in their routine identification efforts. aspergillus niger complex is one of the most common species which is also a oligotroph ; that is, they are capable of growing even in nutrient - depleted environments. it has been in use already for many decades to produce extracellular (food) enzymes and citric acid. in addition, a. niger complex is used for biotransformations and waste treatment. in the last two decades, a. niger a. niger complex, like other filamentous fungi, should be treated carefully to avoid the formation of spore dust. the human external auditory canal is an ideal environment for this fungus to grow and abundance of proteins and carbohydrates and favorable humidity and temperature explain this finding [14, 33 ]. on the other hand, candida possess constitutive hydrolytic enzymes to aid invasion of host tissues and in this investigation the majority of tested candida have been demonstrated to have protease activity. these findings suggest that protease production may play an important role in the pathogenesis of otomycosis caused by candida. it is possible that protease enzymes enhance the ability of candida to colonize the skin and penetrate host cells, which could be important in establishing the infection in the ear. also, despite the fact that many authors did not clearly differentiate between primary otomycosis and otomycosis secondary to otitis media, candida was found more commonly when compared to aspergillus in postoperated cavities or infected middle ears [4, 9, 38 ] and in immunocompromised individuals [8, 39 ]. aspergillus is considered a primary colonizer of the ear canal [17, 27 ]. like skin elsewhere in the body, that of the external auditory meatus has a normal commensal flora such as staphylococcus epidermidis (albus) and corynebacterium spp. in addition when the skin 's natural defense mechanisms break down, as in otitis externa, the resident bacteria multiply because of the more favorable environment and other organisms such as proteus and pseudomonas spp., which are normal commensals of other parts of the body, may then flourish. in our series, staphylococcus aureus, p. aeruginosa, and proteus spp. were dominant bacterial pathogens which is in line with previous reports [1, 32, 33 ]. a. fumigatus was the only one of the aspergilliwhich was not accompanied routinely by s. aureus which is also in line with previous reports. this has been attributed to an antibiotic activity against s. aureus formed by a. fumigatus. in the control group, fungi were seen in 42% of the normal ears. the order of frequency of isolation of fungi from healthy external ear was almost similar to that observed in cases of otomycosis and the isolation rate of aspergilli from healthy external ears was significant (30%). a. niger complex, a. fumigatus complex, a. flavus complex, c. albicans, penicillium sp., and rhizopus were isolated. this leads us to the question : do most normal ear canals harbor fungal commensals ? the answer to this could be in the negative as a majority of the normal ears (58%) did not show any fungus. however, it can be reasonably opined that the presence of fungus in normal ears could be due to a decreased host barrier in the canal skin due to either minor trauma (that may go unnoticed) during ear cleaning, itching (it is usual for patients to itch the ear in case of otalgia due to eustachian catarrh, serous otitis, or acute otitis media), or lacerations due to accidents. factors like poor sanitation, poor nutrition, immunocompromised states (human immunodeficiency virus infection and diabetes mellitus), aberrant anatomy of the canal, or physical factors like increased regional humidity or temperatures may also tilt the balance towards growth of fungus. such patients with fungus in the ears without symptoms or signs can be considered to be predisposed to the condition and may develop frank otomycosis in a course of time if the favoring conditions persist. clotrimazole has an antibacterial effect, and this is an added advantage when treating mixed bacterial - fungal infections. fungicidal creams with ketoconazole or fluconazole may also be applied [6, 41 ]. a readily available and usually effective preparation for candida is tolnaftate, available over the counter for the treatment of athlete 's foot. the major advantage of nystatin is the fact that it is not absorbed across intact skin. nystatin is not available as an otic preparation ; however it can be prepared as a solution or a suspension for the treatment of otomycosis. studies on animals with experimentally induced fungal infections have furnished evidence for the risk of the infections ' spreading to the inner ear and causing serious damage to the organ of corti ; indirect damage to these structures by mycotoxins can not be ruled out. in rare refractory cases of otomycosis due to hiv or other immunocompromised states, or in life threatening condition, parenteral antifungals like amphotericin b or tolnaftate may be used [6, 39 ]. otomycosis is seen across the world with a high incidence especially in tropical countries. in this study we analyzed the growth of fungi and bacteria in 100 otomycotic ears and compared it with 50 healthy ears. in a study conducted in a coastal indian city, aspergillus spp. fungi are also present in a significant number of healthy external auditory canals and their profiles match those in cases of otomycosis. the use of terms primary and secondary otomycosis is important to standardize reporting.	objective. to define otomycosis and determine the predisposing factors and microbiology in primary otomycosis. study design. prospective study of two years and review of the literature. setting. academic department of otolaryngology in a coastal city in india. patients. 150 immunocompetent individuals of whom 100 consecutive patients with a clinical diagnosis of otomycosis are considered as the study group and 50 consecutive patients with no otomycosis are considered as the control group. results and observations. instillation of coconut oil (42%), use of topical antibiotic eardrops (20%), and compulsive cleaning of external ear with hard objects (32%) appeared to be the main predisposing factors in otomycosis. aspergilli were the most common isolates (80%) followed by penicillium (8%), candida albicans (4%), rhizopus (1%), and chrysosporium (1%), the last being reported for the first time in otomycosis. among aspergilli, a. niger complex (38%) was the most common followed by a. fumigatus complex (27%) and a. flavus complex (15%). bacterial isolates associated with fungi in otomycosis were s. aureus, p. aeruginosa, and proteus spp. in 42% of healthy external ears fungi were isolated. conclusion. aspergillus spp. were the most common fungi isolated, followed by penicillium. otomycotic ears are often associated with bacterial isolates when compared to normal ears. fungi are also present in a significant number of healthy external auditory canals and their profiles match those in cases of otomycosis. the use of terms primary and secondary otomycosis is important to standardize reporting.
urban taxi drivers differ from other professional drivers with respect to their risk profiles for work - related low back disorders1. biomechanical studies show that the driving activities within automobiles can impose postural strains on lumbar spines1. reported that back pain is an important health problem for taxi drivers, as well as an urgent occupational safety and health management issue2. back pain is significantly related to the suitability of the driver s seat pan, job stress, and time2. the current study developed unilateral exercises for urban drivers and investigated the effect of these exercises on low back pain (lbp). a 40-year - old male, who complained of lbp on the left side at l35 levels, participated in the study. the objective and methods of the study were explained to the subject prior to his participation. informed consent was also obtained according to the ethical principles of the declaration of helsinki. he had been driving a taxi for 4 h / day in an urban area. an examination revealed that his pelvis was tilted posteriorly. when he performed a forward flexion in the standing position with his knees fully extended, he experienced pain and stiffness in his left lower back. the visual analog scale (vas) score of his back pain was 7. an examiner measured the pelvic inclination with a palpation meter (palm ; performance attainment associates, st. the rotation of the innominate bones in the sagittal plane was also measured with the caliper tips of the palm in contact with the ipsilateral anterior superior iliac spine and posterior superior iliac spine3. in the initial assessment, the anterior pelvic tilt angles were 2 and 8 on the right and left sides (normal range, 11 4), respectively. a dual inclinometer (acumar, lafayette instrument co., lafayette, usa) was used to measure the trunk angles. the initial lumbar flexion angle was 60, and the initial extension angle was 45. an examination revealed that his lumbar was rotated to the left side. this study conducted two 10-day sessions and measured the lbp, pelvic tilt angle, and trunk angle after each session. session 1 involved lumbar extension and flexion exercises, as well as lumbar stability exercises using a ball. session 2 comprised unilateral resistance exercises, which include pushing and holding for 10 s with the left foot on a 45-degree inclined springboard while sitting on a chair with the knee flexed. the unilateral stretching exercise for the quadratus lumborum (ql) muscles was performed by crossing the right leg over the left leg and slowly lowering both legs to the right ; this position was held for 30 s with the arms extended out to the sides of the body. after the first session, the anterior pelvic tilt angles were 4 and 8 on the right and left sides, respectively. the lumbar flexion angle was 68, and the extension angle was 45. lumbar rotation movement was observed during lumbar flexion. after the second session, the anterior pelvic tilt angles were 9 and 10 on the right and left sides, respectively. the lumbar flexion angle was 68, and extension angle was 50. lumbar rotation movement was not observed during flexion. lumbar extension and flexion and lumbar stability exercises that are generally recommended for people suffering from lumbago are also helpful for normal pelvic alignment, as well as in reducing lbp among urban drivers. however, the effects of these exercises are limited. with the driver s seat on the left side in korean cars, the accelerator is located near the right foot, and the brake pedal is located between the right and left feet. urban drivers spend a considerable amount of time on operating the brake pedal while driving around the busy roads of cities1, 2. extending and pushing on the right leg to operate the brake pedal between the right and left feet increase the length of the right leg ; this operation may have caused the right lateral tilt in the right side of the pelvis and one - sided backward tilt of the right pelvis4, 5. therefore, resistance exercises with the left foot pushing a decline board, which was performed by the subject in the current study, were considered to reduce greatly the differences in the angles of the right and left sides of the pelvis as well as the pain therein. moreover, while the right foot moves repeatedly back and forth between the accelerator and the brake pedal, the muscles around the left side of the pelvis stiffen4. in particular, the ql muscles of the waist move in a compensatory manner to maintain the pelvic posture in the sitting position5. the unilateral stretching exercise for the ql muscles performed in the current study is considered to reduce lumbago by improving the right lateral tilt in the right side of the pelvis and reducing the tension of the ql muscles. in conclusion, analyzing car features and performing individual approaches are necessary in providing treatment for urban drivers with lbp.	[purpose ] this study aimed to develop unilateral exercises for urban drivers and investigate the effect of these exercises on low back pain (lbp). [subject and methods ] a 40-year - old male driver, who complained of lbp on the left side at l35 levels, participated in this study. a two - session program was conducted, and lbp, pelvic tilt angle, and trunk range of motion were measured after each session. [results ] after the unilateral exercises, the anterior pelvic tilt angle was improved and the visual analog scale score of back pain decreased. [conclusion ] analyzing car features and performing individual approaches are necessary in providing treatment for urban drivers with lbp.
lichen planus (lp) is a common inflammatory disease affecting the skin, the mucous membranes, the genitalia, the nails and the scalp. prevalence of lichen planus in the general population ranges from 0.1 to 4 % and it is more common in females, especially in the perimenopausal period. pathophysiology of lp involves an immune - mediated reaction, in which an antigen is processed to t - lymphocytes and they, subsequently, attack basal keratinocytes, leading to apoptosis of the cells. several factors have been suggested as possible antigens, including viruses, bacterials and drugs. the typical clinical manifestations of lp are purple to violaceous polygonal papules with sharp borders, usually pruritic, most commonly developing on the extremities and the trunk. less frequently the disease affects the genital area, mucous membranes, palms and soles and nails. mucosal lesions are typified by the presence of reticular white lines, known as wickham striae. the disorder has several clinical variations : annular, hypertrophic, atrophic, ulcerative, bullous, erythrodermic, inverse, linear, follicular, pemphigoides, pigmentosus, follicularis decalvans and actinic lp. the diagnosis of lp is usually established clinically based on the characteristic morphology of the lesions and the coexisting intense pruritus. however, atypical presentations requiring histopathologic confirmation of the diagnosis do exist. dermoscopy allows the visualization of structures located in the epidermis, dermo - epidermal junction and papillary dermis that can not be seen with the naked eye., cumulative evidence suggests that dermoscopy is also meaningful for the evaluation of inflammatory and infectious skin disorders. in the field of papulosquamous dermatoses, dermoscopy has been shown to enhance the differential diagnosis among psoriasis, dermatitis, lp and pityriasis rosea. particularly for lp, dermoscopy brought to light that white crossing lines do not characterize only mucosal lesions, but cover virtually every cutaneous papule of active lp. in this report we present a characteristic example of a patient with misleading clinical manifestations of lp resembling psoriasis. a 61-year - old woman visited our department for evaluation of a three - month, mildly pruritic eruption on the soles and the dorsal surfaces of the feet and hands. clinical examination revealed hyperkeratotic plaques on the dorsal surface of the feet and hands and erythematous hyperkeratotic, partially erosive plaques on the soles.. surprisingly, application of dermoscopy did not reveal the expected psoriatic pattern of regularly distributed dotted vessels and white scales (figure 2). instead, white crossing lines (the so - called wickham striae) were dermoscopically evident, along with dotted and short linear vessels and yellow scales. since the dermoscopic presence of wickham striae is considered highly specific of lp, the dermoscopic findings prompted us to perform a biopsy for histopathologic assessment. histopathology, as shown in figure 3, revealed hyperkeratosis, dense hypergranulosis, vacuolar degeneration of basal cell keratinocytes, band - like lymphocytic infiltration in the upper dermis, as well as presence of colloid bodies, justifying the diagnosis of lp. in the current case, clinical manifestations on the dorsal hands and plantar surfaces were highly suggestive of psoriasis, with eczema and lp included in the differential diagnosis. this was because the lesion deviated from the standard dermoscopic pattern of psoriatic lesions, which are composed of regularly distributed dotted vessels and white - colored scales. although the presence of irregularly arranged dotted vessels and yellow scales were compatible with eczema, the prevailing dermoscopic features were the white crossing lines, corresponding to the so - called wickham striae, which is known as a highly specific criterion of lp. clinical examination is undoubtedly the cornerstone of diagnosis in everyday dermatology practice, and in the majority of our patients, the macroscopic morphology is already enough to establish an accurate diagnosis. this is especially true for widespread inflammatory diseases, where the combination of clinical history, morphology and distribution often points towards a specific diagnosis. however, equivocal clinical manifestations do exist in everyday practice, posing diagnostic doubts and often prompting clinicians to perform diagnostic biopsies. it has been demonstrated that coupling clinical examination with dermoscopy significantly improves the diagnostic performance of clinicians. however, in order to maximize the benefit from dermoscopy in differentiation of inflammatory dermatoses, clinicians have to virtually use their dermatoscope on every lesion. in daily routine furthermore, as shown in the current case, it may change our diagnostic thoughts, saving us from misdiagnosis and potential inappropriate management.	lichen planus (lp) is an inflammatory disease that affects the skin mainly the extremities and the trunk the mucous membranes, the genitalia, the nails and the scalp. the diagnosis of lp is usually established clinically based on the typical morphology and distribution of the lesions in conjunction with the associated itch. we report a patient with lp manifesting highly psoriasiform lesions, that could only be correctly assessed after the application of dermoscopy, which revealed lp - specific findings.
a 67 year old woman was hospitalized in the emergency room due to dysarthria started 4 hours before hospitalization. the patient was diagnosed with systemic sclerosis in 2003 and was taking methylprednisolone 2 mg per day at our clinic 's rheumatic internal medicine department. according to physical examination findings, sensorium was normal where as dysarthria, facial palsy, and right deviant of tongue were observed. blood pressure was 140/80 mmhg, pulse was 102 beats per minute, respiratory rate was 20 times per minute, body temperature was 36.9, and there were no abnormalities in chest auscultation. according to blood test result, number of white blood cells was increased and platelet level was normal. cardiomegaly was observed through simple chest x - ray and acute infarction was observed around the large sulcus and parietal lobe through mri (fig. ct scan of carotid artery and chest to find out the cause of cerebral infarction, lesion suspicious of aortic arch thrombus was observed. in enhanced image was observed, no abnormal observations were found in regional wall motion and heart valves, and floating thrombus was found in ascending aorta (fig. 2). according to hematological test, anti - cardiolibin ab - igg level was 2.0 gpl u / ml (normal : less than 9.9), anti - cardiolibin ab - igm level was 1.0 mpl u / ml (normal : less than 6.9), lupis anticoagulant (-), protein s antigen was 114% (60~150), protein s activity was 93% (58.7~119.2), antiphospholipid ab igg was 1.0 u / ml (less than 9.9), and antiphospholipid ab igm was 2.0 u / ml (less than 9.9) showing normal values. however, protein c activity was 167% (70~130) and protein c antigen was > 160% (72~160) showing increased levels. due to the facts that size of the thrombus was large, location was from ascending aorta to aortic arch, thrombus was highly mobile and cerebral embolism occurred, median sternotomy was done, arterial cannula was inserted through 8 mm vascular graft anastomosed in right axillary artery, venous cannula was inserted in superior and inferior vena cava, started extracorporeal circulation, and decreased central temperature to 22. cardiac arrest was induced by inserting retrograde cardioplegic solution in coronary sinus, right innominate artery and left common carotid artery were cross clamped, and selective antegrade cerebral perfusion through right axillary artery was done eventually leading to circulatory arrest. incision was made in ascending aorta and 2.5 cm - long intraaortic thrombus was completely removed (fig. aortic incision was sutured, right innominate artery and left common carotid artery were unclamped resuming systemic circulation, and weaning from cardiopulmonary bypass was done without complications. selective antegrade cerebral perfusion was done for 15 minutes and cardiopulmonary bypass was done for 128 minutes. based on pathological test of the thrombus, there were no evidence of malignant neoplasm, and the patient recovered without complications and was discharged. factors related with arterial thrombus are arteriosclerosis, arteriodissection, trauma, malignant tumor, and hemostatic disorder. in this case study, the patient had systemic sclerosis from several years ago and was taking methylprednisolone 2 mg per day. there are no clearly known causes of systemic sclerosis, but natural death of cell caused by anti - endothelial cell antibody is suggested as a hypothesis. this usually affects microvessel causing proliferation of endothelial cell, medial thinning, and thickening of basilar membrane. eventual destruction of vessel causes angiodyskinesia, functional disorder of endothelial cell, thickening of vessel wall, activation of coagulation factor, reduction of fiber disassociation, and increase in attachment of platelets and lymphocytes. in cases with systemic sclerosis, thrombus in extremity is relatively common and floating thrombus inside the aorta is known to be rare. thrombus mostly occurs in elder patients, is usually stationary forming atherosclerotic plaque, and often occurs in extremity. patient of this case did not have past history that is known to cause thrombus such as artery detachment, trauma, or malignant tumor. evidences of infection malignant tumor, arteriosclerosis, and inflammatory change were not found after postoperative pathological test of specimen. the patient did not smoke, had no hypertension or diabetes, did not show high parameters in blood test, but only showed higher levels of protein c activity and protein c antigen than normal. however, long - term use of steroid and vessel damage due to systemic sclerosis might be related to thrombus formation. since occurrence in ascending aorta is rare, this case can be considered as meaningful. transesophageal echocardiography is non - invasive and easy method used widely for diagnosing thrombus in heart or aorta. there are several different methods in aortic thrombus treatment such as anticoagulant therapy, thrombolytic therapy, aspiration thrombectomy, and thrombectomy, but there are no evidence that which method is better. in medical treatment, thrombolytic agent and anticoagulant are used. after using anticoagulant with heparin for several days, size reduction of thrombus must be observed and heparin treatment must be maintained until the thrombus is completely gone. in general, when the size of thrombus is large and thrombus is mobile, surgical removal is preferred as primary treatment because of the risk factors such as systemic embolism, repeated embolism, and medical treatment failure. in this case, heparin was used for 2 days after cerebral infarction prior to surgery. thrombectomy was executed since it was highly mobile, large in size, did not change in its size through follow - up ultrasonic test, and repetitive cerebral embolism was possible. this report is written in order to inform a rare case where thrombus in ascending aorta of a patient with systemic sclerosis was surgically treated.	aortic thrombi are important because it can cause the central and peripheral embolizations. aortic thrombi can occur anywhere in the aorta but extremely rare in ascending aorta without atherosclerosis, aneurysm, cardiosurgical or traumatic state. systemic sclerosis (ssc) is an autoimmune disorder of connective tissue and it can involve multisystem. enhanced coagulation pathways, decreased fibrinolysis, and endothelial dysfunction probably contribute to vascular events in ssc. we report a case of a highly mobile thrombus in the ascending aorta, presented as an acute embolic stroke in the patient with systemic sclerosis. surgical removal was performed to prevent recurrent embolic events.
in their analysis on the clinical significance of randomised controlled trials, kraemer and kupfer (2006) advocated the number needed to treat (nnt) and some other parameters to convey the clinical and practical significance of the outcome of a trial. they indicate that such parameters may deviate largely, so nnts ranging from 2 to 100 might be relevant depending on the context. it means that one may treat 2 up to 100 subjects to establish the desired effect in a single person. high nnt may point either to weak diagnostic criteria for a particular therapy or to an ineffective intervention. in their paper on the usefulness of the medical model in psychiatry, shah and mountain (2007) define the medical model as a process whereby informed by the best available evidence, doctors advise on, coordinate or deliver interventions for health improvement. it can be summarily stated as does it work? this definition of a diagnosis refers to utility (usefulness in a particular context), rather than to validity (a true aspect of the real world ; kendell and jablensky 2003). the medical model assumes causality, and that is the basis of rational and evidence - based medicine. the concept of (strict) causality in medicine may originate in classical physics, where force and its consequences are understood as deterministic processes. in medical sciences, the idea of causality is most often expressed in terms of restoration of a steady - state condition. pathological processes lead to a deviation from the steady state, and the intervention is applied by the medical doctor to eliminate the pathological state. so the biological basis of recovery from a medical disorder is the capacity of an organism to restore and maintain its internal milieu or homeostasis. a reversible disorder (or disease) points to the capacity of an organism to eliminate or compensate the pathology, whereas a chronic course is indicative of a failing capacity. we consider psychiatry primarily as a medical discipline and a psychiatric disorder as an undesired condition that a medical doctor should help to eliminate. a diagnosis should then recognise a disorder and the therapy proposed should cure the patient, i.e. let disappear the disorder (or at least helps to have a decent life). psychiatric disorders are primarily concerned with the brain and their manifestation may therefore be more variable than that of somatic disorders, because of the higher complexity of the brain. such complexity may be greater than in neurological disorders. the clinical consequences, including the time course, of the latter disorders are primarily determined by underlying cellular processes. in contrast, organic pathology is absent or relatively insignificant so far in most psychiatric conditions, in particular, in the so - called functional disorders, including depression. we will consider the issue whether functional psychiatric disorders and, in particular, major depressive disorder can be conceptualised in the conventional framework of strict causality and steady - state dynamics. the term evidence - based medicine alludes to a firm scientific basis of the medical practice. often, the evidence is based on clinical trials comparing two or more interventions, including placebo treatment, showing statistically significant different outcomes. the issue is whether a thus defined efficacy allows predicting the outcome of a therapy in the individual patient. the psychiatrist has to propose (or perhaps to decide) which treatment is most adequate for the patient sitting in his office or lying in the hospital bed. this is not meant as a rhetoric question ; we acknowledge that many treatments are effective in the individual patient. in psychiatry (perhaps also in other disciplines of medicine, but that is not our concern), however, there are also treatments that are at least in large cohorts a little more effective than placebo. one may, for instance, refer to the recent discussion on antidepressant drugs (kirsch. 2008 ; turner. 2008 ; moncrieff and kirsch 2005). if an active treatment is hardly better than placebo, it is impossible to make a rational decision about the treatment of the individual patient : this is in fact challenging the assumption of causality. we chose major depressive disorder as a representative functional disorder, because of the availability of illustrative data. another obstacle is the nature of cerebral functioning : we argue that brain processes are not as deterministic as often thought. in line with this contention, we describe nondeterministic time courses of recovery from depression that differ fundamentally from that of reversible somatic disorders. finally, we emphasise that current disease markers or psychological inquiry does not necessarily provide the information required for rational therapeutic strategies. the scope of the present mini - review differs from that of recent related papers, where the philosophical framework and utility or validity of psychiatric diagnosis (shah and mountain, 2007 ; kendell and jablensky 2003 ; kendell 2001 ; kendler 2005 ; angst 2007 ; jablensky 2005) were discussed. parts of this paper were recently presented elsewhere (korf 2008 ; stoyanov. he construed a closed diagnostic system leading to a personally dedicated treatment. despite its apparent rationality, emil kraepelin is the founder of the present diagnostic approaches and his ideas are the basis of current classification proposals (shorter 1997). subsequently a variety of diagnostic systems, including the diagnostic and statistical manual of mental disorders (dsm) and the international classification of diseases (icd), were introduced. these systems were developed as a reaction to the psychoanalytic framework and were aimed not only to serve the clinician with a standardised terminology but also to provide a scientific basis of psychiatry (shorter 1997). it was well realised from the onset onwards that any classification needs a firm scientific basis. many investigators consider such number as exuberant, but there is as yet no scientific argumentation whether such number is too much, too little or just perfect. the arguments are rhetoric instead : do we need also 300 diagnoses of liver diseases ? a way to avoid such fruitless discussions would be to assume that the dsm classification does not define disease entities, but defines (psychological) disorders. but then the question arises : what should be considered as a disorder and what not, and do nonpsychological parameters ever become relevant ? the dsm (for instance) allows some diagnostic variations, as some symptoms are more important for the diagnosis of major depressive disorder (code 296xx) than others : duration of a depressed mood for at least 2 weeks is mandatory, but the presence of only four of the following symptoms is required : disturbed appetite ; weight ; sleep ; psychomotor activity ; energy ; feelings of worthlessness or guilt ; ability to think, concentrate or make decisions ; recurrent thought of death or suicidal ideation, plans or attempts. we now propose a thought an experiment challenging a classification system and so illustrating its scientific status. assume that current classification systems are no ex cathedra formulated diagnostic standards (which they are to a large extent) but rather as a (possibly pre - scientific) hypothesis that may be falsified in the popperian sense. then the question arises when is a parameter sufficiently strong to falsify the dsm ? consider a parameter investigated by the best available methodology found in 80% of the subjects of a cohort and in 20% of a perfectly matched control group [think of, e.g. the dexamethason suppression test (dst) or with a corticotrophin - releasing hormone infusion (dex - crh) ] of melancholic/ major depression (carroll. the carroll (and several later) studies have proposed that the dst might well indicate or reflect pathological mechanisms or processes underlying depression. such reasoning does albeit indirectly most often support the concept of depression as defined by the dsm. the issue here is : could the dst challenge the dsm concept of depression ? not likely. a formal argument is that the dsm does not mention cortisol excretion or dexamethason suppression as a feature of major depression. besides such formal argument, it is more likely that a parameter that is present in a limited number of patients defined with the dsm criteria will be considered as diagnostically useless. another example : consider a symptom or parameter that is responsible for only certain aspects of depression (say, irritability ; russo., an intervention may prove to be therapeutically effective in a cohort, but only in 60% of the subjects. this is, for instance, the case with antidepressants (kirsch. 2008 ; turner. 2008 ; moncrieff and kirsch 2005). apparently also, the therapeutic response is not an argument to redefining a diagnostic classification. another obstacle for a more fundamental discussion on the utility (or better validity, kendell and jablensky 2003) of the dsm is the publication bias. it is generally discouraged to publish psychiatric investigations without adherence to the dsm classification (or another classification system). these considerations together argue that studies on (objective) parameters (e.g. genes, hormones, course of the disorder or response to therapy) are unable to challenge the dsm classification system. is this really bad for psychiatry ? scientifically speaking, there is no argument : adherence to a nontestable classification system hinders progress. it may be even worse (e.g. kendell and jablensky 2003 and references therein) : the dsm classification may obstruct scientific progress, because it forces to compare heterogeneous cohorts (apples and pears) for common characteristics (genes, colour, etc.). but common sense also tells that not every single parameter is sufficiently strong to challenge a psychiatric classification system. and as mentioned, one can not attack the dsm with criteria that were never included. to compare with the medical practice, a first medical diagnosis is a provisional hypothesis about an illness, and further investigations are required to support or to reject the initial diagnosis. we thought that an experiment suggests that the rational medical practice does not apply to a psychiatric diagnostic systems. our reasoning implies that a scientific foundation of psychiatry by using the dsm classification is practically unreachable. to avoid the yes / no argumentation, we suggest a way of challenging current classification systems by searching for diagnostic prototypes (jablensky 2005 ; kendell and jablensky 2003). our suggestion is related to the notion of zones of rarity to indicate natural boundaries between diagnostic entities. this implies a deterministic relationship between an external or internal event and the emerging state of the organism. in that case a rational diagnosis and predictable outcome of a therapy is at least in theory this idea does not necessarily apply to psychiatry, as will be illustrated with recent work on depression. the major pathogenic hypotheses of depression assumes the involvement of life events (kendler. 1999 ; kendler 2008 ; keller. 2007), biogenic amines (in particular, serotonin ; russo. 2007) and stress hormones (cortisol ; holsboer 2000). objective response to a stressful life event should be distinguished from subjective response, because life events are stressful only when perceived as such. the subjective perception depends primarily on the memory of previous experiences, whereas the objective response may be modified by genetic and other biochemical dispositions. it is generally believed that the combination of objective and subjective features of stress coping contribute to the development of depression (e.g. kendler 2008). various aspects of monoamines have been investigated including failing synthesis of brain serotonin or genetically programmed variations of proteins (transporters or catabolic enzymes). close inspection shows that 10% or less of the variance can be explained by the environmental and biochemical factors (russo. for instance, spengler and associates (murgatroyd. 2009) highlighted the crucial role of early life stress events for the emergence of epigenetic marking (hypomethylation) of key regulatory region for the expression of arginine vasopressin in the para ventricular hypothalamic nucleus. this reflects on the function of hypothalamo - pituitary - adrenal axis in the proper adaptation and represents potential model for the vulnerability of the nervous system to environmental and behavioural stimuli. however, such data might point also to the contribution of several more but as yet unknown factors. the question arises whether a more complete knowledge of these factors is possible and, if so, whether their number is sufficient small to construct a useful diagnosis. by far, most of the depressive episodes are reversible : meaning that in the general population with or without professional therapeutic interventions, about 80% recovers (spijker. 2002) we measured and modeled the incidence of recovery of a depressive cohort extracted from the general population with or without comorbid psychiatric or somatic pathology (van der werf. 2006 ; kaptein. it appeared that the rate of recovery follows an exponential time course, indicating that the probability to recover from depression is independent of the length of the depressive episode. in other words, the chance to recover in the first month after the onset of depression is the same than to recover in, for instance, the third month. the exponential function could explain more than 98% of the variance and was applicable irrespective of comorbidities. the model showed that the 2-week criterion of the dsm is rather arbitrary and not a characteristic (i.e. an incubation period) for major depression (van der werf. 1992) suggest that the time - to - recovery curves can be modelled with exponential functions as well. a prospective study on recurrent depression showed that the durations of preceding or subsequent depressive episodes and depression - free intervals were all unrelated (kaptein 2008). the exponential function of recovery from depression can best be explained and modelled by assuming random - mood swings : this means that the brain might transit randomly from one (examples of fast brain - state transitions are sleep stages, fear and happiness. within seconds, we fall asleep or transit from the slow - wave sleep into the paradoxical (rapid eye movement) sleep and vice versa. transitions to or from a depressive state are also fast : a positive response after one night of sleep deprivation may disappear during (or after) a nap of a few minutes (riemann. 1993). the occurrence of fast and random mood transitions questions how deterministic and thus predictable brain transitions are. korf and gramsbergen (2007) argued that the brain operates as an iso - energetic system, meaning that there are minimal energy barriers to initiate or execute neural activities (fig. 1). so neural activities in milliseconds do not depend on energy recruitment but are a manifestation of already present energy resources, designated as potential energy. potential energy is equivalent with trans - membrane ion gradients (i.e. k, nacl) and neurotransmitter pools (i.e. gaba, glutamate) and is (nearly) same all over the brain. the potential energy becomes available with minimal effort : as with a battery by changing a switch. brain - state transitions may appear randomly not only to the external observer but also to the subject himself. 1schematic representation of the concept of iso - energetic brain in relation to complexity of neurobiological processes. to reach iso - energicity, energy metabolism (via glucose and oxygen) once the brain near iso - energetic nondeterministic processes, such as random, stochastic and chaotic processes, become possible, more complex functions are confined to the iso - energetic brain and are thus less deterministic schematic representation of the concept of iso - energetic brain in relation to complexity of neurobiological processes. to reach iso - energicity, energy metabolism (via glucose and oxygen) once the brain near iso - energetic nondeterministic processes, such as random, stochastic and chaotic processes, become possible, more complex functions are confined to the iso - energetic brain and are thus less deterministic conventionally biological systems are considered deterministic, i.e. that a brain state can be predicted from a previous state (goldberger 1996 ; williams 1997 ; gottschalk. the iso - energetic concept allows indeterminacy, so nonpredictable (i.e. random) or near - random (i.e. stochastic) fluctuations are possible. the iso - energetic and some other concepts corroborate the idea that the brain is amenable to chaos theory. in a chaotic (in the mathematical sense) system, this idea has previously been emphasised in studies on bipolar depression and related affective states (gottschalk. 1995 ; mandell and selz 1995 ; pezard. the brain may exert transitions on external or internal signals that are not well predictable in time and in intensity. the concept of an iso - energetic brain implies a rather loose and often untraceable connection between input output and therefore challenges strict causality underlying classical medical models. an iso - energetic brain makes emotional and behavioural reactions to minor external triggers or life events plausible. the type 1 intervention aims to remove the disease - causing agent either by direct challenging its existence in the patient s body or to assist the disease - eliminating activity of the organism. compensation is the type 2 intervention : a component with a similar or identical activity is prescribed or measures are taken to enhance the production of a missing compound. many anti - diabetic treatments or the l - dopa therapy and dopamine agonists in parkinson s disease are examples. the type 3 intervention aims to remove a pathological agent with nonspecific toxins, mechanical devices or irradiation. finally, type 4 treatment is primarily aimed to reduce suffering by, for instance, palliative treatment. each of these treatments is rational and likely to be evidence - based to a certain extent at least. to what category do psychiatric treatments belong ? in depression, the routine treatments include cognitive behavioural therapies and medication (with antidepressant drugs). other regimens include physical exercise, sleep deprivation, light exposure therapy and in severe cases electroconvulsive treatment, alone or combined with the routine treatments. medication has often been thought as type 1 or type 3 interventions, but there is as yet little evidence that deficiencies of brain monoamines actually cause depression. it appeared that low brain serotonin (due to low plasma tryptophan) is associated with failing impulse control and aggression rather than with depression (e.g. russo. cognitive therapies have been developed to correct misconceptions associated with the depression (i.e. to reduce the impact of life events ; beck. whether their reported positive effects are due to a type 4 rather than a type 1 (or perhaps type 3) effect remains to be proven. interventions, such as physical exercise, sleep deprivation, light exposure therapy and electroconvulsive treatment, are certainly not type 1, 2 or 3 treatments. the latter are perhaps type 4 treatments (alleviating suffering), but that indexation may feel as artificial. in view of the transition hypothesis (see previous section), the anti - depressive interventions may facilitate switching from the depressive to the nondepressed state. their effects could be seen as type 1 therapies, as hypo - activity is often a prominent symptom of depression, whereas exposure to light activates or compensates failing mechanisms in seasonal affective disorders, respectively. therefore, we suggest to adding another type of treatment. our type 5 treatment is to facilitate brain transitions this idea might be seen as an extension of type 1 because they assist the organism to battle the ailment, but that is a rather artificial formulation for randomly occurring brain states. there is an ongoing debate on the therapeutic efficacy of antidepressants (see previous sections). in several meta - analyses, their effects appear to be modest as compared to placebo, explaining about 2% of the variance of depressive symptoms on top of placebo. first, the response of depressed subjects to medication varies widely : both fast and clear - cut responses and no response at all have been reported and second, the placebo response is relatively high and variable. apparently no meaningful distinction can be made between subjects who need medication and those who recover without medication (or placebo). is this a matter of lack of knowledge or is this inherent of the current conception of depression ? the placebo - drug controversy may serve as another argument supporting the random mood concept, and, if so, the drug treatment is a type 5 intervention.. 2010) which suggest the application of inhibitors of dna methyl transferase and histone deacetylase (hdac) as potential therapeutic agents in the causal treatments of psychiatric disorders. this is entailed from the assumption that altered patterns of mrna and protein expression are downregulated in the pathogenesis of such disorders like unipolar depression or schizophrenia. therefore, hdac are supposed to activate the mrna expression in these conditions. in short, we argued that perhaps except cognitive psychotherapy so an antidepressive treatment is often not aimed to influence the depressive feelings directly, but to influence the course and severity of a depression. considering the stochastic mood concept, thus assuming near - random brain transitions, a strict causality between diagnosis and therapeutic response must be considered as unlikely. medical disorders, including psychiatric disorders, are diagnosed by the content (e.g. mood), severity, time course and coexistent factors. in the case of depression, for instance, mood characteristics, severity of the depression, duration of the depressive episode and comorbid anxiety, dysthymia or somatic disorders are, among others, important criteria for diagnosis, treatment and prognosis. the diagnosis is mainly based on phenomenology and self - reports (what the psychiatrist sees and hears), together referring to the content of a disorder. such assessments might be biased by the theoretical framework of the therapist and by the feelings of the patient. for instance, the negative attributions of patients about the origin of their depression (feelings of guilt and worthlessness) are well known (beck. for instance, infusing cortisol gave more negative associations in volunteers and patients, suggesting that hypercortisolaemia might bias the patient s report (tops. the most explored biological markers for depression include hormones and associated receptors (e.g. dst or dex - crh), a wide variety of genes, blood or urine levels reflecting metabolic processes, physiological responsiveness and brain imaging (for an extensive review, see mssner. these markers do not inform on the thought contents but may be related to severity, course and comorbidity. most often, markers are explored in cross - sectional study designs by comparing a cohort of depressed patients and a matched nondepressed cohort. one longitudinal study claims that normalisation of the dst or the dex - crh test precedes recovery from depression (carroll. 1981 ; holsboer 2000). despite 30 years of research and strong initial claims, the dst and the dex - crh tests have not contributed significantly to a better diagnosis of depression. it is also not clear to what aspect of depression (severity, course, comorbidity) an abnormal cortisol function is associated. extensive studies have appeared on serotonin - related genes (particularly serotonin transporter regulating genes and their polymorphisms) in relation to depression and life events. claims that severe and repeated life events provoke depression in the s / s genotype subjects (caspi. 2003) have subsequently not been replicated (uhe and mcguffin 2008). there is certain progress in the research focused on the exploration of peripheral markers to define the epigenetic risk for depression. special interest is paid in a pilot study of the promoter methylation of the serotonin transporter gene (olsson., we and others (mssner. 2007) conclude that none of the current candidate markers supports the diagnosis of depression. morphological and functional neuroimaging have the potential to show anatomical brain abnormalities or aberrant physiological responses during a mental task, respectively. morphological changes are usually considered as irreversible, but in depression, reversible changes of regional brain volumes were reported (drevets 2003 ; kronmller. how fast these volumes change is as yet unknown, but most likely not within days or weeks. therefore, we consider morphological changes conditional rather than causing depression (kronmller. 2008), possibly by impairing mood transitions. in this respect, morphological changes are similar to somatic disorders such as cardiovascular heart- and brain - pathology, often associated with depression (de jonge. 2006, 2007). following exposure to emotional stimuli, abnormal responses of brain amygdala, frontal cortex and some meso - limbic areas as detected with functional mri, oxygen pet and water pet were observed (drevets 2003). it is difficult to decide whether imaging results have to be attributed to abnormal processing of the stimuli or that the depressive state overrules brain processing. neuroimaging and endocrine studies have been used to define endo - phenotypes, i.e. subjects who are more susceptible for stress because of certain genes or gene polymorphisms. vulnerable endo - phenotypes have indeed been reported, suggesting that the subjective perception of stress, and by inference of life events, may render individuals more prone to develop depression (e.g. jabbi. psychiatric disorders are characterised by the content (e.g. mood), severity, time course, and coexistent factors. few if any attempts have been made to differentiate between associations with the pathopsychological content or with the course, severity or other characteristics of depression. we have emphasised four major challenges for a rational and causal conceptualisation of functional disorders in psychiatry (table 1). each of these challenges could well lead to the conclusion that a rational and evidence - based psychiatry is theoretically and practically impossible. the present conclusions are mainly based on data of major depression. besides making a critical analysis we start the discussion on strong and weak points of our analysis. table 1some obstacles to reach (strict) causality in psychiatry1. classification systems are not falsifiable, because of (a) heterogeneity of categories (b) inclusion of novel factors and (c) publication bias2. brain functions are at least, in part, not - deterministic, because of (a) iso - energeticity and consequently (b) chaotic / random and stochastic processes3. therapies are not directed to core symptoms / characteristics, because they (a) do not influence subjective feelings (b) influence course and not pathogenesis and (c) are relatively ineffective4. markers and inquiry (a) do not inform on the core symptoms (b) but possibly on course and severity, and (c) inquiry is biased because of subjectivity some obstacles to reach (strict) causality in psychiatry first, we consider some philosophical aspects. more than in somatic medicine the question is whether a psychiatric disorder is a real thing or a construct that does not necessarily refer to a real world. such criticism is not justified, because any scientific theory, diagnosis or hypothesis is not more than a construct that describes phenomena as long as there is no better idea. this philosophical issue touches the problem of irreducibility of real - world processes and objects. an alternative view is that scientific theories and clinical protocols have no or little common basis. real progress is possible only when both the scientific and clinical views converge to unambiguous conceptualisations. as long as that is not the case in psychiatry, one has to live with certain and unavoidable inconsistencies between a medical and scientific framework of psychiatry. we considered classification systems primarily as useful constructs and not so much as describing real world objects. as an alternative to evidence, we considered the term proof and proven to emphasise causality. more exactly, these terms indicate causality of two types : (i) strict linear causality in relatively simple systems useful for lower levels of complexity ; (ii) nonlinear, dynamic causality, applicable to single cells up to the endo - phenotype. there remains the possibility to establish bridge laws (nagel. 1971 ; nagel 1979) between multitude complexities at micro (neuronal) and macro (behavioural) levels (also indicated in fig. 1). in this case, we do not exemplify empirical reduction but we may introduce patchy reduction (kendler 2005) or inter - theoretic reduction between paradigms. the concept of the iso - energetic brain 24 may be seen as a provisional bridge law (nagel 1979) and as a patchy reductive explanation (kendler 2005). in the following, we suggest a strategy to explore some of the discussed issues. the core of our proposal is to re - analyse epidemiological, psychological and laboratory data but not only according to currently accepted diagnostic systems. for instance, genes or biochemical parameters may well be related to severity, course or other features of a disorder (kendell and jablensky 2003). if so, then the same gene variants may be associated not only with depression but also with, e.g. anxiety or schizophrenia or even with nonpsychopathological symptoms as well.. this could give information on the significance of some pathophysiological parameters to evoke particular symptoms or group of symptoms present in different disorders and, ideally, how they can be affected by therapy. many of current ideas on psychopathology are developed from relatively large cross - sectional or longitudinal epidemiological investigations. indeed, such studies may disclose general trends, but the results are not necessarily applicable to a single patient. a firm causal basis for psychiatry (and any medical discipline) requires a reasonable guess about the diagnosis in the single individual. in practice, several relatively nondisorder - specific factors converge to a diagnosis that has implications for further therapeutic regimens. furthermore, we emphasise that a diagnosis has to define a typical condition (disease) of a prototypical individual (patient). one aim of a medical psychiatry could be to define and redefine diagnostic prototypes (kendell and jablensky 2003 ; jablensky 2005 ; nagel 1979 ; schaffner 2004 ; kendler 2008 ; kendler and campbell 2008). following this line ; we suggest that data analysis in epidemiological and other population - based studies might focus to delineate relatively small subcohorts that are defined by a limited number of parameters. these parameters may include not only psychopathological symptoms but also physiological, biochemical or genetic characteristics. and they may be related to severity, time course and other characteristics of the disorder. a similar approach has been elaborated on the distinction of validity and utility of psychiatric diagnosis (kendell and jablensky 2003 ; jablensky 2005 ; schaffner 2004). one criterion is that in a multidimensional space of symptoms a syndrome is defined by detectable discontinuities. so there should be boundaries of rarity, distinguishing syndrome (disease) from sanity or from other syndromes (diseases or disorders ; jablensky 2005 ; schaffner 2004 ; kendler 2008).. the outcome of such exercise could be the following : first, one or several of the diagnostic prototypes are identical or very close to categories of existing diagnostic systems (e.g. the dsm), second, these prototypes are completely different and not easy to reconcile with current diagnostic systems, or third such hypothesised diagnostic prototypes do not exist or can not be recognised as yet. the first outcome provides a strong support for a scientific basis and clinical relevance of or at least a part of the tested diagnostic system. the second outcome forces to reconsider the diagnostic system under investigation and might eventually lead to new diagnostic systems. the third outcome might lead to a temporary anarchy of diagnostic systems as no cues for developing a new system are revealed. future scientific developments may enable more pertinent conclusions in line with the first or second outcome. in the latter case, there is as yet no urgent need to reconsider the diagnostic system under study. the present assay is not aimed to provide a conceptual or philosophical framework of psychiatry, as done recently. these papers (kendler 2005, 2008 ; kendler and campbell 2008) emphasise the multilevel character of explanatory models in psychiatry and warn against too simple monolevel explanations, e.g. at the molecular or psychological level. we agree with this viewpoint as is also illustrated in fig. 1 and acknowledge the implications for research paradigms. our intention is to bring the discussion to a more operational level : what should be done to rationalise psychiatry. the medical model was chosen as starting point in our analysis, because it is in our opinion the best tool to discern shortcomings and weak points. our concern about utility of validity of current diagnostic constructs is shared by others (e.g. angst 2007). our emphasis is on utility, not on validity of diagnostic constructs (kraemer and kupfer 2006). from a scientific point of view, the distinction between validity and utility is rather artificial, because validity of a concept can only be proven by practical testing, in the medicine preferentially by showing utility. the medical model is the basis of evidence - based medicine : to establish causality between diagnosis, interventions and therapeutic effects. we have discussed four major obstacles : first the nonfalsifiable nature of current classification systems, second the nondeterministic character of many cerebral processes, third divergence between therapeutic approaches and pathogenesis and fourth inadequacy of diagnostic tools. one has to appreciate that knowledge about the world is far more than what can be communicated between human beings. one unavoidable shortcoming might be due to the impossibility to describe professional knowledge (or perhaps better professional intuition) of the psychiatrist verbally that said without the previously mentioned theoretical biases. so we might eventually have to consider the possibility that professional experience is an essential element not only in everyday health care but also in evidence - based psychiatry.	considering psychiatry as a medical discipline, a diagnosis identifying a disorder should lead to an effective therapy. such presumed causality is the basis of evidence - based psychiatry. we examined the strengths and weaknesses of research onto the causality of relationship between diagnosis and therapy of major depressive disorder and suggest what could be done to strengthen eventual claims on causality. four obstacles for a rational evidence - based psychiatry were recognised. first, current classification systems are scientifically nonfalsifiable. second, cerebral processes are at least to some extent nondeterministic, i.e. they are random, stochastic and/or chaotic. third, the vague or lack of relationship between therapeutic regimens and suspected pathogenesis. fourth, the inadequacy of tools to diagnose and delineate a functional disorder. we suggest a strategy to identify diagnostic prototypes that are characterised by a limited number of parameters (symptoms, markers and other characteristics). a prototypical diagnosis that may either support or reject particular elements of current diagnostic systems. nevertheless, one faces the possibility that psychiatry will remain a relatively weak evidence - based medical discipline.
the date palm (phoenix dactylifera l.) is of economic importance to the kingdom of saudi arabia which is the second largest producer of dates worldwide. although date palms can grow under a variety of environmental conditions, production is impeded by various biotic and abiotic stress factors. most importantly, water shortage and salinity of the ground water provide abiotic stresses which decrease date production. this is a worldwide problem with some 20% of the world 's cultivated land and approximately 50% of all irrigated land being affected. in consequence, adaptation of crop plants to water deficit and salt stress is of high priority in worldwide programmes for breeding modern varieties (for a review see). for date palm thousands of different cultivars are known which have been selected by the producers mainly for improved crop yield and quality. thus, a high degree of variability is presumably present in date palm germplasms with respect to drought and salinity (desiccation and salt tolerance). the high salinity exhibits negative effects on the critical biochemical processes of the plant : salt stress affects the whole plant as well as tissues and cells. it can lead to water deficit stress, metabolic toxicity, and nutritional deficiencies and finally drastically reduce production. as studied extensively in arabidopsis and rice, three aspects of adaptive responses in plants can be considered under conditions of salt and drought : (a) ion and osmotic homeostasis, (b) growth control / inhibition, and (c) control and repair of stress damage (detoxification). the findings on mechanisms of adaptation to abiotic stresses in model plants such as arabidopsis are relevant to crop plants. transduction of extracellular, abiotic stress signals via the cell wall and membrane into the cytoplasm and subcellular compartments follows various pathways and triggers various responses. two of the principal elements in these pathways of plant cells are (i) intracellular ca ions and (ii) protein kinases. consequently, the sensing of abiotic stress such as drought or salinity results in changes of the phosphorylation status of cellular proteins. as revealed by mutant analysis in arabidopsis, abscisic acid (aba) is another key regulator of signal cascades that are triggered by salt and water deficit. molecular studies in date palm for understanding some basic molecular mechanisms in response to drought and salinity have been rather limited [917 ]. recent date proteome analyses, however, open the way to the identification of important biomarkers. identified an abc superfamily atp - binding cassette transporter as a putative biomarker for male date palms. palms affected by the leaf brittle disease express a manganese - stabilizing 33 kda protein not detectable in healthy plants. the recent sequencing and annotation of date palm genomes opens the possibility for the application of further high throughput technologies to the study of stress - related gene functions in date palm. proteome analysis, for example, opens the possibility to identify date palm proteins involved in transduction network regulation via posttranslational protein modification by phosphorylation / dephosphorylation under abiotic stress conditions as studied for example, in the desiccation - tolerant plant craterostigma plantagineum [22, 23 ]. the effort described here relates to proteome analysis of salinity and water stress - related sensitive protein resulting from the salinity and drought gene expression in leaves of young date palm seedlings. seeds of the cultivar sagie were scarified with sulfuric acid (96%) for 5 min and washed 5 times with sterile distilled water, followed by sterilization with 1% (v / v) mercuric chloride for 3 min, washed 5 times with sterile distilled water, and imbibed for 48 h in distilled water. the seeds were sterilized a second time with calcium hypochlorite (5%, v / v) for 4 min and washed 4 times with sterile distilled water. seeds were germinated between wet layers of tissue papers until the radical reached 1 cm and then transferred to pots containing organic soil and irrigated with tap water and grown in growth chambers under controlled light conditions (12 h light/12 h dark) at 30c until the age of three months. twelve 3-month - old date palm seedlings were selected and divided as follows : 4 seedlings were daily irrigated with distilled water for one month as control, 4 seedlings were subjected to drought (27.5 g / l peg 6000) for one month, and the other 4 seedlings were subjected to salt stress with 16 g / l nacl, according to a modified method of san.. at the end of the stress period, samples were washed with distilled water, frozen in liquid nitrogen, and stored at 80c until use. four replicates of the frozen shoot materials of stressed and control plantlets were ground into a fine powder under liquid nitrogen, and the proteins were precipitated by the addition of 1.8 ml of ice - cold acetone containing 0.07% (v / v) mercaptoethanol. one hundred mg of each of the lyophilized raw extract was dissolved in 400600 l of ief buffer (7 m urea, 2 m thiourea, 2% (w / v) chaps, and 30 mm tris ph 8.0). the mixture was then centrifuged for 10 minutes at 4c at 16,100 g and total soluble protein in the supernatants was quantified using the 2d quant kit (ge healthcare, munich, germany). equal amounts of all single samples were pooled to get dige internal standard (is). amount of 50 g of is was used for each analytical gel and 300 g of is for each preparative gel (necessary for protein identification by ms). 50 g of each protein sample as well as needed amount of internal standard was labeled with fluorescent dyes using the refraction-2dtm labeling kit (nh dyeagnostics gmbh, halle, germany) according to the manufacturer 's protocol. the internal standard was labeled with g100, whereas the single analyzed samples were labeled with g200 or g300 before mixing. 2d gel electrophoresis was briefly performed as follows : samples mixture was separated in the first dimension according to their isoelectric point (pi) using immobilized ph gradient strips (immobiline drystrip, 24 cm, ph 47, ge healthcare) focused by ipgphor 3 (ge healthcare) and in the second dimension according to their molecular weight by sds - page using the ettan dalttwelve gel system (ge healthcare). for preparative gels, glass plates were silanized on one side prior to gel casting and the gels were run in parallel to the analytical gels. the fluorescent scans of the analytical gels were generated using ettan dige imager (ge healthcare) immediately after electrophoresis. preparative gels were stained with 1 mm ruthenium(ii)-tris(bathophenanthroline disulfonate) fluorescence stain and reference markers were attached to the glass plates prior to scanning, thus enabling blind picking of the protein spots after the difference gel electrophoresis (dige) analysis. the preparative gels were scanned directly after destaining and stored wet at 4c before spot cutting. the internal standard included all proteins in the analysis and as it was run on every gel along with all analyzed samples, it was used for spot matching across all the gels. the biological variation analysis (bva) module allowed quantitative comparisons of protein expression across multiple gels. the extended data analysis (eda) was used for multivariate analysis of protein expression data derived from the bva module in order to perform a principal component analysis (pca) and to identify protein spots of interest with differential expression analysis. all automatically identified spots were checked manually to confirm that they are real spots and marked for picking. for protein identification in a single spot, proteins fixed in the polyacrylamide gel plug were reduced, alkylated, and digested with trypsin (promega, mannheim, germany). the resulting peptides were analyzed by nano - hplc (ultimate 3000 hplc system, lc packings, dionex, idstein, germany) coupled to an amazon etd ms ion trap spectrometer (bruker daltonics, bremen, germany) using nano - esi spray. the nano - hplc system and the ion trap spectrometer were controlled using the bruker compass hystar v3.2-sr2 software. the liquid chromatography system was supplied with reversed - phase precolumn (lc packings, dionex) for sample desalting and a 15 cm pepmap 100 reversed - phase c18 column, 75 m inner diameter (lc packings, dionex), for peptide fractionation. the peptides were separated using a 45 min linear gradient from 96% (v / v) solution a (2% (v / v) acetonitrile, 0.1% (v / v) formic acid in high purity water) and 4% (v / v) solution b (98% (v / v) acetonitrile, 0.1% (v / v) formic acid in high purity water) to 50% (v / v) solution a and 50% (v / v) solution b at a flow rate of 300 nl / min. the electrospray was operated in positive ion mode with 4000 v spray voltage and 10 psi gas pressure. the end plate offset of the mass spectrometer was set to 500 v and for the acquisition the standard method proteomics auto msms alternating spectra cid - etd bruker trap control v7.0 was used. raw data files were evaluated using compass data analysis v4.0-sr5 software with embedded search engine mascot search 2.3.01 (matrix science ltd., london, uk). swiss prot (all species) and ncbinr (green plants) databases were involved in the protein search using the following parameters : enzyme trypsin, up to one missed cleavage permitted, no fixed modifications and variable modifications carbamidomethyl (c), oxidation (m) and propionamide (c) allowed, and mass tolerance for both precursor ion and fragment ion 0.3 da. only the protein hit with highest protein score was used for further analysis. when the protein was identified with one peptide only or several proteins with similar protein score were identified in a spot, the spots were excluded from the analysis. the objective of this experiment is comparison of protein spots of salt - stressed and drought - stressed date palm shoots by searching for new protein spots or proteins spots differing in their intensity due to stress. in this experiment, 4 salt stress samples (6164), 4 drought stress samples (7376), and 4 control samples (14) of lyophilized raw protein extracts of date palm were analyzed. samples 6164 represent the date palm seedlings that were exposed to a high concentration of nacl (16 g / l) for one month, whereas samples 7376 represent seedlings that were exposed to a high peg concentration (27.5 all the samples were solubilized in ief1 buffer (7 m urea, 2 m thiourea, 2% chaps, and 30 mm tris ph 8.0) according to the data shown in table 1. after overnight solubilization and subsequent centrifugation, the soluble proteins were quantified using the 2d quant kit. estimated total protein concentrations are listed in table 1. for successful labelling of the samples with g200 or g300, all the samples were diluted with ief 1 buffer to reach the concentration of the sample with lowest concentration (1.1 g/l in this case, table 1). an internal standard was prepared by mixing all 12 samples in the same weight ratio and the standard was labelled with g100. the presence of an internal standard in every gel provided an intrinsic link between samples. each protein spot in a sample was compared to its representative spot within the internal standard on the same gel to generate a ratio of relative protein levels. quantitative comparison of samples between gels was based on the relative change of a sample to its in - gel internal standard. the labelling and mixing scheme for the performed dige experiments is shown in table 2. part of the internal standard was saved before labelling with g100 and this unlabeled part was applied to 2 preparative gels that were in the end used for the protein identification by mass spectrometry. proteins from the extracts were separated in the first dimension according to their protein intensity using ief on immobiline drystrip, 24 cm, ph 47, and in the second dimension according to their molecular weight using sds - page. all of the 6 analytical gels and the 2 preparative gels were run at the same time. the preparative gels were fixed for 1 hour in fixation solution (40% ethanol and 10% acetic acid in water) and then stained with rubps (1 mm in fixation solution) for 20 minutes and destained overnight in fixation solution. the preparative gels were scanned directly after destaining and stored wet at 4c before spot cutting. for dige analysis, spots were detected on all scans and intergel matching was performed using the decyder software. the matching was manually checked and corrected in mismatched regions. in the next step, pca helps to identify some underlying sources of variation and gives first impression on how well experimental groups can be separated. spots that could be localized on 80% of spot maps (gel scans) were included in the analysis (figure 2). scores of the spot maps for salt stress (red circles in figure 2(a)) were localized closely to each other in the graph and thus showed a good reproducibility for the salt stress biological replicates. in addition, scores of the spot maps for controls (green circles in figure 2(a)) had an outlier in spot map for sample 2. other three spot maps were well separated from the salt stress spot maps in a compact group. furthermore, scores of the spot maps for drought stress (blue circles in figure 2(a)) were located between controls and salt stress with an outlier for sample 76. position of other three replicates to each other indicated a lower reproducibility of the drought stress compared to the salt stress. position closer to the control suggested smaller differences in drought stress as compared to salt stress. for this analysis, the standardized abundance of each spot on the gel was calculated for two samples in the gel using the spot intensities of the internal standard. significant differences between the samples were visualized showing blue or red spot contours for higher or lower standardized abundance (threshold 3) of the spots, respectively. figure 3 shows fluorescence scan of the control versus salt stress, figure 4 shows fluorescence scan of control versus drought stress, and figure 5 shows fluorescence scan of the salt stress versus drought stress. several regions with significant spot changes were localized and may indicate potential effects on the proteome due to stress. t - test (p value calculated using student 's t - test), one - way anova (p value calculated using one - way anova statistical test), and average ratio (average ratio between the groups selected in the protein statistics) values were calculated for all matched spots. the spots were filtered for one - way anova value lower than 0.2, t - test value lower than 0.1, and average ratio 2. eda is proteomic software for multivariate analysis of protein expression data derived from bva module. it was used for pca analysis presented in section 1 and to find the protein spots of interest with differential expression analysis. all automatically chosen spots were checked manually if they are real spots and marked for picking (figure 6). using low level statistical parameters, 47 spots were chosen for protein identification, most of them with lower spot volume under stress conditions compared to the standard. detailed results of dige analysis are found in the supplementary excel tables (in supplementary material available online at http://dx.doi.org/10.1155/2015/407165). proteins in the gel plugs were reduced with 10 mm dithiothreitol and alkylated using 55 mm iodoacetamide in 0.1 m to open s - s bridges for action of trypsin. digestion with trypsin (12.5 ng/l of trypsin in 50 mm nh4hco3) was performed overnight at 37c. the resulting peptides were extracted from the gel plugs in two extraction steps : first one with 25 mm nh4hco3 and second one with 5% formic acid. collected extracts were dried down and resolubilized in 2% acetonitrile with 0.1% formic acid in water (ms grade) for ms analysis. the resulting peptides were separated according to their hydrophobicity by nano - hplc (c18 column, ultimate 3000 hplc system, dionex) and sprayed directly into an ion trap spectrometer (amazon etd, bruker daltonics) using nano - esi sprayer. processed ms / ms spectra were used for the protein identification with in - house mascot search server (matrix science software). swiss prot (all species) and ncbinr (green plants) databases were involved in the protein search (figure 7 and table 3). ms results. biological replicates for salt stress showed good reproducibility and were well separated from control samples in the pca score plot. several regions with significant spots changing probably due to the applied stress were localized in the gel scans using differential in - gel analysis (dia, decyder). most of the spots from these regions were decreased in the relative abundance under stress conditions. only low quality statistics allowed the finding of some more intensive spots sensitive to stress. proteins in 47 spots were analyzed by mass spectrometry ; for 8 spots no protein could be identified. levels of atp synthase alpha and beta subunits, unknown protein 18 and some of rubisco fragments were significantly changed under both stress conditions. changes in abundance of superoxide dismutase, chlorophyll a - b binding protein, light - harvesting complex i protein lhca1, rubisco activase, phosphoglycerate kinase, chloroplast light - harvesting chlorophyll a / b - binding protein, phosphoribulokinase, transketolase, rubisco, and some of rubisco fragments were significant only for salt stress. proteome analysis provides one of the best options for the functional analysis of translated regions of the genome. the levels of atp synthase alpha (accession number gi\|158325128) and beta subunits (accession number gi\|292559515), unknown protein (accession number gi\|205830697), and some of rubisco fragments (accession numbers gi\|28195663, gi\|292559516, gi\|3152721, gi\|209417491, gi\|209417489, gi\|55785631, and gi\|16565309) were significantly changed under both stress conditions compared to the control which indicates that these protein subunits are associated with drought and salinity stress [2527 ]. in the current study, changes in abundance of superoxide dismutase, chlorophyll a - b binding protein, light - harvesting complex i protein lhca1, rubisco activase, phosphoglycerate kinase, chloroplast light - harvesting chlorophyll a / b - binding protein, phosphoribulokinase, transketolase, rubisco, and some of rubisco fragments were significant only in salt stress condition. this result is consistent with many salinity stress tolerance studies in other organisms [2830 ]. the superfamily of light - harvesting chlorophyll a / b - binding (lhc) proteins in higher plants and green algae is composed of more than 20 different members associated with photosystem i (psi) or photosystem ii (psii). in this study, chlorophyll a - b binding protein (cab) and light - harvesting complex i protein lhca1 were upregulated. accumulation of cab and lhca1 in psi exposed to salt and drought stress might represent one of the strategies to prevent or lower light stress - induced damage [25, 26, 28 ]. the accumulation of chl has been proposed as one of the potential biochemical indicators of salt tolerance in different crops. it was also proposed that these proteins might have a protective function within psii under stress conditions either by binding free chlorophyll molecules and preventing the formation of free radicals and/or by acting as sinks for excitation energy, because under stress conditions, a mobile pool of lhcb1 and lhcb2 moves from psii to psi due to the reversible phosphorylation of lhc proteins by a thylakoid - bound kinase [26, 32 ]. decrease in the initial activity and activation state of rubisco as a result of drought stress was encountered in mediterranean species. in the present study, the expression of superoxide dismutases (sod) the superoxide dismutases are metalloenzymes that catalyze the dismutation of ion superoxide into oxygen and hydrogen peroxide (sods) and constitute the first line of defence against reactive oxygen species. the superoxide radical is a reactive oxygen species (ros) whose production increases under abiotic and biotic stresses, including drought [28, 35 ]. meanwhile, alscher. stated that sods play a critical role in protecting plant tissues from ros. roveda - hoyos and fonseca - moreno reported that eight proteins were overregulated in wheat leaves. in response to salt stress, among them a protein complex of psii, a protein oee2 (oxygen - evolving enhancer protein 2), and superoxide dismutase. the change in regulation of phosphoribulokinase in the current study is also correlated with salt stress. the expression of prk (phosphoribulokinase) in ice plant was affected under salt stress conditions. the amount of mrna was declined by a factor of approximately three within days, followed by an increase to approximately prestress levels. in conclusion, proteome of salinity and drought - stressed palm seedlings was compared with that of nonstressed plants of the same age. the ms analysis of 47 sensitive protein spots showed that 12 proteins could be identified, 3 of them were significantly changed in both stresses, and 9 proteins were significantly changed only in salt - stressed plants. proteins could not be identified in 8 spots, whereas in 26 spots rubisco and its fragments were identified.	this study was carried out to study the proteome of date palm under salinity and drought stress conditions to possibly identify proteins involved in stress tolerance. for this purpose, three - month - old seedlings of date palm cultivar sagie were subjected to drought (27.5 g / l polyethylene glycol 6000) and salinity stress conditions (16 g / l nacl) for one month. dige analysis of protein extracts identified 47 differentially expressed proteins in leaves of salt- and drought - treated palm seedlings. mass spectrometric analysis identified 12 proteins ; three out of them were significantly changed under both salt and drought stress, while the other nine were significantly changed only in salt - stressed plants. the levels of atp synthase alpha and beta subunits, an unknown protein and some of rubisco fragments were significantly changed under both salt and drought stress conditions. changes in abundance of superoxide dismutase, chlorophyll a - b binding protein, light - harvesting complex1 protein lhca1, rubisco activase, phosphoglycerate kinase, chloroplast light - harvesting chlorophyll a / b - binding protein, phosphoribulokinase, transketolase, rubisco, and some of rubisco fragments were significant only for salt stress.
lamotrigine is a new, anti - epileptic drug used in the treatment of partial and generalized seizures and maintenance treatment of bipolar i disorder. common adverse effects of lamotrigine include headache, nausea, vomiting, tremors, anxiety and insomnia. the major adverse drug reaction leading to lamotrigine discontinuation is skin rash, observed in 3 - 10% of users. lamotrigine related rashes are mostly simple maculopapular exanthems, observed within first eight weeks of treatment and resolve promptly on withdrawal of the drug. however, in a small percentage of patients, lamotrigine can cause serious adverse cutaneous reactions like stevens - johnson syndrome and toxic epidermal necrolysis. we report two cases of toxic epidermal necrolysis resulting from lamotrigine therapy and discuss the risk factors associated with the development of serious reactions, with emphasis on the importance of adhering to the recommended dosage guidelines. a 47-year - old female presented with fever, oral erosions and blisters all over the body for ten days. patient was a known epileptic, on valproic acid 300 mg / day since four months. two weeks prior to onset of symptoms she had been initiated on lamotrigine 50 mg twice daily by her physician. examination revealed multiple, flat target - like lesions, diffuse erythema, flaccid blisters and erosions over face, trunk and extremities including palms and soles [figure 1 ]. body surface involvement was around 60%. the diagnosis of lamotrigine induced toxic epidermal necrolysis was established using the naranjo adverse drug reaction probability scale. patient was admitted to intensive care unit, and lamotrigine and valproic acid were discontinued. after a tumultuous course in the hospital extending up to 45 days, patient made an eventual recovery but was left with pigmentary changes, nail dystrophy, bilateral ectropion and corneal scarring. erythematous papules, bullae, erosions and separation of necrotic epidermis a 26 year old female was under treatment with valproic acid 750 mg / day, propranolol 10 mg / day, clomipramine 10 mg / day, and risperidone 2 mg / day for bipolar mood disorder and obsessive compulsive disorder since two years. four weeks prior to presentation, she had been initiated on lamotrigine at 25 mg / day, which was rapidly increased to 50 mg bd over 2 weeks and 100 mg bd over another 2 weeks, at which time she developed skin rash and oral erosions. examination revealed a rash typical of toxic epidermal necrolysis with erythematous macules, papules, flaccid blisters, targetoid lesions, and erosions over face, trunk and palms involving around 30% of body surface area [figure 2 ]. genital and oral erosions with hemorrhagic crusting over lips and severe conjunctival congestion were observed. a causality assessment of this adverse event revealed a probable association between lamotrigine and toxic epidermal necrolysis based on the naranjo probability scale. prednisolone was tapered gradually, and the patient made an uneventful recovery and was discharged. multiple targetoid lesions, bullae, and erosions on the face and hemorrhagic crusting on the lips antiepileptic drugs are frequently associated with idiosyncratic adverse skin reactions, which are a leading cause of withdrawal of the drug. among the traditional antiepileptic drugs, aromatic compounds like phenytoin, carbamazepine and phenobarbitone of the nine newer antiepileptics introduced in the past decade, lamotrigine has been most associated with rash, in up to 3 - 10% of users in clinical trials. most of the rashes with lamotrigine are benign and resolve promptly on discontinuation of treatment. the incidence of rash due to lamotrigine, associated with hospitalization in clinical trials was three of 1000 in adults (0.3%) and one of 100 in children (1%). co - administration of valproic acid with lamotrigine significantly increases the risk for development of serious adverse cutaneous reactions. the elimination half life of lamotrigine when used as monotherapy is 25 - 30 hours and it is more than doubled to 60 hours when combined with valproic acid. it has also been suggested that a high starting dose and a rapid dose escalation may increase the occurrence of skin rashes with lamotrigine. the risk of allergic reactions is decreased when treatment is started at a low dose and the doses are increased gradually following the recommended dosage guidelines, possibly because slow titration may allow desensitization to occur. both of our patients were on valproic acid when lamotrigine was initiated as an add - on therapy. high starting doses were employed and dose was also rapidly increased in both the cases. history of a rash attributed to another antiepileptic drug results in a five times greater increase in the rate of an antiepileptic drug rash. hirsch. have concluded that a rash with another antiepileptic drug is the greatest risk factor for developing a rash with lamotrigine. in the first patient the phenomenon of cross reactivity may be a general feature of patients predisposed to drug rashes. also a patient with a prior medication related rash would be more vigilant and likely to notice a drug related rash from a new medication. females have a higher risk for antiepileptic skin reactions compared to males ; probably due to hormonal factors. a causality assessment using naranjo probability scale was performed in both the patients that revealed a probable association between lamotrigine and toxic epidermal necrolysis in both of the cases. our second patient was on several medications (valproic acid, propranolol, clomipramine, risperidone) since two years. these drugs have a very low potential for causing serious drug reactions like toxic epidermal necrolysis. the rates of skin rash with valproic acid are very low as compared to lamotrigine. thus it is unlikely that they were responsible for causing toxic epidermal necrolysis in the second patient. the international prescribing guidelines regarding use lamotrigine as an adjunctive treatment with valproic acid, recommend that lamotrigine should be initiated at a dose of 12.5 mg once daily for first two weeks, followed by 25 mg once daily for next two weeks, and increased thereafter by 25 mg every other week until a maintenance dose of 100 - 200 mg daily. it must be pointed out, however, that serious reactions can occur with recommended dosing and titration schedules, and without concurrent valproic acid. increased vigilance is thus required when initiating lamotrigine in a patient and treatment should be discontinued if any rash appears. when lamotrigine is prescribed as an adjunctive treatment with valproic acid a 47-year - old female presented with fever, oral erosions and blisters all over the body for ten days. patient was a known epileptic, on valproic acid 300 mg / day since four months. two weeks prior to onset of symptoms she had been initiated on lamotrigine 50 mg twice daily by her physician. examination revealed multiple, flat target - like lesions, diffuse erythema, flaccid blisters and erosions over face, trunk and extremities including palms and soles [figure 1 ]. body surface involvement was around 60%. the diagnosis of lamotrigine induced toxic epidermal necrolysis was established using the naranjo adverse drug reaction probability scale. patient was admitted to intensive care unit, and lamotrigine and valproic acid were discontinued. after a tumultuous course in the hospital extending up to 45 days, patient made an eventual recovery but was left with pigmentary changes, nail dystrophy, bilateral ectropion and corneal scarring. a 26 year old female was under treatment with valproic acid 750 mg / day, propranolol 10 mg / day, clomipramine 10 mg / day, and risperidone 2 mg / day for bipolar mood disorder and obsessive compulsive disorder since two years. four weeks prior to presentation, she had been initiated on lamotrigine at 25 mg / day, which was rapidly increased to 50 mg bd over 2 weeks and 100 mg bd over another 2 weeks, at which time she developed skin rash and oral erosions. examination revealed a rash typical of toxic epidermal necrolysis with erythematous macules, papules, flaccid blisters, targetoid lesions, and erosions over face, trunk and palms involving around 30% of body surface area [figure 2 ]. genital and oral erosions with hemorrhagic crusting over lips and severe conjunctival congestion were observed. a causality assessment of this adverse event revealed a probable association between lamotrigine and toxic epidermal necrolysis based on the naranjo probability scale. prednisolone was tapered gradually, and the patient made an uneventful recovery and was discharged. multiple targetoid lesions, bullae, and erosions on the face and hemorrhagic crusting on the lips antiepileptic drugs are frequently associated with idiosyncratic adverse skin reactions, which are a leading cause of withdrawal of the drug. among the traditional antiepileptic drugs, aromatic compounds like phenytoin, carbamazepine and phenobarbitone of the nine newer antiepileptics introduced in the past decade, lamotrigine has been most associated with rash, in up to 3 - 10% of users in clinical trials. most of the rashes with lamotrigine are benign and resolve promptly on discontinuation of treatment. the incidence of rash due to lamotrigine, associated with hospitalization in clinical trials was three of 1000 in adults (0.3%) and one of 100 in children (1%). co - administration of valproic acid with lamotrigine significantly increases the risk for development of serious adverse cutaneous reactions. the elimination half life of lamotrigine when used as monotherapy is 25 - 30 hours and it is more than doubled to 60 hours when combined with valproic acid. it has also been suggested that a high starting dose and a rapid dose escalation may increase the occurrence of skin rashes with lamotrigine. the risk of allergic reactions is decreased when treatment is started at a low dose and the doses are increased gradually following the recommended dosage guidelines, possibly because slow titration may allow desensitization to occur. both of our patients were on valproic acid when lamotrigine was initiated as an add - on therapy. high starting doses were employed and dose was also rapidly increased in both the cases. history of a rash attributed to another antiepileptic drug results in a five times greater increase in the rate of an antiepileptic drug rash. hirsch. have concluded that a rash with another antiepileptic drug is the greatest risk factor for developing a rash with lamotrigine. in the first patient the phenomenon of cross reactivity may be a general feature of patients predisposed to drug rashes. also a patient with a prior medication related rash would be more vigilant and likely to notice a drug related rash from a new medication. females have a higher risk for antiepileptic skin reactions compared to males ; probably due to hormonal factors. a causality assessment using naranjo probability scale was performed in both the patients that revealed a probable association between lamotrigine and toxic epidermal necrolysis in both of the cases. our second patient was on several medications (valproic acid, propranolol, clomipramine, risperidone) since two years. these drugs have a very low potential for causing serious drug reactions like toxic epidermal necrolysis. the rates of skin rash with valproic acid are very low as compared to lamotrigine. thus it is unlikely that they were responsible for causing toxic epidermal necrolysis in the second patient. the international prescribing guidelines regarding use lamotrigine as an adjunctive treatment with valproic acid, recommend that lamotrigine should be initiated at a dose of 12.5 mg once daily for first two weeks, followed by 25 mg once daily for next two weeks, and increased thereafter by 25 mg every other week until a maintenance dose of 100 - 200 mg daily. it must be pointed out, however, that serious reactions can occur with recommended dosing and titration schedules, and without concurrent valproic acid. increased vigilance is thus required when initiating lamotrigine in a patient and treatment should be discontinued if any rash appears. when lamotrigine is prescribed as an adjunctive treatment with valproic acid	anti - epileptic drugs can be associated with a wide spectrum of cutaneous adverse reactions ranging from simple maculopapular rashes to more severe and life threatening reactions like stevens - johnson syndrome and toxic epidermal necrolysis. these rashes are well documented with older antiepileptic drugs like phenytoin, phenobarbitone and carbamazapine. lamotrigine is a newer, unrelated antiepileptic drug that causes skin rashes in 3 - 10% of new users. higher starting dose or rapid escalation, concurrent treatment with valproic acid, and a previous history of a rash with other antiepileptic drugs are well recognized risk factors for lamotrigine related serious rashes. we report two patients with toxic epidermal necrolysis, resulting from concomitant use of lamotrigine and valproic acid. it is emphasized that clinicians adhere to the recommended dosage guidelines and adopt a slow dose titration when initiating treatment with lamotrigine.
	ovarian cancer is the leading cause of death among women with gynecological malignancies. acquired resistance to chemotherapy is a major limitation for ovarian cancer treatment. we report here the first use of nanoscale metal organic frameworks (nmofs) for the co - delivery of cisplatin and pooled small interfering rnas (sirnas) to enhance therapeutic efficacy by silencing multiple drug resistance (mdr) genes and resensitizing resistant ovarian cancer cells to cisplatin treatment. uio nmofs with hexagonal - plate morphologies were loaded with a cisplatin prodrug and mdr gene - silencing sirnas (bcl-2, p - glycoprotein [p - gp ], and survivin) via encapsulation and surface coordination, respectively. nmofs protect sirnas from nuclease degradation, enhance sirna cellular uptake, and promote sirna escape from endosomes to silence mdr genes in cisplatin - resistant ovarian cancer cells. co - delivery of cisplatin and sirnas with nmofs led to an order of magnitude enhancement in chemotherapeutic efficacy in vitro, as indicated by cell viability assay, dna laddering, and annexin v staining. this work shows that nmofs hold great promise in the co - delivery of multiple therapeutics for effective treatment of drug - resistant cancers.
the hospitals ' duty is beyond providing clinical and specialized services, and making plans for delivering health care services and promoting health is one of their important duties (1). in each country, from 40% to 70% of health budget is absorbed by and spent in the hospitals and usually one to three percent of working population is employed in them (2). on the other hand, the employees in hospitals suffer from high levels of job stress (3). therefore, the plan of health promoting hospitals (hph) was launched more than a decade ago (45). this plan refers to the fact that the hospital activities should be aimed not only at therapeutic and diagnostic activities (6) but also at disease prevention and health promotion, and the hospital services should be targeted to the needs of the people (7). hence, developing a new strategy for hospital services and paying attention to the health promotion programs in the hospitals are essential in the present century, today, the hospitals are required which consider health promotion programs as a key service (89). the issue of hph is paid more attention due to the increasing prevalence of lifestyle - related diseases and also chronic diseases, the changes in public expectations, the increasing number of chronic patients who need continuous support, and the number of hospital staff who daily exposure to psychological pressures and health risks (10). on the other hand, health promotion is a concept that should be considered as a behavioral challenge (11). in this context, and given the current problems in hospitals, 20 hospitals were selected officially by who from 11 european countries for a pilot project of ephp (european pilot hospital project of hph) in warsaw in april 1993 (12) and the international network of health promotion hospital was established (13). these hospitals not only provide comprehensive and high quality medical and nursing services but also develop an organizational structure and culture assuming an active role for patients and staff in health promotion programs and actively cooperate with the community in health activities (2). hospitals with the health promotion plan should take several measures to implement health promotion programs such as using organizational development processes, forming a joint committee of health promotion projects, providing regular reports by subgroups of the health promotion project, providing feedback, using public relations techniques, reporting and documentation systems (14). in the model of hph, provided by the research and development center of hph in taiwan (11), at first, the preliminary measures, including formation of a project implementation team and a health promoting hospital committee were taken and, then, the project planning and implementation were made. in many health promotion programs implemented in hospitals, other health promotion projects for implementing health promotion programs in the hospitals focus on managerial methods, sharing and using resources (17), and collaboration with society organizations (18). finally, many hospitals in the world have implemented the who hph program and have achieved cost - effectiveness and quality assurance of health services (1920). however, only the peripheral levels of the health network system in iran play the role of prevention and hospitals are responsible for performing the traditional role of diagnosis and treatment and there is no clear plan to deliver health promotion services in the hospitals. although some of these services such as nutritional counseling are provided sporadically in some hospitals in iran, there has not been any defined structure for becoming a hph and very few studies (18, 21) have been conducted in this field. therefore, this study aimed to determine factors affecting the establishment of a health promoting hospital in iran using factor analysis method. this study was done in 2013, as an applied, cross - sectional and descriptive - analytical study in four steps, as follows : to develop a conceptual model of hph, the models of establishing and implementing health promotion programs, the executable models and the experiences of who - european pilot hospital project were reviewed and finally, the main dimensions of the model as a conceptual model were extracted. after determining the main dimensions of implementing health promotion project in hospitals, the validity and reliability of this questionnaire were approved using expert judgment and cronbach 's alpha (22). first, four medical universities of medical sciences in four provinces of iran (tehran, guilan, isfahan, shiraz) were randomly selected using cluster sampling method. then, in the selected medical universities, a sample of 268 people was determined using the findings of pilot study and the following formula, assuming =0.1, d= 0.1 and p = q= 0.5, and was randomly selected among faculty members, managers and experts who had scientific and academic, administrative and managerial experiences in health promotion. n=(z2)(pq)d2 n=(1/64)2(pq)0/01s2=268 the measurement model was tested using confirmatory factor analysis (cfa) via amos graphics (18). cfa is a structural equation modeling technique which is used to determine the model 's goodness of fit. in order to handle any missing data, evaluation of each model in cfa is based on considering a variety of fit indices. there are three categories of fit indices for model evaluation, including : 1) absolute fit indices, including gfa (goodness of fit), agfi (adjusted goodness of fit) and rmsea (root mean square error of approximation) ; 2) comparative (incremental) fit indices, including nfi (normed fit index) and nnfi (non - normed fit index) ; and 3. it has been suggested that researchers should report at least two indices from each category. if cfi, gfi, nfi, nnfi, ifi, rfi, and agfi are higher than 0.90 and rmsea and rmsri are less than 0.050, the studied model will have desirable and appropriate fitness. to develop a conceptual model of hph, the models of establishing and implementing health promotion programs, the executable models and the experiences of who - european pilot hospital project were reviewed and finally, the main dimensions of the model as a conceptual model were extracted. after determining the main dimensions of implementing health promotion project in hospitals, a questionnaire was designed. the validity and reliability of this questionnaire were approved using expert judgment and cronbach 's alpha (22). first, four medical universities of medical sciences in four provinces of iran (tehran, guilan, isfahan, shiraz) were randomly selected using cluster sampling method. then, in the selected medical universities, a sample of 268 people was determined using the findings of pilot study and the following formula, assuming =0.1, d= 0.1 and p = q= 0.5, and was randomly selected among faculty members, managers and experts who had scientific and academic, administrative and managerial experiences in health promotion. the measurement model was tested using confirmatory factor analysis (cfa) via amos graphics (18). cfa is a structural equation modeling technique which is used to determine the model 's goodness of fit. in order to handle any missing data, evaluation of each model in cfa is based on considering a variety of fit indices. there are three categories of fit indices for model evaluation, including : 1) absolute fit indices, including gfa (goodness of fit), agfi (adjusted goodness of fit) and rmsea (root mean square error of approximation) ; 2) comparative (incremental) fit indices, including nfi (normed fit index) and nnfi (non - normed fit index) ; and 3. it has been suggested that researchers should report at least two indices from each category. if cfi, gfi, nfi, nnfi, ifi, rfi, and agfi are higher than 0.90 and rmsea and rmsri are less than 0.050, the studied model will have desirable and appropriate fitness. most of the studied sample were managers (0.42), male (62%), in the 4150 yr age group (46%), in the 2130 yr job experience group (48.2%), and had a master 's degrees (70%) (table 1). six dimensions were determined as the dimensions of hph establishment which were as follows (table 2) : society and community assessment consisting of community needs assessment and epidemiological assessment (i.e. the assessment of population health issues and disease patterns) as its sub-dimensions.policy consisting of setting policies, implementing health promotion projects, attracting adequate funds and determining the related investors and sponsors for the implementation of health promotion projects, and mentioning the concept of health promotion in the hospital mission and goals as its sub-dimensions.management consisting of designing the projects and interventions required for a hph, creating and providing strategic leadership (providing technical support for implementing projects), and developing strategies for implementing health promotion projects using data collected from society and policy dimensions.dissemination which means publishing health promotion programs and interventions and their results at international conferences and news media and arranging business meetings on health promotion in the hospital.technique. in order to implement a hph program, hospitals should have appropriate structures and techniques which the simplest one of them is the formation of a project team. another appropriate structure for implementing health promotion programs is a combination of project committees, project management, and project coordinator.evaluation. the purpose of this step is monitoring and evaluation of progress of each activity related to the implementation of health promotion programs. table 2:the means and standard deviations (sds) of hph model dimensionssocietypolicymanagementdisseminationtechniqueevaluation2.9.893.3.543.11.043.3.582.8.924.2.53 society and community assessment consisting of community needs assessment and epidemiological assessment (i.e. the assessment of population health issues and disease patterns) as its sub - dimensions. policy consisting of setting policies, implementing health promotion projects, attracting adequate funds and determining the related investors and sponsors for the implementation of health promotion projects, and mentioning the concept of health promotion in the hospital mission and goals as its sub - dimensions. management consisting of designing the projects and interventions required for a hph, creating and providing strategic leadership (providing technical support for implementing projects), and developing strategies for implementing health promotion projects using data collected from society and policy dimensions. dissemination which means publishing health promotion programs and interventions and their results at international conferences and news media and arranging business meetings on health promotion in the hospital. technique. in order to implement a hph program, hospitals should have appropriate structures and techniques which the simplest one of them is the formation of a project team. another appropriate structure for implementing health promotion programs is a combination of project committees, project management, and project coordinator. the purpose of this step is monitoring and evaluation of progress of each activity related to the implementation of health promotion programs. the means and standard deviations (sds) of hph model dimensions among the dimensions of hph model, technique (2.80.92) and evaluation (4.20.53) had the highest and lowest means, respectively (table 2). moreover, society (0.97) and policy (0.74) had the highest regression coefficient (effects) and management had the lowest one (fig. 1). according to the results of model 's goodness of fit indices, all indices were within the acceptable range showing that a significant amount of variance had been assigned by the model. therefore, the conceptual model used in the present study was a valid and acceptable model (table 3). the hph model fit summary incremental index of fit / comparative fit index / chi - square /degree of freedom / parsimony normed of fit index / parsimony comparative fit index / root mean square error of approximation the results of the present study showed that the hph model had dimensions, including society, policy, management, dissemination, technique, and evaluation. in the current study, society with the highest regression weight (0.97) had the greatest impact on the health promotion in hospitals indicating the importance of this dimension (23). community - based health care requires the society members ' involvement in improving their health. the reason for recognizing and understanding the community is that by collecting data from the assessment of community covered by the hospital, administrators and policy - makers can determine the health problems and preferences in the society. in an intervention program implemented in a hospital in south africa to move it towards a health promoting hospital, the needs assessment of staff, patients and their relatives was carried out at first, which is similar to the health and epidemiological needs assessment of the society groups in the present study (16). the precede - proceed model is used to assess the perceptions of society members and to recognize the society 's health issues (24), which is similar to the results of present study. the planned approach to community health focuses on the society 's facilities and possibilities assessment. in the present study, also, the assessment of society 's status has been one of the dimensions (24). in some diseases such as cancer (a specific disease), according to patients ' survival rate and the importance of increasing their longevity, it is necessary to determine the information needs of the patients ' health promotion (25). in the current study, the toronto health promotion model (26) has studied the legal and political environment, stakeholders, population health needs, previous assessments, and the overall outlook for each project. in the present study, also, the political environment and its assessment has been paid attention. bangkok declaration (27) considers the existence of policy - making and participation as a central principle of national development in order to improve health and health equity. this can also be seen in the current study as the dimension of policy. for implementing hph programs, access to adequate budget (2831)and the health promotion policy (28, 31) are important. using data obtained from the society and making the required policies, hospitals can develop and implement programs and interventions for health promotion (the dimension of management). miseviciene and colleagues (32) have focused on the development of a strategic plan for implementing health promotion programs, which is similar to the dimension of management in the present study. in order to implement this dimension, providing strategic leadership (providing technical support for implementing projects) has been emphasized in the present study, which is similar to the results of lee and colleagues ' study (33). in addition, johnson and baum (29) in their study concluded that top management support was important and necessary for implementing hph programs. to establish a hph, in fact, an efficient and effective hph is dependent on organizational and managerial prerequisites such as project management, etc. (35), which is similar to the findings of the present study. in preston green hospital, the project management principles and appropriate organizational structure and processes have been used in order to achieve the objectives of health promotion (14). in the present study, the effect of project implementation techniques on health promotion in the hospital with 0.53 loading factor was significant. the administrators of rudolf stiftung hospital have taken several measures to implement health promotion programs in their hospital such as employing the techniques of project implementation (23), consistent with the results of present study. the results of other studies have supported the organizational structures as a prerequisite for establishing a comprehensive hph (36), which confirm the results of current study. in the 2-year evaluation of the implementation and effects of a hph program in scotland, the existence of a framework and structure for implementing the health promotion in hospitals has been emphasized (37). the hph coordinator is the minimum structure required for the membership of hospitals in the hph network (45). the existence of a structure and framework for implementing health promotion programs in the present study has been referred to as technique. the health promotion program in hospitals in taiwan (15) has had four phases, including the formation of a project implementation team, formation of a health promotion committee, project planning and implementation, and evaluation of the effects of health promotion programs. the administrators of be jing hospital (28) have considered the dimensions of policy, reorientation of health care services, society, and health skills as the health promotion activities (28) which are similar to the dimensions of the current study conceptual model, as well as the design and implementation phases of health promotion in the proposed model of kouranie hospital (14). in the present study, dissemination (publishing health promotion programs and activities and their results at international conferences and news media and arranging business meetings on hph programs) with a good loading factor (0.64) had a high impact on the implementation of health promotion programs in the hospital. the results of a study in taiwan have shown the similar results and have indicated that dissemination in the hph local and international conferences plays an important role in the establishment of a hph (38). therefore, it can be said that if a hospital implement the health promotion programs well but is weak in the dissemination, it has not been able to introduce itself as a health promoting hospital. in a seven - dimension model developed by burke. (39), which shows the needs for changing the organizational capacities to become a health promoting hospital, the development of mission and strategies, leadership and managerial activities have also been mentioned, which are similar to the results of the present study in which mentioning the concept of health promotion in the hospital mission and goals and developing strategies have been stated in the dimension of policy, and leadership and managerial activities in the dimension of management. like other studies, in the current study, the existence of planning and a specific technique and framework such as project management technique have been concluded as a dimension. some of obstacles to the implementation of health promotion programs include inefficient project management, the lack of communication, coordination and integration, and the lack of planning, guidelines, standards and frameworks for implementing health promotion programs (18, 29, 37, 40). all factors mentioned above have been stated in the present study as the dimensions of implementing health promotion programs in hospitals. the final dimension in the present study, the evaluation of health promotion programs and interventions, has been confirmed by the model of evaluating health promotion projects presented in the preston hospital (14). although remarkable advances have been made in medical technologies, because of the increasing number of chronic patients who need permanent support and employees who are daily subjected to the psychological pressures, hospital managers and administrators should make plans to develop and implement health promotion programs and ensure healthy workplace in hospitals. the results of the present study showed that paying attention to six dimensions for establishing a hph is essential among which the most and least important ones were society and community assessment and management, respectively. it should be noted that considering all of these dimensions without making systematic planning (40) and organizational changes, providing health education for patients and determining factors affecting the establishment of a hph will not be effective after the researchers explained the purpose and procedures of the study to the participants, they consented to participate in the study. ethical issues (including plagiarism, informed consent, double, etc.) have been completely observed by the authors.	background : hospitals are the central entity of each health care system and health promoting hospitals (hph) was launched by who in 1988. however, there has not been any accurate and detailed model for establishing a hph in iran up to now. therefore, this study aimed to determine factors affecting the establishment of a health promoting hospital in iran using factor analysis method.methods:this applied, cross - sectional and descriptive - analytical study was conducted in iran in four steps. confirmatory factor analysis (cfa) was used for determining factors affecting the establishment of a hph.results:society (0.97) and policy (0.74) had the highest regression weights (effects) and management had the lowest one.conclusion:community assessment was the most important dimension of proposed conceptual model for establishing a hph.
calcium phosphate ceramics have seen extensive clinical application as synthetic bone fillers and graft extenders [13 ]. the biocompatibility as well as osteoconductive and osteoinductive properties of these ceramics have been well documented [412 ]. bone tissue engineering research has capitalized on these qualities, making porous calcium phosphate ceramics a popular choice of scaffold [1316 ]. porous ceramics for medical applications have been manufactured for decades using a variety of traditional methods. conversion of natural structures, such as coral [17, 18 ], and trabecular bone [19, 20 ] yield porous ceramics with organic architectures that appear very similar to that of the bone that is being replaced. synthetic manufacturing methods such as foaming [2123 ], dual - phase mixing and the slip - casting of polymer foams and particles [2527 ], may also be used to produce porous ceramics. however, conversion and synthetic techniques result in highly complex macroporous structures that are difficult to define quantitatively. despite the complex nature of the porous ceramics produced by conventional means, quite some information is available regarding the influence the porous structure has on osteoconduction [2835 ], bmp induced osteogenesis [26, 3638 ], and osteoinduction [11, 39 ]. rapid prototyping (rp), also termed free form fabrication, refers to a variety of technologies capable of producing three - dimensional (3d) physical constructs directly from 3d computer aided models. in recent years, rapid prototyping has been proposed for the production of both scaffolds with controlled porous architectures [4042 ] as well as porous implants with patient specific geometries [4345 ]. several rapid prototyping techniques have been developed to produce ceramic scaffolds for bone tissue engineering research [4649 ]. the aim of the current study was to produce porous ceramic scaffolds from different calcium phosphate materials with sufficiently similar macroporous architectures as to be able to reasonably eliminate the macroporous architecture as a confounding variable in future tissue engineering studies. the scaffolds were produced by casting four different calcium phosphate materials into identical molds produced using a rapid prototyping technique. the resulting macroporous structures as well as the chemistry before and after manufacture were evaluated. briefly, the scaffolds specifications called for an interconnecting network of 400 m square cross - section channels oriented along the orthogonal axes and separated from each other and the exterior by 400 m. six, four, and three channels were incorporated in the x, y, and z axis directions, resulting in overall design dimensions of 5.2 3.6 2.8 mm, respectively. 1. molds, with cavities for the production of six scaffolds each, were designed using the rhinoceros computer aided design software (robert mcneel & associates, usa). the mold model was scaled to account for shrinkage of approximately 20% during thermal processing demonstrated previously by our hydroxyapatite ceramics. this resulted in pre - thermal processing scaffold dimensions of 6.5 4.5 3.5 mm in the x, y, and z axis directions. multiple copies of the mold were produced using a modelmaker ii rapid prototyping system (solidscape inc. 1schematic of the designed scaffolds including the three orthogonal planes used to define the scaffold surfaces schematic of the designed scaffolds including the three orthogonal planes used to define the scaffold surfaces ceramic scaffolds were manufactured to achieve four conditions through combinations of calcium phosphate ceramic compositions and sintering temperatures as outlined in table 1. hydroxyapatite powder (ha, merck, germany), beta - tricalcium phosphate powder (tcp, merck, germany) and biphasic calcium phosphate powder (bcp, wt% 85/15 ha / tcp, isotis sa) were obtained commercially. the ha and tcp raw powders were calcined by heating from ambient to 900c at a rate of 100c / hour and then cooled naturally with no dwell period. aqueous slurries of ha, tcp, and bcp powders were prepared as previously described for the production of cast plates. in brief, the slurry components detailed below were slowly admixed until a homogenous blend was achieved. the ha and tcp slurries consisted of 67.1 wt% calcined ha powder, 28.6 wt% demineralized water, 2.6 wt% ammonia solution (25%, merck), 1.5 wt% deflocculant (dolapix, aschimmer & schwarz gmbh, germany). once a homogeneous blend was obtained, a cmc binder was added (0.15 wt%, pomosin bv, the netherlands) to the ha slurry and the slurry further mixed until homogeneous. the bcp slurry consisted of 56.4 wt% ceramic powder, 37.6 wt% demineralized water, 3.9 wt% ammonia solution, and 2.1 wt% deflocculant. all slurries were stored in covered beakers until there use within the same day.table 1scaffold dimensions, shrinkage, volume, weight, and apparent porosity of scaffolds compared to the solids in slurry and sintering temperature during manufacturingmaterialsolids in slurry (wt%)sintering temp. (c)exterior dimensions (mm sd)srinkage (from as molded, %) volume (mm sd)weight (mg sd)apparent porosity (% sd)ha h67.11250x : 5.05 0.05x : 22.3547.92 0.8776.16 2.8949.65 1.76y : 3.48 0.03y : 22.68z : 2.73 0.03 ha l67.11150x : 6.14 0.05x : 5.4884.63 3.1773.82 5.4072.39 1.27y : 4.23 0.13y : 6.04z : 3.26 0.11bcp56.41150x : 5.45 0.05x : 16.2158.96 1.3848.86 2.3373.72 1.03y : 3.73 0.06y : 7.86z : 2.90 0.06tcp67.11150x : 6.05 0.04x : 6.8880.53 3.0569.49 4.5472.54 1.10y : 4.15 0.13y : 7.86z : 3.21 0.09 scaffold dimensions, shrinkage, volume, weight, and apparent porosity of scaffolds compared to the solids in slurry and sintering temperature during manufacturing the molds were filled using a simple vacuum device. france) were divided in half and the filter paper removed to expose the perforated interior surface. the open face of a mold was carefully placed against the interior surface of a filter half and secured by circumferentially wrapping with wax laboratory film. the mold / filter constructs were attached to 50 ml syringes and flushed with demineralized water. during casting, the beakers containing slurry were placed on a porex vibrating table (renfert, germany). this assisted in mold filling by imparting shear energy and thus lowering the viscosity of the pseudoplastic (shear thinning) slurries. the molds were filled by submerging the open face of each mold in slurry and then drawing vacuum pressure using the syringe. the molds were then placed on a sheet wax laboratory film and the syringes and filter halves removed. the molds were allowed to air dry overnight at room temperature and were then further dried for 24 h at 50c in air. excess slurry from each ceramic composition was processed identically to the molded ceramics to serve as controls when examining material chemistry and to further examine the previously observed influence of the rapid prototyping wax on the material composition. debinding and sintering of the ceramics were performed in two steps in a high temperature furnace (nabertherm 1400, germany). debinding of all ceramics was performed by heating at a rate of 0.5c / minute to 400c and then cooling naturally with no dwell period. the ceramics were then sintered using a 600 min heating phase with a 480 min dwell period at the final sintering temperature followed by natural cooling. one set of ha scaffold was sintered at 1250c, designate ha h, while a sintering temperature of 1150c was used for a second set of ha scaffolds, designate ha l, as well as all of the tcp and bcp scaffolds. excess ceramic, occasionally present on the scaffold faces corresponding to the open sides of the molds, was removed using a rotary polisher (labopol-5, struers, denmark) with 1200 grit waterproof silicon carbide paper (struers). the ceramics were cleaned by ultrasound for 15 min each in acetone, 100% ethanol and deionized water, and then dried in air at 50c. the exterior scaffold dimensions were measured using a digital caliper (cd-15c, mitutoyo ltd., uk) and used to calculate the shrinkage resulting from the combined debinding and sintering processes. scanning electron microscopy (sem, xl 30 esem - feg, philips, the netherlands) was used to examine the macro - architecture and surface micro - structure of the scaffolds. the dimensions of the macroporosity were measured in each of the orthogonal planes (fig. 1). the apparent porosity of the scaffolds was determined by comparing the apparent density of each scaffold (dry weight / measured volume) and the theoretical density of ha (3.156 g / cm), tcp (3.14 g / cm3), and bcp (85% ha, the chemistry of raw ceramic powder, calcined ceramic powder, non - molded sintered ceramic and molded scaffolds were evaluated by x - ray diffraction (xrd, miniflex, rigaku, japan). finally, the potential contamination of the ceramics by residues from the wax mold material was investigated by performing energy - dispersive x - ray spectroscopy (edx, xl 30 esem - feg, philips, the netherlands) on the surface of cast and non - cast (not exposed to wax mold material) ceramics specimens. briefly, the scaffolds specifications called for an interconnecting network of 400 m square cross - section channels oriented along the orthogonal axes and separated from each other and the exterior by 400 m. six, four, and three channels were incorporated in the x, y, and z axis directions, resulting in overall design dimensions of 5.2 3.6 2.8 mm, respectively. 1. molds, with cavities for the production of six scaffolds each, were designed using the rhinoceros computer aided design software (robert mcneel & associates, usa). the mold model was scaled to account for shrinkage of approximately 20% during thermal processing demonstrated previously by our hydroxyapatite ceramics. this resulted in pre - thermal processing scaffold dimensions of 6.5 4.5 3.5 mm in the x, y, and z axis directions. multiple copies of the mold were produced using a modelmaker ii rapid prototyping system (solidscape inc. 1schematic of the designed scaffolds including the three orthogonal planes used to define the scaffold surfaces schematic of the designed scaffolds including the three orthogonal planes used to define the scaffold surfaces ceramic scaffolds were manufactured to achieve four conditions through combinations of calcium phosphate ceramic compositions and sintering temperatures as outlined in table 1. hydroxyapatite powder (ha, merck, germany), beta - tricalcium phosphate powder (tcp, merck, germany) and biphasic calcium phosphate powder (bcp, wt% 85/15 ha / tcp, isotis sa) were obtained commercially. the ha and tcp raw powders were calcined by heating from ambient to 900c at a rate of 100c / hour and then cooled naturally with no dwell period. aqueous slurries of ha, tcp, and bcp powders were prepared as previously described for the production of cast plates. in brief, the slurry components detailed below were slowly admixed until a homogenous blend was achieved. the ha and tcp slurries consisted of 67.1 wt% calcined ha powder, 28.6 wt% demineralized water, 2.6 wt% ammonia solution (25%, merck), 1.5 wt% deflocculant (dolapix, aschimmer & schwarz gmbh, germany). once a homogeneous blend was obtained, a cmc binder was added (0.15 wt%, pomosin bv, the netherlands) to the ha slurry and the slurry further mixed until homogeneous. the bcp slurry consisted of 56.4 wt% ceramic powder, 37.6 wt% demineralized water, 3.9 wt% ammonia solution, and 2.1 wt% deflocculant. all slurries were stored in covered beakers until there use within the same day.table 1scaffold dimensions, shrinkage, volume, weight, and apparent porosity of scaffolds compared to the solids in slurry and sintering temperature during manufacturingmaterialsolids in slurry (wt%)sintering temp. (c)exterior dimensions (mm sd)srinkage (from as molded, %) volume (mm sd)weight (mg sd)apparent porosity (% sd)ha h67.11250x : 5.05 0.05x : 22.3547.92 0.8776.16 2.8949.65 1.76y : 3.48 0.03y : 22.68z : 2.73 0.03 ha l67.11150x : 6.14 0.05x : 5.4884.63 3.1773.82 5.4072.39 1.27y : 4.23 0.13y : 6.04z : 3.26 0.11bcp56.41150x : 5.45 0.05x : 16.2158.96 1.3848.86 2.3373.72 1.03y : 3.73 0.06y : 7.86z : 2.90 0.06tcp67.11150x : 6.05 0.04x : 6.8880.53 3.0569.49 4.5472.54 1.10y : 4.15 0.13y : 7.86z : 3.21 0.09 scaffold dimensions, shrinkage, volume, weight, and apparent porosity of scaffolds compared to the solids in slurry and sintering temperature during manufacturing france) were divided in half and the filter paper removed to expose the perforated interior surface. the open face of a mold was carefully placed against the interior surface of a filter half and secured by circumferentially wrapping with wax laboratory film. the mold / filter constructs were attached to 50 ml syringes and flushed with demineralized water. during casting, the beakers containing slurry were placed on a porex vibrating table (renfert, germany). this assisted in mold filling by imparting shear energy and thus lowering the viscosity of the pseudoplastic (shear thinning) slurries. the molds were filled by submerging the open face of each mold in slurry and then drawing vacuum pressure using the syringe. the molds were then placed on a sheet wax laboratory film and the syringes and filter halves removed. the molds were allowed to air dry overnight at room temperature and were then further dried for 24 h at 50c in air. excess slurry from each ceramic composition was processed identically to the molded ceramics to serve as controls when examining material chemistry and to further examine the previously observed influence of the rapid prototyping wax on the material composition. debinding and sintering of the ceramics were performed in two steps in a high temperature furnace (nabertherm 1400, germany). debinding of all ceramics was performed by heating at a rate of 0.5c / minute to 400c and then cooling naturally with no dwell period. the ceramics were then sintered using a 600 min heating phase with a 480 min dwell period at the final sintering temperature followed by natural cooling. one set of ha scaffold was sintered at 1250c, designate ha h, while a sintering temperature of 1150c was used for a second set of ha scaffolds, designate ha l, as well as all of the tcp and bcp scaffolds. excess ceramic, occasionally present on the scaffold faces corresponding to the open sides of the molds, was removed using a rotary polisher (labopol-5, struers, denmark) with 1200 grit waterproof silicon carbide paper (struers). the ceramics were cleaned by ultrasound for 15 min each in acetone, 100% ethanol and deionized water, and then dried in air at 50c. the exterior scaffold dimensions were measured using a digital caliper (cd-15c, mitutoyo ltd., uk) and used to calculate the shrinkage resulting from the combined debinding and sintering processes. scanning electron microscopy (sem, xl 30 esem - feg, philips, the netherlands) was used to examine the macro - architecture and surface micro - structure of the scaffolds. the dimensions of the macroporosity were measured in each of the orthogonal planes (fig. 1). the apparent porosity of the scaffolds was determined by comparing the apparent density of each scaffold (dry weight / measured volume) and the theoretical density of ha (3.156 g / cm), tcp (3.14 g / cm3), and bcp (85% ha, 15% tcp). the chemistry of raw ceramic powder, calcined ceramic powder, non - molded sintered ceramic and molded scaffolds were evaluated by x - ray diffraction (xrd, miniflex, rigaku, japan). finally, the potential contamination of the ceramics by residues from the wax mold material was investigated by performing energy - dispersive x - ray spectroscopy (edx, xl 30 esem - feg, philips, the netherlands) on the surface of cast and non - cast (not exposed to wax mold material) ceramics specimens. the manufacturing process resulted in scaffolds with remarkably similar structural appearances (fig. 2). scaffold dimensions, shrinkage, volume, weight and apparent porosity values are summarized in table 1. the bcp scaffolds also demonstrated considerable shrinkage but maintained a high apparent porosity similar to the low sintering temperature ha and tcp scaffolds. the low sintering temperature ha and tcp scaffolds exhibited the lowest shrinkage. shrinkage in the z - direction and volumetric shrinkage were not calculated since the respective surfaces were manually polished to remove excess ceramic and therefore do not represent the as cast properties. in order to evaluate whether the various treatments influenced the ratio of macroporosity to total porosity, computer models of the porous scaffolds were created using the measured exterior and macropore dimensions in tables 1 and 2, respectively. table 3 shows the volumes approximated by the computer models for the various treatments and compares the resulting macroporosity to the measured apparent porosity.fig. note the similarity of the scaffold structures and the differences in the scaffold colorstable 2pore dimensions by orthogonal plane (fig. 1)materialpore dimensions (m sd)x y planex z planey z planeha hx : 286 15x : 353 28y : 394 24y : 280 16z : 339 17z : 376 30 ha lx : 414 44x : 470 37y : 484 29y : 416 34z : 496 21z : 486 27bcpx : 366 24x : 444 47y : 432 37y : 377 18z : 433 20z : 414 42tcpx : 405 43x : 460 36y : 474 29y : 408 33z : 486 21z : 476 26table 3scaffold volumes and macroporosity calculated form computer models compared to the measured total apparent porosity and other porosity (difference between macro and apparent porosity)materialtotal volume (mm)material volume (mm)pole volume (mm)macro - porosity (%) apparent porosity (%) other porosity (%) ha h47.9831.7816.1933.7549.6515.90 ha l84.6750.1934.4840.7272.3931.67bcp58.9534.8224.1440.9473.7232.78tcp80.6048.0832.5140.3472.5432.20 the four ceramic compositions all in 25 well plates. note the similarity of the scaffold structures and the differences in the scaffold colors pore dimensions by orthogonal plane (fig. 1) scaffold volumes and macroporosity calculated form computer models compared to the measured total apparent porosity and other porosity (difference between macro and apparent porosity) sem images of the resulting scaffolds are shown in fig. 3. these scaffolds are discussed in the following text using the axes and orthogonal planes depicted in fig. 1. a distinctive texture of parallel ridges and valleys was observed in sem micrographs on all vertical scaffold surfaces, i.e., surfaces parallel to the x z and y z planes. this texture is an impression of the rapid prototyped mold and a consequence of the layer - by - layer manufacturing of the mold. the cross - sectional geometry of the channels was dependant upon the orientation of the channel. channels running in the x- and y - directions were square in cross section with textured vertical surfaces and smooth horizontal surfaces. rows top to bottom ha h, ha l, bcp, and tcp. first column perspective view of scaffolds at 50 magnification (bar 50 m). fourth column scaffold surfaces at 1000 magnification (bar 20 m) sem micrographs of the four scaffold materials. rows top to bottom ha h, ha l, bcp, and tcp. first column perspective view of scaffolds at 50 magnification (bar 50 m). second column scaffold structures at 100 magnification (bar 200 m). note the regular surface texture on the scaffolds. fourth column scaffold surfaces at 1000 magnification (bar 20 m) the surface microporosity of the scaffolds, as observed by sem, varied with material composition and sintering temperature (fig. 3). the bcp scaffolds exhibited a spectrum of surface microporosity features from approximately 1 to 10 m in size. the surface features of the low sintering temperature ha scaffolds were similar to the bcp with perhaps somewhat less and smaller surface microporosity, approximately 0.55 m. the tcp material, in contrast to the other materials sintered at low temperatures, appeared very similar to the high sintering temperature ha with very little surface microporosity. the xrd patterns for the different ceramic chemistries, shown in fig. 4, were generally as expected. figure 4a shows the patterns for the ha ceramic raw powder, calcined powder, non - molded material sintered at 1150c, scaffolds sintered at 1150c, non - molded material sintered at 1250c, and scaffolds sintered at 1150c. several peaks associated with tcp formation were observed in the patterns for the cast ha scaffold materials at both the 1150 and 1250c sintering temperatures (vertical lines in fig. the bcp ceramics also demonstrated these tcp peaks in the cast scaffolds (fig. 4b, vertical lines). the tcp ceramics exhibited changes, relative to the raw powder, that were consistent with the calcination and sintering process temperatures (fig. the xrd patterns for the four scaffold conditions are shown in fig. 5 for clarity. edx of the surfaces of both cast and non - cast ceramic specimens showed identical spectra consistent with the calcium phosphate materials. 4xrd patterns of a ha h (1250) and ha l (1150), b bcp, and c tcp. shown are xrd patterns of the raw powder, calcined powder and molded ceramics (scaffolds). xrd patterns of non - molded ceramics are also shown in (a) for the ha h (1250) and ha l (1150) materials. vertical dotted lines indicate additional peaks associated with beta - tcp formation that are only present in the molded specimens (scaffolds)fig. 5xrd patterns of the four scaffold materials. vertical dotted lines indicate beta - tcp peaks which form in the ha and bcp material as a result of the molding process xrd patterns of a ha h (1250) and ha l (1150), b bcp, and c tcp. shown are xrd patterns of the raw powder, calcined powder and molded ceramics (scaffolds). xrd patterns of non - molded ceramics are also shown in (a) for the ha h (1250) and ha l (1150) materials. vertical dotted lines indicate additional peaks associated with beta - tcp formation that are only present in the molded specimens (scaffolds) xrd patterns of the four scaffold materials. vertical dotted lines indicate beta - tcp peaks which form in the ha and bcp material as a result of the molding process the present study demonstrates the application of computer aided design and rapid prototyping technologies for the production of ceramic scaffolds from different chemistries but with defined, virtually identical, macro - architectures. other than producing macroporosities with pore dimension in the range suggested in the literature for osteoconduction, i.e., between 50 and 500 mm [4, 28, 30, 31, 34 ], we did not attempt to produce optimal or ideal porous structures. the purpose of this study was to manufacture porous scaffolds in which the macroporous architecture was designed and sufficiently similar to be able to reasonable exclude the macroporous architecture as a confounding variable in future research studies. the material chemistries and thermal processing methods employed in this study were chosen to provide continuity with materials used in past and ongoing research [4954 ]. although the visual appearance of the scaffolds was similar with regard to structure, there were differences in shrinkage and therefore the macroporous dimensions. as expected from our previous work, a sintering temperature of 1250c resulted in a shrinkage of just over 22% for the ha material compared to approximately 6% shrinkage for ha and tcp sintered at 1150c. the relatively large shrinkage of 1617% for the bcp scaffolds, also sintered at 1150c, can almost completely be accounted for by the lower solids loading of the bcp slurry (56.4 wt%) compared to the ha and tct slurries (67.1 wt%). the lower solids loading was necessary to achieve appropriate rheological properties for the casting of scaffolds from slurries of the non - calcined bcp powder. interestingly, the apparent porosity of bcp scaffolds was very similar to the tcp and low sintering temperature ha scaffolds despite the much higher shrinkage (table 2). comparing the porosity resulting from the measured macroporous structure to the total apparent porosity, table 3, reveals that the a much greater proportion of the apparent porosity of the high sintering temperature ha is likely due to the macroporosity compared to the lower sintering temperature materials. the bcp, tcp and low sintering temperature ha all had similar proportions of macroporosity despite the much higher shrinkage of the bcp material. the texture exhibited on the vertical surfaces of the scaffold (fig. 1), as well as the rounded nature of the macropores in the z - direction, are a consequence of the rapid prototyping technique used to manufacture the molds. this technique jets molten droplets of wax material, which flatten and spread when they strike the surface, to build each layer of the molds. as molds are built up vertically layer by layer, this results in a texture on the vertical surfaces (x z and y z planes) which is subsequently cast into the ceramic. the rounded corners of channels running in the z - direction (cross - sections parallel to the x y plane) result from the coalescing or pooling of adjacent droplets prior to solidification, resulting in rounding of both inside and outside corners within the printed layers. these rounded mold corners are then cast into the resulting ceramic scaffolds and observed in channels running parallel to the z - direction. ha and bcp scaffolds indicated that a tcp phase had been introduced. since the xrd patterns of the non - molded specimens did not show the tcp phase and the molded and non - molded materials were treated identically with the exception of the molding process, it is likely that the presence of tcp phase after molding results from the exposure of the ha and bcp materials to the wax mold material itself, despite elemental analysis of cast and non - cast specimens demonstrated there was no direct contamination of the ceramics by the mold material. the mechanism for this is not clear but is consistent with our previous findings for ha materials. in conclusion, we have demonstrated a rapid prototyping method for fabricating ceramic scaffolds with virtually identical, 3-dimensional, macroporous architectures from different calcium phosphate ceramics. scaffolds produced by this method will not only enhance research aimed at optimizing macroporous architectures and material compositions but will improve many other aspects of tissue engineering research by eliminate differences in macroporous structure as a confounding variable.	calcium phosphate ceramics, commonly applied as bone graft substitutes, are a natural choice of scaffolding material for bone tissue engineering. evidence shows that the chemical composition, macroporosity and microporosity of these ceramics influences their behavior as bone graft substitutes and bone tissue engineering scaffolds but little has been done to optimize these parameters. one method of optimization is to place focus on a particular parameter by normalizing the influence, as much as possible, of confounding parameters. this is difficult to accomplish with traditional fabrication techniques. in this study we describe a design based rapid prototyping method of manufacturing scaffolds with virtually identical macroporous architectures from different calcium phosphate ceramic compositions. beta - tricalcium phosphate, hydroxyapatite (at two sintering temperatures) and biphasic calcium phosphate scaffolds were manufactured. the macro- and micro - architectures of the scaffolds were characterized as well as the influence of the manufacturing method on the chemistries of the calcium phosphate compositions. the structural characteristics of the resulting scaffolds were remarkably similar. the manufacturing process had little influence on the composition of the materials except for the consistent but small addition of, or increase in, a beta - tricalcium phosphate phase. among other applications, scaffolds produced by the method described provide a means of examining the influence of different calcium phosphate compositions while confidently excluding the influence of the macroporous structure of the scaffolds.
the peptides fdnpvyqktt, fdnpvy, fdnpvsqktt, fdnpvs, fdnpva, fdnpvf, and fdnpvl were synthesized using standard fmoc chemistry with a free cooh terminus and an acylated nh2 terminus. crude peptides were purified by reverse - phase hplc on a c18 column (vydac, hesperia, ca), equilibrated in h2o with 0.1% trifluoroacetic acid, and eluted with a linear gradient of acetonitrile. the identities of the peptides were confirmed by mass spectrometry and amino acid analysis. concentrated stock solutions of peptides were made by dissolving the peptide in d2o and adjusting the ph to 6.0 with aliquots of concentrated naoh. the coat proteins were then gel - filtered over a superdex 200 column (pharmacia biotech, inc., clathrin and ap fractions were pooled and loaded directly onto an anion exchange column (source q 15 ; pharmacia biotech, inc.) equilibrated in 20 mm ethanolamine, ph 9.0, and eluted with a steep linear gradient of 1 m nabr. clathrin and ap fractions were pooled and concentrated using centriprep 30 (amicon corp., easton, tx). concentrated proteins were polymerized (25) by extensive dialysis against nmr sample buffer (40 mm d4-malonic acid [cambridge isotope labs, andover, ma ], ph 6.0, 0.2 mm edta, 0.2 mm dtt, and 3 mm nan3). protein concentration of the polymerized cage complex was determined using the bradford reagent (bio - rad laboratories, hercules, ca). for preparation of cages composed of clathrin or clathrin and ap2, an additional anion exchange chromatography step was performed to separate clathrin from ap2 before gel filtration. ap-2 containing fractions from the second anion exchange step were then pooled on the basis of coomassie blue stained sds - page. the clathrin fraction was divided in half, and all of the ap-2 sample was added to one half of the clathrin. equal total protein amounts of clathrin or clathrin / ap-2 cages were added to each nmr sample, which contained clathrin (9.7 mg / ml) or clathrin (8.5 mg / ml) and ap-2 (1.2 mg/ ml). sample purity was confirmed by coomassie blue stained sds - page gels and by western blotting with an antibody against -adaptin (100/1 ; sigma chemical co., st. the clathrin terminal domain (residues 1579) was expressed as a glutathione - s - transferase (gst) fusion protein in escherichia coli bl21 cells, and was prepared as described (17). glutathione agarose beads (sigma chemical co.) with the bound gst construct were then washed with thrombin cleavage buffer (50 mm tris, 2.5 mm cacl2, 150 mm nacl, ph 7.4). the beads were incubated overnight with gentle agitation in the presence of thrombin (sigma chemcial co.). dtt was added to 10 mm and allowed to incubate an additional 1 h before eluting the cleaved terminal domain construct from the beads. the clathrin hub construct (residues 10741483) was expressed as a hexahistidine construct in e. coli bl21(de) cells as described (29). bacterial lysates were incubated with ni - nta agarose (qiagen inc., valencia, ca) overnight and eluted with 250 mm imidazole in 50 mm tris, ph 7.8. the eluted polypeptides were then loaded directly onto an anion exchange column equilibrated in 30 mm tris, 1 mm edta, ph 7.8, and eluted with a linear gradient of 0.5 m kcl. fractions were pooled on the basis of coomassie blue stained sds - page gels. because the hub polypeptide aggregated in the nmr sample buffer, both the terminal domain and hub polypeptides were dialyzed against 40 mm kpo4, 150 mm kcl, 0.2 mm edta, 3 mm nan3, and 0.2 mm dtt ph 7.2. all spectra were acquired at 500 mhz and 25c in sample buffer containing 10% d2o on a varian unity 500 spectrometer with a 5999.7-hz spectral width using 3-(tetramethylsilyl) propionic acid as a chemical shift reference. 1d spectra were acquired with 128 transients of 64,000 points zero filled to 131,072 points (see fig. 1) or 64 transients of 32,000 points zero filled to 65,536 points (see figs. 4 and 5). line - broadening was measured as the difference of peak widths at half height using the felix 2.30 (biosym technologies) program. the broadening is reported as the line - width of the resonance peak in the presence of cages minus the line - width in the absence of cages. crosspeak intensities were calculated in arbitrary units of volume using the felix 2.30 program from spectra acquired at 60-, 100-, 140-, and 220-ms mixing times. the spectra were acquired in phase - sensitive mode (48) with 2 256 fids of 1024 complex points each. a spectral width of 5999.7 hz the relative kd for wild - type and position 807 mutant peptides from clathrin terminal domains was obtained from measurements of the peak intensities of the phe (3, 5) aromatic protons for each of the peptides in the presence and absence of protein. the free and bound populations were calculated from the following equation : \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\frac{1}{i_{o}}=\frac{p^{f}}{i_{f}}+\frac{p^{b}}{i_{b}}\end{equation}\end{document } where i is the intensity of the free (if), the bound (ib), and observed (io) resonance, and p and p are the free and bound populations, respectively. since peak intensities are inversely proportional to the transverse relaxation rate (1/t2), the population of the free and bound peptide can be estimated from the change in intensity of a given peptide resonance upon addition of clathrin terminal domain if the t2 of the resonance in the free and bound forms is known. the t2 for a protein is approximately proportional to the molecular weight, and from the line - widths observed in 20-kd proteins (3), the expected t2 for a 55-kd protein is 10 ms. the t2 calculated from the line - widths of several resonances of the free peptides was 100 ms. the values of ib were estimated to be if/10, assuming that the t2s of the free and bound resonances are 100 and 10 ms, respectively. therefore, the calculated populations of p and p were then used to obtain kds assuming standard protein ligand equilibrium. since the kds were based on t2 estimates, their absolute values are likely to be inaccurate. however, the relative values of the kds can be compared because they were all obtained by the same method. distances were calculated only for interresidue noes seen in 60 ms mixing time data sets. sequential h to nh noes were not used because they contained contributions from the free peptide. noe distance restraints were calculated from both wild - type hexa- and decapeptide either by using bins of weak (w ; 1.82.3), medium (m ; 1.83.5), and strong (s ; 1.85.0) peaks, or by measured noe volumes calibrated individually for each dimension with the average volume of the phe, tyr, and pro (/) noes corresponding to 1.75. structures were calculated by simulated annealing performed with the programs insightii and nmrchitect (biosym technologies, san diego, ca) incorporating either type of restraint. in short, peptides were heated to 1,000 k, and then the distance and chiral restraints were slowly increased over 30 ps. the peptides were then slowly cooled to 300 k while increasing covalent and nonbond forces over 32 ps. the final structures were then obtained by iteratively minimizing to a final derivative of 0.01 kcal / mol / and simulating 30 ps of restrained dynamics. removal of pseudoatom restraints for tyr (,) and phe() was based on the geometry of an initial set of structures. the final set of 30 restraints was (denotes pseudoatom and distance is denoted as s, m, or w) : ac(me)::f(h1), w ; f(h1) : : d(hn), w ; f(h2)::d(hn), w ; d(h2)::n(hn), w ; n(h1)::p(hs), w ; n(h2)::p(hs), m ; n(h1)::p(hr), w ; n(h2)::p(hr), w ; n(hn) : : p(hr), w ; p(hr)::v(hn), m ; p(hs)::v(hn), w ; p(hs)::v(hn), w ; v(hn)::y(hn), s ; v(hn)::y(h1), w ; v(h1)::y(h1), w ; v(h1) : : y(h1), w ; ac(me)::d(hn), w ; ac(me)::n(h21), w ; ac(me) : : n(h22), w ; v(hn)::n(h1), w ; v(hn)::n(h2), w ; n(h1)::y(h2), w ; n(h2)::y(h1), w ; p(hs)::y(h1), w ; p(hs)::y(h1), w ; p(hs) : : y(hn), m ; p(hr)::y(hn), w ; p(hr)::y(h1), w ; p(hr)::y(h1), w ; the peptides fdnpvyqktt, fdnpvy, fdnpvsqktt, fdnpvs, fdnpva, fdnpvf, and fdnpvl were synthesized using standard fmoc chemistry with a free cooh terminus and an acylated nh2 terminus. crude peptides were purified by reverse - phase hplc on a c18 column (vydac, hesperia, ca), equilibrated in h2o with 0.1% trifluoroacetic acid, and eluted with a linear gradient of acetonitrile. the identities of the peptides were confirmed by mass spectrometry and amino acid analysis. concentrated stock solutions of peptides were made by dissolving the peptide in d2o and adjusting the ph to 6.0 with aliquots of concentrated naoh. the coat proteins were then gel - filtered over a superdex 200 column (pharmacia biotech, inc., clathrin and ap fractions were pooled and loaded directly onto an anion exchange column (source q 15 ; pharmacia biotech, inc.) equilibrated in 20 mm ethanolamine, ph 9.0, and eluted with a steep linear gradient of 1 m nabr. clathrin and ap fractions were pooled and concentrated using centriprep 30 (amicon corp., easton, tx). concentrated proteins were polymerized (25) by extensive dialysis against nmr sample buffer (40 mm d4-malonic acid [cambridge isotope labs, andover, ma ], ph 6.0, 0.2 mm edta, 0.2 mm dtt, and 3 mm nan3). protein concentration of the polymerized cage complex was determined using the bradford reagent (bio - rad laboratories, hercules, ca). for preparation of cages composed of clathrin or clathrin and ap2, an additional anion exchange chromatography step was performed to separate clathrin from ap2 before gel filtration. ap-2 containing fractions from the second anion exchange step were then pooled on the basis of coomassie blue stained sds - page. the clathrin fraction was divided in half, and all of the ap-2 sample was added to one half of the clathrin. equal total protein amounts of clathrin or clathrin / ap-2 cages were added to each nmr sample, which contained clathrin (9.7 mg / ml) or clathrin (8.5 mg / ml) and ap-2 (1.2 mg/ ml). sample purity was confirmed by coomassie blue stained sds - page gels and by western blotting with an antibody against -adaptin (100/1 ; sigma chemical co., st. the clathrin terminal domain (residues 1579) was expressed as a glutathione - s - transferase (gst) fusion protein in escherichia coli bl21 cells, and was prepared as described (17). glutathione agarose beads (sigma chemical co.) with the bound gst construct were then washed with thrombin cleavage buffer (50 mm tris, 2.5 mm cacl2, 150 mm nacl, ph 7.4). the beads were incubated overnight with gentle agitation in the presence of thrombin (sigma chemcial co.). dtt was added to 10 mm and allowed to incubate an additional 1 h before eluting the cleaved terminal domain construct from the beads. samples were concentrated and dialyzed overnight against nmr sample buffer with 100 mm kcl. the clathrin hub construct (residues 10741483) was expressed as a hexahistidine construct in e. coli bl21(de) cells as described (29). bacterial lysates were incubated with ni - nta agarose (qiagen inc., valencia, ca) overnight and eluted with 250 mm imidazole in 50 mm tris, ph 7.8. the eluted polypeptides were then loaded directly onto an anion exchange column equilibrated in 30 mm tris, 1 mm edta, ph 7.8, and eluted with a linear gradient of 0.5 m kcl. fractions were pooled on the basis of coomassie blue stained sds - page gels. because the hub polypeptide aggregated in the nmr sample buffer, both the terminal domain and hub polypeptides were dialyzed against 40 mm kpo4, 150 mm kcl, 0.2 mm edta, 3 mm nan3, and 0.2 mm dtt ph 7.2. all spectra were acquired at 500 mhz and 25c in sample buffer containing 10% d2o on a varian unity 500 spectrometer with a 5999.7-hz spectral width using 3-(tetramethylsilyl) propionic acid as a chemical shift reference. 1d spectra were acquired with 128 transients of 64,000 points zero filled to 131,072 points (see fig. 1) or 64 transients of 32,000 points zero filled to 65,536 points (see figs. 4 and 5). line - broadening was measured as the difference of peak widths at half height using the felix 2.30 (biosym technologies) program. the broadening is reported as the line - width of the resonance peak in the presence of cages minus the line - width in the absence of cages. crosspeak intensities were calculated in arbitrary units of volume using the felix 2.30 program from spectra acquired at 60-, 100-, 140-, and 220-ms mixing times. the spectra were acquired in phase - sensitive mode (48) with 2 256 fids of 1024 complex points each. a spectral width of 5999.7 hz the relative kd for wild - type and position 807 mutant peptides from clathrin terminal domains was obtained from measurements of the peak intensities of the phe (3, 5) aromatic protons for each of the peptides in the presence and absence of protein. the free and bound populations were calculated from the following equation : \documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation}\frac{1}{i_{o}}=\frac{p^{f}}{i_{f}}+\frac{p^{b}}{i_{b}}\end{equation}\end{document } where i is the intensity of the free (if), the bound (ib), and observed (io) resonance, and p and p are the free and bound populations, respectively. since peak intensities are inversely proportional to the transverse relaxation rate (1/t2), the population of the free and bound peptide can be estimated from the change in intensity of a given peptide resonance upon addition of clathrin terminal domain if the t2 of the resonance in the free and bound forms is known. the t2 for a protein is approximately proportional to the molecular weight, and from the line - widths observed in 20-kd proteins (3), the expected t2 for a 55-kd protein is 10 ms. the t2 calculated from the line - widths of several resonances of the free peptides was 100 ms. the values of ib were estimated to be if/10, assuming that the t2s of the free and bound resonances are 100 and 10 ms, respectively. therefore, the calculated populations of p and p were then used to obtain kds assuming standard protein ligand equilibrium. since the kds were based on t2 estimates, their absolute values are likely to be inaccurate. however, the relative values of the kds can be compared because they were all obtained by the same method. distances were calculated only for interresidue noes seen in 60 ms mixing time data sets. sequential h to nh noes were not used because they contained contributions from the free peptide. noe distance restraints were calculated from both wild - type hexa- and decapeptide either by using bins of weak (w ; 1.82.3), medium (m ; 1.83.5), and strong (s ; 1.85.0) peaks, or by measured noe volumes calibrated individually for each dimension with the average volume of the phe, tyr, and pro (/) noes corresponding to 1.75. structures were calculated by simulated annealing performed with the programs insightii and nmrchitect (biosym technologies, san diego, ca) incorporating either type of restraint. in short, peptides were heated to 1,000 k, and then the distance and chiral restraints were slowly increased over 30 ps. the peptides were then slowly cooled to 300 k while increasing covalent and nonbond forces over 32 ps. the final structures were then obtained by iteratively minimizing to a final derivative of 0.01 kcal / mol / and simulating 30 ps of restrained dynamics. removal of pseudoatom restraints for tyr (,) and phe() was based on the geometry of an initial set of structures. the final set of 30 restraints was (denotes pseudoatom and distance is denoted as s, m, or w) : ac(me)::f(h1), w ; f(h1) : : d(hn), w ; f(h2)::d(hn), w ; d(h2)::n(hn), w ; n(h1)::p(hs), w ; n(h2)::p(hs), m ; n(h1)::p(hr), w ; n(h2)::p(hr), w ; n(hn) : : p(hr), w ; p(hr)::v(hn), m ; p(hs)::v(hn), w ; p(hs)::v(hn), w ; v(hn)::y(hn), s ; v(hn)::y(h1), w ; v(h1)::y(h1), w ; v(h1) : : y(h1), w ; ac(me)::d(hn), w ; ac(me)::n(h21), w ; ac(me) : : n(h22), w ; v(hn)::n(h1), w ; v(hn)::n(h2), w ; n(h1)::y(h2), w ; n(h2)::y(h1), w ; p(hs)::y(h1), w ; p(hs)::y(h1), w ; p(hs) : : y(hn), m ; p(hr)::y(hn), w ; p(hr)::y(h1), w ; p(hr)::y(h1), w ; p(h)::y(hn), w. clathrin triskelions and aps purified from bovine brain were assembled into membrane - free polyhedral cages using standard methods (25). we prepared synthetic hexa- and decapeptides corresponding to the wild - type ldl receptor internalization motif (fdnpvy and fdnpvyqktt) and to an internalization defective motif (fdnpvs and fdnpvsqktt). 1, g and h) side chain regions from one- dimensional h nmr spectra are shown for peptide samples (2.5 mm) mixed with nmr sample buffer alone, or buffer containing clathrin cages (20 mg / ml). the wild - type and mutant peptides exhibited characteristic narrow h line - widths in the absence of cages (fig. 1, a, d, and g, and data not shown). adding cages to wild - type hexa- and decapeptides caused a pronounced increase in the line widths, indicative of an interaction between the peptide and the cages and a consequent slower effective tumbling time for the peptide (fig. 1, b, c, and h ; 36). by contrast, the cage - induced line - broadening of mutant peptides was markedly less than that of wild - type for both the hexa- and decapeptides, indicating a weaker interaction (fig. 1, e and f). as a control for nonspecific binding or viscosity - induced broadening, we examined the decapeptides in the presence of 20 mg / ml bsa. neither peptide displayed appreciable line - broadening (data not shown). noe spectroscopy (noesy) is another method of detecting interactions between peptides and large protein complexes (26). the hexa- and decapeptides have inefficient cross - relaxation due to their small size, and thus have noe spectra relatively devoid of cross - peaks (data not shown). a peptide interacting with a large protein complex will develop stronger noes because cross - relaxation is more efficient in the bound state. the resulting spin populations are then maintained in the free state where they are observed as an increase in the number and intensity of noe crosspeaks (10, 11). as shown in fig. 2 (a and c), the noesy spectra of both the deca- and the hexa- wild - type peptides in the presence of cages contain numerous cross - peaks, many of which are strong. in contrast, only a few cross - peaks are observed in the noesy spectra of the mutant peptides in the presence of cages (fig. the large difference in number and intensity of noes at a short mixing time (60 ms) is consistent with the line - broadening results (fig. 1), and indicates that the wild - type peptides have higher affinity for the cages. transferred noes were not observed with either wild - type or mutant decapeptide in solutions with 20 mg / ml bsa, even at long mixing times (300 ms ; data not shown). when a peptide binds to a macromolecule, the effective tumbling rates are slower for protons in the region of the peptide involved in binding, since protons in other regions are likely to experience fast internal motions. thus, cross - relaxation is more efficient in the regions immobilized by binding, resulting in faster noe buildup rates for a given fixed distance. to analyze the mobility of residues of the wild - type decapeptide bound to the cages, we first compared the build - up rates of transferred noes corresponding to covalently fixed equivalent proton distances. the similarity in noe buildup rates (measured with 60-, 100-, 140-, and 220-ms mixing times) for the geminal protons from residues f, n, p, and y indicates that these residues behave isotropically in the bound state (fig. this phenomenon was also apparent in the similar line - broadening of the tyr (h, 3.45 hz) and phe (h, 3.60 hz) ring protons (fig. 1). by contrast, the buildup rates for h/h noes decrease towards the cooh terminus of the peptide. val has the most rapid buildup rate, indicating that it is immobilized the most, then thr9, and then thr10 (fig. this trend was also observed in line - broadening of the val (h1, 5.20 hz ; h2, 3.82 hz), thr9 (h, 3.18), and thr10 (h, 2.95 hz) protons (compare fig. 1, g and h). these results indicate that the decapeptide residues outside the internalization motif have greater mobility than those within the motif. therefore, the same residues known to be critical for internalization in vivo were immobilized in the presence of cages. the internalization motif peptide could be interacting with multiple proteins in the lattice. to distinguish between the contributions of ap2 and clathrin, we prepared clathrin cages composed of either clathrin alone or clathrin plus purified ap2. 4 shows that the wild - type hexapeptide in the presence of clathrin cages displayed distinct line - broadening (compare fig. the mutant peptide, by contrast, showed much less line - broadening (compare fig. 4 d with fig. some additional line - broadening was seen when the wild - type peptide was mixed with cages composed of both clathrin and ap2 (compare fig. however, it is likely that the broadening arose primarily from clathrin (see discussion). the mutant peptide (y807s), by contrast, showed much less line - broadening (compare fig. these results suggest that the ldl receptor interacts with clathrin through the fxnpxy motif during receptor clustering in coated pits. we examined further the interaction between the hexapeptides and clathrin using recombinant portions of the clathrin molecule. the terminal domain of clathrin (residues 1579) expressed as a gst fusion protein was purified, and thrombin was cleaved from gst. clathrin hubs (residues 10741483) were expressed as a hexahis - tagged polypeptide, and were purified by a ni resin and anion exchange. line - broadening measurements were first made using the terminal domain at the acidic ph used in the cage assays. b) seen when peptide is incubated in the presence of clathrin cages. the mutant peptide, by contrast, displayed much less broadening (compare fig. equimolar amounts of either bsa or gst under the same conditions had no effect (data not shown). we saw a similar degree of line - broadening at neutral ph (compare fig. 5 f), but no appreciable broadening was detected when the hub region was substituted for the terminal domain at neutral ph (compare fig. these results show that the ldl receptor tail selectively interacts with the terminal domain of clathrin. the amino acid requirements at position 807 for ldl receptor internalization (14) have been found to correlate with the propensity of peptides from that region to form a reverse turn (2). we examined position 807 mutants for their ability to interact with clathrin terminal domains by measuring the change in peak intensity of 1d proton resonances for each peptide. because peak heights are inversely proportional to the effective molecular weight, they can be used to calculate the relative binding constant for each peptide as it interacts with the terminal domain (see materials and methods). table i shows that the relative affinity of each peptide for the clathrin terminal domain strongly correlated with the predicted turn propensity (2) as well as the ability of receptors bearing these mutations to internalize ldl (14). thus, peptides with tyr and phe at position 807 had the highest affinity, the greatest turn propensity, and the highest internalization rate, while a leu at this position had an intermediate affinity and both ser and ala had the lowest affinities. transferred noes have been used previously to determine the conformation of peptides bound to proteins (26, 27, 49). these results have later been confirmed from crystal structures of the complexes (40, 57, 62). we used data from transferred noe spectra to determine the structure of the fdnpvy motif bound to clathrin cages. similar noe results were obtained when cages were replaced with recombinant terminal domains (data not shown). structures were calculated by simulated annealing (37) using 30 nontrivial transferred noe restraints. among these, 23 noes correspond to the residues npvy, including the strong nh / nh noe between val and tyr characteristic of a turn conformation (fig. 6 a), and other short and intermediate range, structurally diagnostic noes. a superposition of the peptide backbones for the 21 structures with distance violations of < 0.1 obtained among 30 calculated structures is shown in fig. 6 b. all resulting structures had an extended nh2 terminus followed by a type 1 turn around residues npvy. this result is more likely to arise from a lack of proton density in the extended structure of this region than from increased mobility, since line - broadening and transferred noe build - up rates indicate that phe is immobilized (see above). the 21 calculated structures had a backbone root mean square deviation of 0.61, indicating that a single conformational form of the peptide was bound by the cages. based on this information, a representative structure is shown in fig. 6 c. the surprising conclusion of this study is that clathrin may be the molecule in coated pits responsible for capturing the ldl receptor during receptor - mediated endocytosis. four stringent criteria were used to distinguish specific from nonspecific interactions between the clustering motif peptide and clathrin. first, changing tyrosine 807 to serine, which matches the mutation that prevents ldl receptor clustering in coated pits (13), substantially reduced interaction of the peptide with the cages. second, the wild - type peptide bound to the cages in a single conformation, and had the predicted (2, 12) reverse turn conformation. third, binding was limited to those residues that lie within the fxnpxy motif. fourth, once clathrin was identified as a candidate molecule, the wild - type peptide was found to interact with the terminal domain, but not with the hub portion of recombinant clathrin. furthermore, the relative affinity of terminal domains for peptides with various position 807 substitutions correlated strongly with both the ldl receptor internalization index (14) and the predicted turn propensity (2). these results suggest that the receptor internalization rates depend on the affinities of the hexapeptide motif for clathrin, and that the affinity depends on a reverse turn conformation. the apparent affinity we measured between the hexapeptides and clathrin terminal domains is in the same range as that reported for the interaction between yxx peptides and the subunit of ap (4, 44). it is hard to judge the functional significance of these affinities. within the cell, ldl receptors probably exist as multimers interacting with the rigid clathrin lattice in a two - dimensional plane (52). in this environment, the receptors will have a restricted rotational and translational mobility as they interact with a multivalent, nearly crystalline set of terminal domains. assuming that a 76-nm diameter clathrin - coated vesicle contains 60 clathrin triskelions (21), we calculated the concentration of terminal domains to be at minimum 1.5 mm. this result suggests the effective concentration of terminal domains in the region where receptor cluster is quite high, so the relatively low - affinity interactions we have measured should be sufficient to mediate clustering. clathrin terminal domains have been implicated before in receptor clustering (17, 18). the binding of -arrestin to the phosphorylated cytoplasmic tail of the 2-adrenergic receptor appears to be required for receptor migration to coated pits. the binding of -arrestins to clathrin is stoichiometric, clustering in coated pits is coincident with receptor internalization, and expression of -arrestin mediates internalization (18). the -arrestin binding site has been localized to the first 100 amino acids of the clathrin heavy chain (17). structural studies show that the terminal domain of clathrin is optimally positioned in an assembled lattice to interact with the cytoplasmic tails of receptors (53). however, none of these studies have sufficient resolution to determine the location of the terminal domains in an assembled clathrin coat. although clathrin terminal domains clearly interact with the npxy clustering motif, we have not ruled out the possibility that aps can bind to the cytoplasmic portion of the ldl receptor, or that they can modulate the interaction of the nxpy motif with clathrin. the presence of ap2 in our clathrin cage preparations moderately enhanced line - broadening of the nmr spectra compared with clathrin alone, suggesting that the wild - type peptides may have a higher affinity for cages containing ap2 (fig. the bound peptide displayed a well - defined conformation in the presence or absence of aps, so broadening must be due to a single type of binding site for the hexapeptide. therefore, most likely the enhanced broadening is not due to introduction of additional hexapeptide binding sites present in the aps. our results stand in contrast to previous studies showing that purified ap2 binds substoichiometrically to a fusion protein containing the cytoplasmic tail of the ldl receptor (42). the fusion protein used in this study contained 31 residues from the cii protein, a thrombin cleavage site, 112 residues of myosin light chain, a second thrombin cleavage site, and the 50-residue ldl receptor tail (42). interestingly, the cii fragment contains a dileucine motif that can directly interact with aps (20, 43). since a fusion protein containing an internalization - defective ldl receptor tail (e.g., y807c) was not tested, these studies did not rule out the possibility that the interaction with ap2 was mediated by the dileucine motif. moreover, there is compelling in vivo evidence that ap2 does not cluster ldl receptors. depletion of intracellular k and hypertonic treatment of cells both cause reversible loss of clathrin lattices from the cell surface (22, 28), which is accompanied by ldl receptor unclustering (22, 28). if receptor clustering were primarily dependent on ap2, then ldl receptors should have remained clustered under these conditions. an important prediction from these results is that ldl receptors should be sorted and concentrated during clathrin - coated vesicle formation regardless of whether budding occurs from golgi, endosome, or surface membranes. coated vesicles purified from adrenal glands are enriched in masked ldl receptors four to fivefold (33). these isolated vesicles contain coated vesicle derived from both the plasma membrane and the golgi apparatus that can be separated on the basis of size (5). ligand blotting indicates that both populations of vesicles are equally enriched in ldl receptors relative to the plasma membrane (5). therefore, even though there are no quantitative em studies showing ldl receptors concentrated in golgi - coated vesicles, these biochemical experiments suggest that sorting does occur. ap2 does appear to be responsible for capturing receptors that contain yxx (39) and dileucine internalization motifs (20, 43). two - hybrid screen (38, 39) and surface plasmon resonance analysis (4, 39) both show that yxx interacts with the chain of ap2. the properties of yxx motif are distinct from those for fxnpxy since fxnpxy motifs did not bind the subunit, and a combinatorial library showed no preference for this motif (4). dileucine motifs also bind clathrin ap2 and ap1, yet receptors with this motif do not compete with receptors containing yxx during internalization (31). receptor internalization by coated pits can therefore be mediated by at least three distinct motifs (two tyrosine - based and one dileucine - based), each of which is captured by a different binding site in the lattice. tyrosine phosphorylation alters the binding specificity of both yxx and npxy, although phosphorylation is not essential for binding to all phosphotyrosine binding (ptb) domains (6, 59). pyxx binds sh2 domains (46) while npxpy binds proteins that contain a ptb domain (51). the structures of both pyxx/sh2 (16, 54, 55) and npxpy / ptb domain (15, 60, 61) complexes have been solved. the structure of the latter confirms transferred noe data (61) showing that the tyrosine in the bound npxpy is in a tight turn preceded by an extended region. since we found that bound fxnpxy is in a tight turn (fig. 6 c), it is possible the simple addition of phosphate or specific expression of a ptb domain blocks coated pit clustering. the conformation of yxx bound to the chain of ap has not been determined, but if the phosphate is also what changes binding specificity, then the bound conformation of yxx should be an extended, two - pronged plug rather than a tight turn. this study demonstrates the power of nmr spectroscopy to identify the molecules in coat protein complexes responsible for sorting membrane proteins during vesicular traffic. at the same time, this method provides dynamic structural information about the targeting sequence when it is bound. internalization motifs can function at multiple steps along an internalization pathway, differentially using clathrin and ap subunits to generate different migratory patterns. the specificity of clathrin for fxnpxy and aps for yxx appears to be as distinct as ptb and sh2 domains are for their phosphorylated counterparts. yxx and dileucine motifs may target receptors to plasma membrane, late endosome, lysosome, and tgn structures via combinatorial interactions with ap complexes and possibly other membrane coats. by contrast, the fxnpxy motif may be needed for efficient clathrin - mediated recycling of ldl receptors between the plasma membrane and endosomes. based only on the primary sequence of these motifs, it has not been possible to predict the cellular distribution of receptors. nmr spectroscopy should be useful to characterize further the conformational determinants of sorting motifs that ultimately determine receptor sorting patterns. differential line - broadening of internalization peptides in the presence and absence of clathrin - ap cages. corresponding regions of 1-dimensional h nmr spectra are shown at absolute intensity for 2.5 mm peptide with or without 20 mg / ml assembled clathrin / ap cages. (a f) aromatic region of the spectrum from wild - type peptides fdnpvyqktt alone (a), fdnpvyqktt plus cages (b), fdnpvy plus cages (c), and y807s mutant peptides fdnpvsqktt alone (d), fdnpvsqktt plus cages (e), and fdnpvs plus cages (f). (g and h) upfield region of wild - type peptide, fdnpvyqktt, alone (g) and with cages (h). the resonances of the aromatic protons are at 7.29, 7.33, and 7.38 ppm for phe and 6.81 and 7.12 ppm for tyr and for the methyl protons at 0.83 and 0.89 ppm for val(), 1.25 ppm for thr9(), and 1.18 ppm for thr10(). differential broadening of the wild - type and mutant hexapeptides in the presence of assembled clathrin cages reconstituted with and without ap-2. corresponding regions of 1-dimensional h nmr from the aromatic region of the spectra are shown at absolute intensity. the indicated peptide (final concentration 1.5 mm) was added to buffer alone or buffer plus 9.7 mg / ml protein : fdnpvy alone (a), plus clathrin cages (b), plus clathrin and ap-2 cages (c), fdnpvs alone (d), plus clathrin cages (e), or plus clathrin and ap-2 cages. nmr samples were prepared as described for fig. 1. because of differing concentrations of peptide and protein, 1. differential line - broadening of internalization peptides in the presence and absence of recombinant clathrin terminal domains and hubs. corresponding regions of 1-dimensional h nmr from the aromatic region of the spectra are shown at absolute intensity. samples contained 1.0 mm peptide with or without 175 m clathrin terminal domain td(1579) at ph 6.2 as follows : fdnpvy alone (a), plus td(1579) (b) or fdnpvs alone (c), plus td(1579) (d). the interactions of the wild - type hexapeptide (1 mm) with 200 m of either td(1579) or clathrin hub(10741483) were compared at ph 7.2 in 150 mm kcl. samples contained : fdnpvy alone (e), plus td(1579) (f), or plus hub(10741483) (g). noesy spectra of wild - type and mutant internalization motif peptides in the presence of clathrin - ap cages. corresponding expansions from the aliphatic / amide + aromatic region of noesy spectra are presented as contour plots at the same level from data acquired with 60 ms of mixing time. all samples contained 20 mg / ml cages and 2.5 mm peptide : fdnpvy (a), fdnpvs (b), fdnpvyqktt (c), and fdnpvsqktt (d). noe buildup curves from the sample containing the wild - type decapeptide with cages are shown for the intraresidue h/h noes of phe (squares), asn (diamonds), pro (circles), and tyr (triangles ; a), and for the intraresidue h/h noes of val(h1) (squares), val(h2) (diamonds), thr9 (circles), and thr10 (triangles ; b). crosspeak intensities were calculated in arbitrary units of volume using the program felix 2.30 from spectra acquired at 60, 100, 140, and 220 ms mixing times. the relative kd calculated from phe(3,5) aromatic proton peak intensities as described in the experimental procedures. using noe magnitudes as an indicator for turn propensity, scaled to that for the npvy peptide (2). (a) contour plot of the transferred noe between tyr(hn) (7.71 ppm) and val(hn) (8.07 ppm) from the hexapeptide with a 60-ms mixing time. (b) the superimposed backbones of 21 calculated structures obtained by simulated annealing. (c) a single representative structure of the acfdnpvy peptide bound to clathrin - ap cages.	previously the hexapeptide motif fxnpxy807 in the cytoplasmic tail of the ldl receptor was shown to be essential for clustering in clathrin - coated pits. we used nuclear magnetic resonance line - broadening and transferred nuclear overhauser effect measurements to identify the molecule in the clathrin lattice that interacts with this hexapeptide, and determined the structure of the bound motif. the wild - type peptide bound in a single conformation with a reverse turn at residues npvy. tyr807ser, a peptide that harbors a mutation that disrupts receptor clustering, displayed markedly reduced interactions. clustering motif peptides interacted with clathrin cages assembled in the presence or absence of ap2, with recombinant clathrin terminal domains, but not with clathrin hubs. the identification of terminal domains as the primary site of interaction for fxnpxy807 suggests that adaptor molecules are not required for receptor - mediated endocytosis of ldl, and that at least two different tyrosine - based internalization motifs exist for clustering receptors in coated pits.
the enzymes that synthesize pe in yeast, worms, and flies have mammalian counterparts. first, lodged in the inner membrane, the enzyme phosphatidylserine decarboxylase converts phosphatidylserine to pe (fig. 1). pe synthesized in mitochondria can spread via mitochondrial associated membranes to other cellular compartments. second, the kennedy pathway consists of 3 enzymes that convert the metabolite ethanolamine into pe ; the last enzyme in this pathway is embedded in the membranes of the endoplasmic reticulum (er) (fig. phosphatidylserine decarboxylase may synthesize most of the pe, whereas in other cells the kennedy pathway may synthesize the most of the pe. figure 1.scheme for phosphatidylethanolamine (pe) synthesis in mitochondria and the endoplasmic reticulum (er) in yeast. cdp, cytidine diphosphate ; cho, choline ; eta, ethanolamine ; p - eta / p - cho, phosphorylated eta / choline ; pm, plasma membrane. eki1, eta kinase ; ect1, eta - phosphate cytidylytransferase ; ept1, eta / cho phosphotransferase. scheme for phosphatidylethanolamine (pe) synthesis in mitochondria and the endoplasmic reticulum (er) in yeast. cdp, cytidine diphosphate ; cho, choline ; eta, ethanolamine ; p - eta / p - cho, phosphorylated eta / choline ; pm, plasma membrane. eki1, eta kinase ; ect1, eta - phosphate cytidylytransferase ; ept1, eta / cho phosphotransferase. we found that the psd1 deletion mutant has low pe, intense er stress, and a slight growth defect compared to wild - type cells. when -synuclein is expressed in these pe - deficient cells, -synuclein forms intracytoplasmic foci, the level of -synuclein is 3-fold higher than identically treated wild - type cells, and growth is severely inhibited. treating such cells with exogenous ethanolamine boosted the level of pe through the kennedy pathway, which eliminated these various defects due to initially low pe. these findings were replicated in transgenic c. elegans worms in which -synuclein was expressed in each of the worm 's 6 dopaminergic neurons. such worms exhibit a gradual age - dependent loss (t1/2 = 3.6 d) of the dopaminergic neurons due to the expression of -synuclein. rnai depletion of psd-1 in neurons that express -synuclein accelerated the age - dependent cell loss (t1/2 = 2.3 d), whereas rnai depletion of psd-1 in neurons lacking a - syn expression did not affect cell loss over time. ethanolamine rescued cell loss due to the expression of -synuclein in the neurons with psd-1 depleted (t1/2 = 7.9 d). ethanolamine even rescued the age - dependent degeneration that occurs in worms that express -synuclein but without rnai depletion of psd-1. given that stimulating pe synthesis through the kennedy pathway ameliorates -synuclein toxicity in worm neurons, we asked whether the kennedy pathway might be associated with other neurological diseases. a class of drosophila mutants that exhibit a transient paralysis following a brief mechanical shock was identified in a behavioral screen and reported in 1971. subjecting eas flies to a brief mechanical shock (bang or vortex) triggers a transitory seizure that persists for several seconds, but then the flies recover. tanouye and colleagues later characterized the eas mutant and showed that the paralytic phenotype was due to a mutation in the gene for ethanolamine kinase, which phosphorylates ethanolamine to yield phosphoethanolamine (fig. 1). biochemical analysis of whole fly lipid extracts showed that the eas flies lacked ethanolamine kinase activity and had a deficit of pe compared to wild type flies. low pe in the eas mutant might cause membrane leakiness or decrease ion channel activity, which in either case leads to the transient paralytic phenotype. we suggest that chronically low pe in the membranes of the endoplasmic reticulum will likely lead to chronic stress in this compartment, which, over time, can lead to neuronal failure.table 1 shows the effects of low pe on different organisms. table 1.low pe harms membranesgenecommentsreferencephosphatidylserine decarboxylaseyeast : deletion of psd1 causes intense er stress, -synuclein foci, protein trafficking defects, pe / pc ratio decreases by 70% (vs wild - type). eta rescues.worms : knocking down psd-1 with rnai enhances the toxicity of -synuclein expressed in worm neurons ; eta rescues. eta also protects from -synuclein - induced neurodegeneration in the absence of psd-1 knockdown.ref. 6ethanolamine kinaseflies : i) null - activity mutant of eas : a brief mechanical shock induces transient paralysis. pe decreases, and the pe / pc ratio decreases by 18% (vs wild - type). ii) null - activity mutant of eas : increases triglycerides and causes tachycardia, cardiac relaxation problems, and srebp activation.i) ref. 8 ii) ref. if lipid disequilibrium is indeed a problem for pd and/or epilepsy, then one would expect that lipid - related genes would be identified from sequencing studies. mutations in fasn (fatty acid synthase) and plcb1 (phospholipase c isoform 1) were recently identified from a small number of individuals with a severe form of childhood epilepsy. mutations in gba (codes for enzyme that cleaves glucose from glucocerebroside) have been linked to a higher risk of pd, and single nucleotide polymorphisms in pla2g6 (calcium - independent phospholipase a2) and in srebp-1 (sterol - regulatory binding protein) have been identified in large genome - wide association studies of individuals with pd. curiously, the enzymes coded by plcb1 and pla2g6 degrade phospholipids to yield important lipid second messengers. while these sequencing results are tantalizing, clearly more sequencing needs to be done to establish whether mutations in lipid metabolism genes are significant risk factors for epilepsy and pd. in conclusion, how a disruption in phospholipid homeostasis alters the function of the endoplasmic reticulum and how it triggers protein aggregation are relatively unexplored areas. a deeper understanding of the role of phospholipid homeostasis can come about from lipidomic profiling by mass spectrometry of postmortem brain tissue and from magnetic resonance imaging of the brain to detect phospholipid metabolites combined with other imaging techniques to detect the proteins such as aggregated -synuclein.	we recently reported that knocking down the enzyme phosphatidylserine decarboxylase, which synthesizes the phospholipid phosphatidylethanolamine (pe) in mitochondria, perturbs the homeostasis of the human parkinson disease (pd) protein -synuclein (expressed in yeast or worms). in yeast, low pe in the psd1 deletion mutant induces -synuclein to enter cytoplasmic foci, the level of this protein increases 3-fold compared to wild - type cells, and the mutant cells are severely sick. the metabolite ethanolamine protects both yeast and worms from the deleterious synergistic effects of low mitochondrial pe and -synuclein. here we highlight a drosophila mutant called easily shocked thought to be a model of epilepsy that can not use ethanolamine to synthesize pe. we also highlight recently identified mutated genes associated with defective lipid metabolism in pd and epilepsy patients. we propose that disruptions in lipid homeostasis (synthesis and degradation) may be responsible for some cases of pd and epilepsy.
breast cancer is the most common malignancy after lung cancer and the leading cause of cancer - related deaths among women worldwide. although the incidence of breast cancer has been increasing, male breast cancer is a rare and uncommon disease, accounting for only 0.5% to 1% of all cases. male patients are diagnosed at late stages and usually have a worse prognosis, mainly due to the lack of screening for disease prevention and less awareness of pathognomonic symptoms. nonetheless, there have been several studies where male sex was not reported as an independent prognostic factor for worse clinical outcome and there were no meaningful differences between the 5- and 15-year excess mortalities of men and women. among the histological subtypes of breast cancer, invasive lobular carcinomas have a high capacity to metastasize to uncommon sites such as the skin, pleura, peritoneum, ovaries, and the gastrointestinal (gi) tract. in such cases, the stomach is the most affected segment of the digestive tract, detected in 3% to 18% of patients with metastatic disease. on the other hand, importantly, metastatic spread to the stomach may occur many years after the initial treatment for breast cancer, and it may be extremely difficult to distinguish from a primary gastric cancer based on clinical, endoscopic, radiological, and histopathological features. however, this discrimination is imperative to establish the best treatment strategy for our patients. herein, we report the first case of ductal breast carcinoma metastatic to the stomach mimicking primary linitis plastica in a male patient. a 65-year - old man presented to the emergency room of santa maria della misericordia hospital (perugia, italy) with the primary complaints of hematemesis and epigastric pain in april 2015. twelve years ago, he had undergone total mastectomy with complete axillary dissection for invasive ductal carcinoma. postoperative histology showed a completely excised invasive ductal carcinoma with the following immunohistochemistry (ihc) profile : estrogen receptor (er), 90% ; progesterone receptor (pr), 0% ; ki-67, > 10% ; and human epidermal growth factor receptor 2 (her2), 1 + (figure 1). the patient was treated with four cycles of adjuvant doxorubicin and cyclophosphamide, followed by 5 years of tamoxifen with no signs of recurrence. in august 2011, the relapse was observed at the surgical mastectomy site and the patient underwent radical surgical excision of the nodule, which was diagnosed as metastasis from invasive ductal breast carcinoma with the same ihc profile as the primary tumor (er, 100% ; pr, 0% ; ki-67, 14% ; her2, 1 +). following these findings, the patient was treated with 12 cycles of doxorubicin, cyclophosphamide, and paclitaxel, followed by tamoxifen with no signs of recurrence until september 2014, when a positron emission tomography / computed tomography (pet / ct) image showed new bone lesions. subsequently, tamoxifen treatment was stopped and the patient was started on anastrozole ; he showed stable disease on the subsequent follow - up visits and instrumental evaluations. at the time of hospitalization in april 2015, the patient had undergone an esophagogastroduodenoscopy, which had revealed a borrmann type 4 tumor that was characterized by diffuse thickening and sclerosis of the gastric wall and marked hypertrophy of the mucosal folds. a subsequent pet / ct scan showed wall thickening of both the gastric antrum and the body of the stomach with a significant 18f - fluorodeoxyglucose (fdg) uptake, indicating linitis plastica (figure 2). based on these findings, the patient was diagnosed with primary gastric adenocarcinoma, although the possibility of a metastatic progression of invasive ductal carcinoma to the stomach could not be eliminated. however, considering the poorer prognosis associated with primary diffuse gastric carcinoma compared to oligometastatic breast cancer and the different treatment strategies associated with these diagnoses, after a multidisciplinary discussion, it was decided that surgery was the most suitable treatment strategy. therefore, the patient underwent gastrectomy with roux - en - y esophagojejunal anastomosis with both diagnostic and therapeutic intention. on macroscopic examination, the stomach measured 163 mm with wall thickening (up to 10 mm) of the antrum and corpus. the internal surface of the thickened wall showed a diffuse loss of mucosal rugae in an 88-mm region without mucosal ulceration. microscopic examination revealed diffuse growth of a poorly differentiated carcinoma with a full - thickness extension through the stomach wall. the neoplasm appeared to grow from the serous surface toward the overlying gastric mucosa, which appeared normal but infiltrated. foci of dysplasia were not found in the surrounding gastric mucosa, leading to the suspicion that the gastric involvement was of metastatic origin. the tumor cells had large vesicular nuclei with prominent nucleoli, and they were arranged in solid and tubular patterns of growth (figure 3a, b). the mitotic count was elevated (up to 20 mitosis/10 high power fields). importantly, the tumor cells showed immunore - activity for cytokeratin 7 (ck7), e - cadherin, gross cystic disease fluid protein 15 (gcdfp15), and gata binding protein 3 (gata3), consistent with the findings for metastatic breast carcinoma of no special type (figure 3c - e). the er, pr, ck20, and synaptophysin statuses were negative, whereas ihc staining for her2 revealed a 3 + positivity (figure 3f). a pet / ct scan performed after the gastric resection showed no fdg uptake (figure 2 g, h). following diagnosis of her2 positive gastric metastasis of breast carcinoma, the patient received six cycles of docetaxel plus trastuzumab, thus achieving stable disease. presently, the patient is receiving maintenance therapy with trastuzumab, without any signs of progression. breast cancer metastases to the gi tract are very rare occurrences ; the most common metastatic sites are the bones, liver, lungs, and brain. the estimated overall incidence of breast cancer metastasis to the stomach during either long - term follow - up or post mortem analyses is 2% to 18%, with 90% to 94% of the patients having concurrent breast cancer metastases. importantly, metastases to the gi tract can represent the first manifestation of metastatic breast cancer, as well as late recurrence even years after the diagnosis of the primary neoplasm, similar to the present case. of note, the potential of metastasis to the stomach is higher among patients with lobular histologies. in this respect, a retrospective analysis conducted over a period of 15 years demonstrated that 83% of patients with gastric metastases had lobular histologies. these findings were confirmed in a larger series, which demonstrated that the lobular type accounted for at least 83% to 85% of the cases with gastric metastases form breast cancer. the biological mechanism underlying the unusual spread of lobular breast carcinoma to the gi tract is not clearly understood ; however, some authors have hypothesized that it might be linked to a specific tropism of the lobular cells. intriguingly, in the mixed histology subtype (ductal and lobular), only the lobular component potentially metastasizes to the stomach. clinically, metastatic gastric linitis plastica is indistinguishable from a primary gastric linitis plastica, similar to the present case. in fact, the clinical presentation of gastric involvement is completely a specific ; it commonly manifests as epigastric pain, indigestion, lack of appetite, and occasional gastric obstruction and bleeding. although rare, linitis plastica represents the most frequent type of gastric metastasis from lobular breast cancer. conversely, the evidence for the association of ductal breast carcinoma with gastric metastases is insufficient, and its association with linitis plastica is very rare as it usually manifests as a polyp, ulcer erosion, or stenosis. in the present case, the patient showed tumor recurrence in the gi tract, presenting as secondary linitis plastica from a primary breast ductal carcinoma, as the second case reported in medical literature, and the first ever described case among male patients. in order to obtain an accurate differential diagnosis between breast cancer metastasis to the stomach and primary gastric cancer, ihc analysis is strongly recommended, because clinical, radiological, and endoscopic findings are generally unhelpful. importantly, even though the detection of er and pr expression in biopsy samples and surgical gastric specimens is indicative of breast cancer metastasis to the stomach, early studies with first - generation antibodies documented er and pr positivity in 32% and 12% of the cases, respectively, in patients with primary gastric cancer. later, van velthuysen. demonstrated that second - generation anti - er antibodies are sensitive and specific biomarkers for determining breast origin, because they are not usually expressed by gastric cancer cells. unfortunately, they are unhelpful in the case of er - negative primary breast cancer, and loss of er expression in the metastatic site of breast cancer has been widely reported, as observed in our patient. therefore, in such cases, er and pr do not represent suitable biomarkers to discriminate between breast cancer metastasis to the stomach and primary gastric cancer. in the present study, the authors provide an ihc panel of selected antibodies that allowed a proper differential diagnosis (ck20, ck7, er, pr, e - cadherin, gcdfp15, and gata3). gcdfp15 has been proven an accurate biomarker for identifying a malignant lesion of breast origin, yielding 55% to 76% sensitivity, and 95% to 100% specificity. additionally, combined ihc for ck7 and ck20 represents another useful tool, as breast carcinomas are ck7 positive in 90% of cases versus the 50% to 55% of primary gastric cancers, whereas ck20 is negative in all breast carcinomas and highly positive in gastric, colorectal, and pancreatic carcinomas. notably, our panel also included gata3, a member of the gata family of zinc - finger dna binding proteins, which is currently considered a reliable, sensitive, and specific immunomarker for the diagnosis of breast cancer, as it was found only in breast and urothelial carcinomas but not in other tumors. aberrant e - cadherin expression is common in invasive ductal carcinomas that develop distant metastases. intriguingly, distant metastases consistently express e - cadherin, often more strongly than the primary tumor. when breast cancer metastasis to the gi tract is suspected, positive ihc for ck7, gcdfp15, and gata3 can effectively confirm the diagnosis, particularly in case of ck20 negativity. in addition, e - cadherin expression further corroborates a ductal histology, whereas e - cadherin loss is peculiar of invasive lobular carcinomas. in the present case, discordance was observed between the hormone receptor statuses of primary (er, 90% ; pr, 0%) and metastatic breast cancer (er, 0% ; pr, 0%). loss of er and pr during disease progression is a well - recognized phenomenon, representing a consequence of disease progression or prolonged endocrine therapy. concerning the her2 status, we reported discordance between primary invasive ductal carcinoma and gastric recurrence as well. as opposed to the primary tumor, the immunohistochemical staining for her2 on gastric specimens revealed 3 + positivity that supported targeted therapy with trastuzumab, which in turn resulted in stable disease. data on treatment are extremely limited because of the lack of randomized trials and the lack of reports ; however, majority of the patients with gastric metastases from breast cancer usually receive chemotherapy or hormone therapy, based on the assessment of hormone receptor statuses in metastatic tissues. in case of isolated gi tract involvement, surgery should be considered where appropriate, although it is often reserved for palliation purposes in cases of intestinal obstruction or bleeding. conversely, in the multi - metastatic setting, surgery does not improve survival compared to systemic treatment. of note, to the best of our knowledge, trastuzumab - based therapy for her2 positive gastric metastasis from breast cancer has never been reported previously. beyond the rarity of the case and its presentation, this report further enlarges the growing body of evidence suggesting that hormone receptor and her2 status should be determined for all recurrences, in case the procedure is technically feasible and not unreasonably invasive, since any change in er, pr, and her2 statuses might grant new therapeutic possibilities for the patient, allowing us to tailor the treatment in the most accurate manner.	breast cancer metastases to the gastrointestinal tract are very rare occurrences. among the histological subtypes of breast cancer, invasive lobular carcinomas have a high capacity of metastasis to uncommon sites including the stomach. conversely, there has not been sufficient evidence supporting the gastric metastasis of invasive ductal carcinoma. herein, we report a unique case of metastatic ductal breast carcinoma mimicking primary linitis plastica in a male patient, particularly focusing on the clinical and pathological features of presentation. moreover, we propose a immunohistochemical panel of selected antibodies including those for cytokeratin 20, cytokeratin 7, estrogen receptor, progesterone receptor, e - cadherin, gross cystic disease fluid protein 15, and gata binding protein 3 for an accurate differential diagnosis.
skeletal muscle presents unique features that allow it to respond to several exogenous stimuli. this characteristic is named plasticity. exercise and nutrition are examples of such stimuli that may promote adaptive responses in skeletal muscle in terms of structure and function [24 ]. for example, there are some reports describing that mechanical stimuli, particularly resistance exercise, may induce histological changes such as fiber type transition and profile and increase in cross - sectional area, and alterations in muscle function [6, 7 ]. branched - chain amino acids (bcaa), especially leucine, are also well - known nutrients that may influence the adaptive response of skeletal muscle. leucine supplementation has been described as a potential nonpharmacological tool able to stimulate both muscle anabolism and decrease catabolism [8, 9 ] and to modulate glucose homeostasis. furthermore, leucine can act synergistically with exercise to improve the efficiency and effectiveness of these adaptive responses. currently, there are some cellular pathways that partially explain why bcaa supplementation may promote such responses in skeletal muscle. most of these consistent evidences were observed on incubated cells, which have contributed to elucidate important mechanisms regarding amino acids modulation of skeletal muscle protein turnover. however, we have to consider that such conditions are considerably different from the human body. although experimental animals (rodents) represent an in vivo model, it may also present distinct results when compared to humans. recently, our group observed that due to differences in muscle metabolism, rodents may respond differently from humans to amino acids supplementation. although the same signaling pathways are found in rodent and human cells the response of these models to amino acids supplementation present individualities that may compromise the extrapolation of results. the mammalian target of rapamycin (mtor) pathway is a signal - dependent cascade that responds to a variety of stimuli ranging from growth factors and mitogens to amino acid deprivation and hypoxic stress. it has been well characterized that mtor pathway has a pivotal role in modulating protein translation initiation through eukaryotic initiation factors (eifs) and kinases, which in turn alter the phosphorylation status and activity of several proteins in this cellular pathway. amino acids supplementation is involved in signaling to upstream proteins, responsible for sensing and triggering (mtor, human vacuolar protein sorting 34 (hvps4), calcium - related proteins), as well as downstream proteins, responsible for ribosome initiation complex formation (eif4e, eif4e - binding protein 1 (4e - bp1), eif4f complex) [15, 16 ]. additionally, it has been shown that bcaa can also interact with the proteolytic machinery (ubiquitin proteasome system ups) in order to attenuate muscle wasting. this response may partially involve the protein kinase akt / pkb, which also participates in glucose homeostasis and muscle hypertrophy. regarding the proteolytic machinery, akt / pkb is known to phosphorylate the transcription factor forkhead box class - o (foxo), which translates the two majority genes (or e3 ligases) of muscle atrophy : atrogin-1 and muscle ring - finger protein-1 (murf-1) [12, 18, 19 ], to phosphorylate mtor and stimulate protein synthesis, and to modulate glucose transporter 4 (glut4) to the sarcolemma. in view of this, these cellular pathways (synthesis and degradation) are not distinct and may be controlled by amino acids through indirect genomic and nongenomic actions. although much attention has been given to the role of amino acids in these pathways, the responsiveness of skeletal muscle to these nutrients may be limited. for instance, amino acids infusion stimulate muscle protein accretion until it reaches a plateau. this condition, known as anabolic resistance to amino acidsthe inability of skeletal muscle to maintain or increase its protein mass by appropriate nutritional stimulation occurs because skeletal muscle protein synthesis is refractory to hyperaminoacidemia. thus, it appears that the optimal action of amino acids on skeletal muscle growth occur in combination with other exogenous stimuli (e.g., exercise) or in situations characterized by disruption of organic homeostasis (e.g., cancer, diabetes, muscle disuse, sepsis, chronic heart failure). in this context, the inflammatory status has a considerable role and the innate immune system (responsible for cytokines and chemokines production) should be carefully considered. the focus of this paper is to discuss the possible metabolic and cellular roles of bcaa supplementation on the inflammatory status of skeletal muscle and the effects on protein synthesis and degradation. it is possible that, in some conditions, the administration of these amino acids could exert an anti - inflammatory role or indirectly modulate the inflammatory status and balance of the system and/or the muscle cell in order to favor the biological response and tissue adaptation. the healing of injured muscle is composed of sequential but overlapped phases of injury, inflammation, regeneration, and fibrosis. injury and inflammation predominate the first few days after injury, followed by regeneration. when there is a severe injury the muscle does not recover completely and forms fibrotic tissue approximately two weeks after injury (figure 1). the inflammatory response is an important phase of the natural healing process. during this phase there is a release of several types of cytokines and growth factors to increase the permeability of blood vessels and chemotaxis of inflammatory cells, such as neutrophils and macrophages. these cells contribute to the degradation of damaged muscle tissue by releasing reactive oxygen species (ros) and producing proinflammatory cytokines [2427 ] such as tumor necrosis factor alpha (tnf-), interleukin-1 (il-1), and il-6 that regulate the inflammatory process [28, 29 ]. the role of these cells is quite complex and they can promote both injury and repair. a detailed discussion of their action is beyond the scope of this paper and has been reviewed elsewhere. some systemic inflammatory cytokines such as tnf- and il-1 have direct catabolic effects on skeletal muscle. the cytokine tnf- plays a key role in the skeletal muscle wasting present in chronic diseases, such as cancer, sepsis, and rheumatoid arthritis, conditions in which a raise in the plasma tnf- concentration have been described [27, 31 ]. tnf- impairs muscle protein synthesis [32, 33 ] by destabilizing myogenic differentiation and altering transcriptional activity and increases muscle loss [35, 36 ] by targeting proteins to the ubiquitin - proteasome- mediated degradation pathway [3739 ]. have shown that exposure of myoblasts to tnf- inhibits their differentiation [40, 41 ]. the release of ros induced by tnf- induces degradation of the inhibitor-b (ib), which allows the nuclear factor - kappab (nf-b) to translocate to the nucleus and to activate the transcription of several b - dependent genes such as those encoding proinflammatory cytokines, and breakdown of myod and myogenin (regulators of the transition from proliferation to differentiation) in the proteasome. although most research has focused on the muscle wasting effects of tnf-, under specific conditions this cytokine can also promote muscle protein synthesis and stimulate satellite cell proliferation and differentiation [38, 42 ]. among the factors that mediate the different effects of tnf- on protein synthesis or degradation are the state of cell differentiation and the concentration of tnf-. chen. have shown that the effects of tnf- on myogenesis and muscle regeneration are concentration dependent : a low concentration of tnf- (0.05 ng / ml) promoted the differentiation of cultured myoblasts while higher concentrations (0.5 and 5 ng / ml) inhibited it. furthermore, differences in the expression of the tnf- receptor on the surface of different cell types may explain the variable effects of this cytokine. in primary myotubes, low doses of tnf- (1 ng / ml) stimulated maximal protein synthesis, while a much higher dose (50 ng / ml) was required to stimulate maximal protein synthesis in c2c12 myotubes. therefore, the effects of tnf- depend on the concentration and exposure duration : low concentrations help the repair process, while high and prolonged exposure impairs the regeneration process. it is possible that other factors, such as insulin growth factor 1 (igf-1) and inflammatory cytokines mediate the effects of tnf- on protein synthesis / degradation, but their roles are still unclear [43, 44 ]. elevated levels of tnf- have also been implicated in sarcopenia, the age - related loss of muscle mass, strength, and function. it is a key factor contributing to loss of functional mobility, frailty, and mortality in the elderly [46, 47 ]. inflammation, which generally increases with age, is a key factor contributing to sarcopenia, and high level of tnf- is partly responsible for the decrease in muscle protein synthesis that occurs in the elderly [4851 ]. have found elevated levels of tnf- mrna and protein in the skeletal muscle of elderly (81 1 years) when compared to young (23 1 years) men and women. the same authors also showed that resistance exercise decreased tnf- expression in the elderly group, suggesting that tnf- contributes to the age - related muscle wasting, and that resistance exercise may attenuate this process by suppressing tnf- expression. it is well known that gram - negative infection (or the administration of lipopolysaccharides) causes loss of skeletal muscle protein. the decrease in muscle mass results from increases in the rate of proteolysis and decreased rates of protein synthesis [53, 54 ]. a decrease in mtor activity may explain, at least in part, the impaired muscle protein synthesis. have shown that a combination of lps and ifn - gamma dramatically downregulated the autophosphorylation of mtor and its substrates s6k1 and 4e - bp1 via an increased expression of inos (nos2) and excessive production of nitric oxide (no). studies in mice have shown that overexpression of il-6 may increase muscle atrophy. however, under certain conditions il-6 furthermore, al - shanti. demonstrated that il-6 in combination with tnf- stimulate growth of myoblasts. therefore, the role of il-6 in regulating muscle mass appears to be concentration dependent : when overexpressed it may stimulate muscle atrophy, whereas its insufficiency inhibits muscle growth. it mainly controls the expression of genes involved in the immune response, but it also regulates the expression of genes outside the immune system and is therefore able to influence several aspects of normal and disease physiology [59, 60 ]. the classic form of nf-b, a heterodimer of the p50 and p65 subunits, is retained in the cytoplasm through interactions with ib inhibitory proteins. inducing stimuli lead to the phosphorylation and degradation of ib by ib kinases (ikk), allowing nf-b to enter the nucleus and regulate gene expression. nf-b activation causes severe muscle wasting and nf-b is a key factor in cytokine induced loss of skeletal muscle. exercise may activate several signaling cascades and increase the production of ros, which activate nf-b [42, 64 ] in muscle [65, 66 ]. the exercise induced increase in nf-b induces acute - phase proteins and also proinflammatory genes that facilitate the regenerative response in damaged tissues. the involvement of nf-b in muscle damage has been shown in several reports where exhaustive exercise has caused increases in nf-b binding activity. roberts. have also shown that the inflammatory response to exercise is attenuated by chronic training, demonstrating that the activity of nf-b can be seen as a beneficial mediator of exercise - induced adaptations to cellular stress. a training program can also exert an inhibitory effect on nf-b dna binding [70, 71 ]. regular physical training leads to several adaptations in the vascular, oxidative, and inflammatory systems, suggesting that transcriptional regulators of the various nitric oxide synthase (nos) isoforms by nf-b play a key role in training - induced adaptations [72, 73 ]. lima - cabello. have demonstrated that the effects observed after a bout of acute exercise on the nf-b signaling were attenuated by submaximal eccentric exercise training for 8 weeks. also, the levels of nnos, inos and enos expression and nitrotyrosine formation decreased when compared to the acute group. unlike other amino acids, the most active enzyme system for bcaa transamination is found in the skeletal muscle rather than the liver. the first reaction involved in the catabolism of bcaa is the reversible reaction of transamination by isoenzymes bcat (branched - chain aminotransferase) found in both cytosol and mitochondria, which convert amino acids into their respective keto acids (branched - chain keto acids bcka), being the -ketoisocaproic acid (kic) for leucine, -keto--methylvaleric acid (kmv) for isoleucine, and -ketoisovaleric acid (kiv) for valine. the bckas formed may undergo oxidative decarboxylation reactions and/or be released in the blood stream and taken up by different tissues, where they are resynthesized to bcaa or oxidized. it is well known that the amino group from bcaa can be incorporated into the -keto - glutarate (-kg) producing glutamate through glutamate dehydrogenase (gdh). the glutamate can lose the amino group for oxalacetate (oaa) through glutamate - oxalacetate aminotransferase (got) producing aspartate to be used in the purine cycle for regeneration of adenosine monophosphate (amp) from inosinic acid. glutamate can also be metabolized by glutamine synthetase producing glutamine through atp - dependent incorporation of nh3 (figure 2). therefore, the amino group released from bcaa could be easily reincorporated by bcka (reamination) to produce bcaa or directed to the liver to be oxidized. alanine can also be synthetized from bcaa since the glutamate - pyruvate aminotransferase can produce pyruvate which can be transaminated to alanine through pyruvate aminotransferase [78, 79 ]. however, this reaction appears to occur only in situations characterized by absence of energy (i.e., fasting) since skeletal muscle also presents high concentrations of alanine. the intracellular pool of amino acids can be derived from biosynthesis (i.e., nonessential amino acids) or from transfer across the plasma membrane (i.e., essential amino acids). transfer across biological membranes can occur through active (na - dependent) or passive transport (na - independent) due to their ionic nature. in some instances the process of transfer involves not only the entry but exit of amino acids (exchange). the transport system of glutamine is called system a (na - dependent) and the one of leucine is called system l (l ; na - independent), which are integrated. the glutamine entry in the cell the requirement of glutamine is increased (i.e., catabolic illness), theoretically the intracellular content of glutamine is decreased. the decrement in intracellular pool of glutamine may impair leucine transport to inside the cell. on the other hand, the increase of glutamine transport outside the cell may favor the entry of leucine into the cell. if leucine transport is stimulated, the final result of catabolism could increase the availability of glutamine to the cell through glutamate. however, there are no studies evaluating the effects of leucine and glutamine supplementation under inflammatory conditions. bcaa can indirectly modulate the inflammatory status of muscle cells through glutamine production but this reaction appears to occur only in situations characterized by high glutamine consumption and/or decreased glutamate concentrations (i.e., catabolic illnesses, cancer, burning, and sepsis). ehrensvard. first described the importance of glutamine to survival and proliferation of cells such as kidney, intestine, liver, specific neurons in the central nervous system (cns), pancreatic cells, and cells of the immune system. it is widely known that cells of the immune system such as lymphocytes, macrophages, and neutrophils use high rates of glutamine and many functional parameters of immune cells such as t - cell proliferation, b - lymphocyte differentiation, macrophage phagocytosis, antigen presentation, and cytokine production, plus neutrophil superoxide production and apoptosis are enhanced by glutamine. under pathological conditions, which increase the activity of these cells glutamine is extremely used as substrate [8385 ]. it has already been demonstrated that the availability of glutamine influences the production of cytokines such as interleukin- (il-) 2 in cultured rodent lymphocytes and, il-2, il-10, and interferon- (ifn-) in cultured human lymphocytes [87, 88 ]. studies have also demonstrated that glutamine may play an important role on nf-b signal transduction pathways, contributing to the attenuation of local inflammation [8992 ]. when inhibited in the cytoplasm nf-b, the ibs are phosphorylated by the action of specific protein kinases, such as the ikb kinase complex (ikk) at two serine residues with addition of ubiquitin by ubiquitin ligase and degraded by 26s proteasome complex resulting in liberation of nf-b. activated nf-b then binds to the cognate dna - binding sites inducing gene transcription that regulates the innate and adaptive immune response (i.e., t - cell development, maturation, and proliferation) [9395 ]. regarding skeletal muscle remodeling, nf-b acts as foxo, a transcription factor of murf-1 gene which promotes sarcomeric degradation by ups. several cytokines also have their gene expression modulated by nf-b (i.e., tnf- and il-1). it was demonstrated that il-1 presents significant correlation with skeletal muscle cross - sectional area and, therefore, can be considered as an atrophic modulator. nf-b also promotes the transcription of the inducible isoform of nitric oxide synthase (inos) which leads to insulin resistance through nitrosylation of the insulin receptor (ir). under such conditions, the mtor translation pathway has impaired signal transduction through proteins involved in translation initiation such as insulin receptor substrates (irs), akt, and 4e - bp1. counteracting such effects, bcaa (especially leucine) has demonstrated to be a strong nutritional stimulus able to increase skeletal muscle protein synthesis and attenuate protein degradation. for example, hamel. demonstrated that leucine presents one of the strongest inhibitory effects upon ups in muscle cells when compared to the other essential amino acids (for details about the antiproteolytic effects of leucine, please see zanchi. and nicastro. furthermore, it has already been demonstrated that bcaa can stimulate the phosphorylation of proteins involved in the mtor pathway such as akt, mtor, 4e - bp1, eifs, p70s6k in order to improve the protein turnover of the cell [100, 101 ]. since bcaa do not present kinase nor phosphatase activity thus, bcaa can directly modulate the protein turnover of the muscle cell in order to counteract the catabolic and anti - anabolic effects of the inflammatory stimulus. additionally, under pathological conditions, bcaa may influence the inflammatory status of the cell through glutamine production. however, this reaction appears to occur only in situations characterized by a high need of glutamine synthesis. skeletal muscle cells continuously produce reactive oxygen species (ros), which can be generated by various cell organelles and enzymes, such as mitochondria, nad(p)h oxidases, xanthine oxidoreductases, and nitric oxide synthases, whereas their biological activity is opposed by an array of endogenous enzymatic and nonenzymatic antioxidants. normally ros play important physiological roles in skeletal muscle homeostasis and function [104, 105 ]. however, a disturbance in the state of the well - balanced control of oxidant production and antioxidant activity, known as oxidative stress, in turn, is commonly observed during aging and is characteristic of several pathological conditions such as cancer, diabetes, muscle disuse, sepsis, and chronic heart failure. it has been reported that this oxidative stress directs muscle cells into a catabolic state and that chronic exposure leads to wasting. oxidative damage may contribute to skeletal muscle dysfunction and oxidants may stimulate expression and activity of skeletal muscle protein degradation pathways [38, 109 ]. there are several evidences showing that the generation of ros is one mechanistic link between inflammation and skeletal muscle dysfunction and degradation. ros produced by infiltrating immune cells may cause direct injury to muscle tissue or activate catabolic signaling. alternatively, inflammatory cytokines can interact with muscle receptors to initiate catabolic signaling wherein ros are key mediators of this response, acting as second messengers [107, 110, 111 ]. accordingly, overexpression of tnf- in transgenic mice and single intraperitoneal doses of this cytokine promotes muscle wasting that can be attenuated by antioxidants [103, 108 ]. on the other hand, ros activate transcription factors (e.g., nf-b and ap-1) and upregulate expression of proinflammatory genes such as tnf-, il-6 and c - reactive protein, which are involved in the pathogenesis of inflammation [113, 114 ]. although administration of bcaa has been investigated as a tool that could exert an anti - inflammatory role or indirectly modulate the inflammatory status in order to favor the biological response and tissue adaptation, less is known about the relationship between this strategy and oxidative stress modulating skeletal muscle structure and function. there are some emerging reports describing that ros modulate the efficiency and effectiveness of the adaptive responses of skeletal muscle induced by some bcaa, especially leucine [115, 116 ]. regarding bcaa supplementation and oxidative stress, an interesting study has shown that this nonpharmacological strategy increases expression of genes involved in antioxidant defense and reduces ros production in cardiac and skeletal muscles in middle - aged mice, which was accompanied by preserved skeletal muscle fiber size, enhanced physical endurance and increased average life span. of interest, bcaa - mediated effects were even more remarkable in middle - aged mice submitted to long - term exercise training (running 30 to 60 min 5 days / week for 4 weeks). in young animals (46 months old), aging has been described as a condition characterized by anabolic resistance to nutrients, especially amino acids, which impairs muscle protein synthesis and contributes to muscle wasting. such resistance is partially associated to oxidative stress and low - grade inflammation and may be attenuated by chronic anti - inflammatory treatment.. demonstrated that older adults who received omega-3 fatty acids for 8 weeks increased the hyperaminoacidemia - hyperinsulinemia - induced muscle protein synthesis when compared to the control group (corn oil), which was accompanied by greater phosphorylation of mtor and p70s6k. therefore, the anti - inflammatory action of nutrients such as omega-3 may attenuate anabolic resistance in order to favor amino acid - induced muscle protein synthesis. concerning bcaa supplementation, marzani. demonstrated that old rats supplemented with leucine and with an antioxidant mixture (rutin, vitamin e, vitamin a, zinc, and selenium) showed higher protein synthesis when compared to old - control animals and that these effects could be mediated through a reduction in the inflammatory state, which decreased with antioxidant supplementation. under inflammatory conditions, such as aging, anabolic resistance occurs mainly because of elevated proinflammatory cytokines. thus, antioxidant supplementation may attenuate anabolic resistance and therefore favor leucine action on skeletal muscle protein turnover. it is well accepted that their catabolic reactions can be easily modulated through alterations in metabolic demands, such as in inflammatory status. however, it is unknown if bcaa can directly modulate the status of proteins involved in inflammatory pathways and if this effect could reflect on protein turnover. since glutamine is highly consumed by inflammatory cells, it appears to be a mediator of bcaa and inflammation but this reaction is dependent of glutamate content and gdh activity in skeletal muscle. future studies should address the effects of bcaa, glutamine and the amino acids transporter activity under proinflammatory conditions.	skeletal muscle protein turnover is modulated by intracellular signaling pathways involved in protein synthesis, degradation, and inflammation. the proinflammatory status of muscle cells, observed in pathological conditions such as cancer, aging, and sepsis, can directly modulate protein translation initiation and muscle proteolysis, contributing to negative protein turnover. in this context, branched - chain amino acids (bcaas), especially leucine, have been described as a strong nutritional stimulus able to enhance protein translation initiation and attenuate proteolysis. furthermore, under inflammatory conditions, bcaa can be transaminated to glutamate in order to increase glutamine synthesis, which is a substrate highly consumed by inflammatory cells such as macrophages. the present paper describes the role of inflammation on muscle remodeling and the possible metabolic and cellular effects of bcaa supplementation in the modulation of inflammatory status of skeletal muscle and the consequences on protein synthesis and degradation.
with the advent of the hpv vaccine, data on genotype - specific prevalence of hpv infection in sexually active female population would be useful to predict the potential benefits of hpv vaccination and to monitor the impact of vaccination on hpv type replacement. a recent meta - analysis estimated that the worldwide hpv prevalence in the cervix of women with normal cytology is about 10.4%. hpv-16 is the most common hpv type both in women with normal cervical cytology and in those with cervical lesions or cancer, followed by hpv-18 in europe, central and south america, hpv-52 and hpv-58 in asia, and hpv-53 and hpv-52 in northern america [1, 2 ]. the pattern of hpv type distribution may vary even among countries and regions, being related to sexual habits and migrations of people, as suggested by detection of hpv types typically found in asia and africa and also in southern europe [2, 3 ]. this cross - sectional study describes hpv prevalence and type distribution in cervical samples from women resident in southern areas of italy, that is, apulia region and naples area, who were referred for opportunistic screening and for gynaecological care for evaluation of suspected hpv - related lesions. from 2005 to 2008, a total of 654 genital swabs, which were collected from women aged from 17 to 60 years and living in apulia region (provinces of lecce, brindisi, taranto and bari) and naples city in the south of italy, were analyzed. women were referred to laboratory dr. pignatelli (lecce - italy, departments of virology & molecular biology and cyto - hystopatology) for opportunistic screening and for evaluation of hpv - associated lesions, so this patient population does not represent the general population of women attending public screening programs. the in force italian privacy law did not allow to collect any data about life - style, sexual partners ' number, and so forth. most samples (87%) were cervical swabs, 3.5% were vulvar swabs, 3% vaginal, and 6.5% other genital swabs. total dna was purified from 150 l cellular suspension by using an abi prism 6100 system (applied biosystems inc., monza, italy) and eluted in a final volume of 120 l ; 10 l of each dna sample were used for hpv pcr analysis. hpv pcr was performed using the spf10 primers (innolipa hpv genotyping, innogenetics srl pomezia, italy), which amplify a 65-bp fragment of the l1 open reading frame and allow detection of at least 28 different hpv types by hybridization on oligonucleotide probes [4, 5 ], as described in the commercial kit protocol. appropriate negative and positive controls were included in each session to monitor cross - contamination and efficiency of dna purification and pcr amplification. the inno - lipa hpv genotyping system can identify the following hpv types : hpv-6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69/71, 70, 73, 74 and 82. hpv types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 were defined high - risk (hr) hpv types, whereas the other types were defined low - risk (lr) hpvs. recently, hpv classification has been revised and hpv-26, 53, 66, 73, and 82 have been downgraded from the category of probably carcinogenic (group 2a) to possibly carcinogenic (group 2b) [7, 8 ]., the strips were visually interpreted using the reference guide provided, according to the manufacturer 's protocol. the association between hpv infection and clinical variables was assessed through the chi - square test. hpv dna was identified in 300 of 654 samples (45.9%) ; 175 (58.3%) samples had single infection and 125 (41.7%) had multiple infection. among hpv - positive samples, 267 (89.0%) had hr - hpv infection (table 1). hpv-6 and hpv-16 were the most commonly detected types, both in single and multiple infections, followed by hpv-51, hpv-31, hpv-66, and hpv-53. hpv-16 was significantly more frequent in single infection, whereas other hr - hpv types (i.e., hpv-56, hpv-58, and hpv-68) were more frequently identified in multiple infection (table 1). hpv dna detection rate decreased with age, placing on 56.1% for women under 40 and on 37.7% in older ones (chi - square trend test, p <.0001), as well as the prevalence of multiple hpv infections tended to decrease with age, probably due to selection by immunity or hpv type fitness, as suggested by the different distribution of hpv types in different age groups. in the 231 women who performed both hpv typing and pap test, (n = 208) and asc - us (n = 11) and in 83.3% of lsil (n = 12 ; no cases of hsil were identified). detection of hr - hpvs was common (80%) both in normal and abnormal pap smears. hpv dna was detected in 56.1% of biopsies with negative histology, 60.9% of which had hr - hpvs. the prevalence of hpv - dna (and hr - hpv dna) detection progressively increases with the worsening of histological pattern, with 100% cin-2/3 cases positive for hr - hpvs, including hpv-16, hpv-18, hpv-31, and hpv-51 (table 2). this study on a female population from the south of italy undergoing hpv testing for opportunistic screening or for evaluation of genital lesions shows a relatively high prevalence of hpv infection, 40% of which involved multiple hpv types. the most frequent hpv types were hpv-6, hpv-16, hpv-51, hpv-31, hpv-53, and hpv-66, with a different distribution in young versus older women, suggesting the existence of a natural selection of hpv types which preserve a better fitness, such as hpv-16 and hpv-31. hr - hpvs were detected in all cin-2/3 samples, with hpv-16, hpv-18, hpv-31, and hpv-51 as the most frequent types. however, hr - hpv types were detected also in a high rate in women with a negative pap test or with a negative histology. these findings are in agreement with other studies on hpv infection and type distribution in italian women from different regions and in other european countries. in fact, a high prevalence of hpv infection, mostly caused by hr - hpv types, has been observed also in other italian regions [1018 ]. all studies concordantly demonstrate that hpv-16 is by far the most common type, followed by hpv-66, hpv-31, hpv-51, hpv-52, hpv-53, and hpv-58 (the rating order varies among series), while hpv-18 and hpv-45 are less common [1118 ]. at variance with studies on women participating in organized cervical screening programs, hpv-6 was frequently detected in our patients, who were often referred for suspected condylomas. similar hpv type distribution has been recently reported from other european countries, such as denmark and germany, where hpv-16 was shown to be the most common hpv type, followed by hpv-31, hpv-51, hpv-52, hpv-53, and hpv-66. in the present study, hr - hpvs, including hpv-16, hpv-18, hpv-31, and hpv-51, were detected in 100% of cin2/3 lesions. the same distribution of hr - hpv types was identified in high - grade cervical lesions investigated in other italian regions [12, 18, 21 ] and european countries [19, 20, 22 ]. the results of this paper provide information on hpv type distribution in women from southern italy and, by a revision of the recent literature, indicate that the epidemiology of hpv infection in these areas is not different from other italian regions and from other european countries. besides hpv-16 and hpv-18, which are targeted by the currently available hpv vaccines, other hr - hpvs, such as hpv-31 and hpv-51, appear to be diffused in the population of sexually active women and to be associated with high - grade cervical lesions. these hpvs should be taken into consideration in the development of enhanced vaccines with larger type coverage.	human papillomavirus (hpv) type - specific distribution was evaluated in genital samples collected from 654 women from the south of italy undergoing voluntary screening and correlated with cyto - histological abnormalities. hpv dna was detected in 45.9% of the samples, 41.7% of which had multiple infection and 89.0% had high - risk hpv infection. the prevalence of hpv infection and the rate of multiple infections decreased with age, suggesting natural selection of hpv types with better fitness. in line with other italian studies, the most common hpv types were hpv-6 and hpv-16, followed by hpv-51, hpv-31, hpv-53, and hpv-66, in women with both normal and abnormal cytology. cervical intraepithelial lesions grade 2 or 3 were associated with high - risk hpv-16, hpv-18, hpv-31, and hpv-51 infection. these data indicate that prophylactic hpv vaccination is expected to reduce the burden of hpv - related cervical lesions in this population, but also suggest the potential utility of new vaccines with larger type coverage.
xanthogranulomatous diseases of the orbit are a group of rare disorders of unknown etiology and pathogenesis affecting the skin and subcutaneous tissues of the periorbital areas and the ocular adnexa. the group displays considerable heterogeneity with some patients manifesting only local symptoms and signs at the face, and others manifesting systemic disorders, while its severity spans from an indolent disease to an invasive disease that may even lead to death.1 diagnosis is difficult and in some cases clinicopathological investigation fails to provide a classification more specific than non - langerhans histiocyte proliferative disorder. below we present a case of a man with long - standing periorbital xanthogranuloma who was treated with cyclophosphamide (cyc) and methylprednisolone (mp). a 53-year - old caucasian man presented to our outpatient rheumatology clinic for bilateral periorbital swelling. it had first started 10 years earlier with gradual swelling of both upper and lower eyelids and cheeks. the swelling was painless with a yellow orange discoloration without other abnormalities of the overlying skin. it was so voluminous that it limited the opening of the eyes and distorted the patient s physiognomy. two years prior to the periorbital swelling he had developed bronchial asthma and allergic rhinitis with nasal polyps. his family history also revealed hypertension and cardiovascular events. a few years before presenting to us, he had been investigated in a dermatologic hospital where he had an eyelid biopsy performed, consistent with xanthogranuloma. he was treated with immunoglobulin infusions, corticosteroids per os and isotretinoin without sustained improvement. there was obvious swelling of all four eyelids, the cheeks and temples which consisted of a painless, soft, slightly lobular tissue (figure 1). the patient s vision and ocular motility were not affected, although the opening of the eyes was limited. laboratory investigation showed eosinophilia (total eosinophils 1085/l) and elevated acute phase reactants (erythrocyte sedimentation rate 53 mm / hour ; c - reactive protein 9 mg / l [normal < 6 mg / l ]). urinalysis, liver and kidney biochemistry, serum lipid profile and thyroid hormones were normal. immunological tests showed elevated igg immunoglobulin (2500 mg / dl, normal 7511560 mg / dl), a slightly depressed iga and normal ige levels. x - rays of the chest and the long bones were normal. a magnetic resonance imaging (mri) of the orbits demonstrated eyelid swelling, mucosal thickening of all sinuses and abnormal signal and contrast uptake of the temporal, masseter and pterygoid muscles bilaterally corresponding to infiltration by a pathological process moreover, the oculomotor muscles were swollen and infiltrated with pathological signal intensity (figure 2). computed tomography (ct) of the lungs showed small interstitial nodules, bronchiolitis - like opacities and hilar lymph node enlargement (up to 2 cm). a new biopsy of the left upper eyelid showed large areas of xanthogranulomatous infiltration throughout the reticular dermis into the subcutaneous fat. the granulomatous infiltrate was composed of lymphocytes, epithelioid cells, foamy histiocytes and giant cells, many of them of touton type. the lymphocytes were numerous and nodular lymphocytic aggregates were occasionally present (figure 3). clinical differential diagnosis included xanthelasma, thyroid disease, sarcoidosis, allergic granulomatosis, non - langerhans histiocytoses (juvenile xanthogranuloma, papular xanthoma, xanthoma disseminatum), periocular xanthogranuloma with adult - onset asthma (pxaoa), necrobiotic xanthogranuloma (nxg) and erdheim chester disease (ecd). immunological and histopathologic studies excluded the first four diagnoses. considering that the patient suffered from an infiltrative nonmalignant non - langerhans histiocytic disorder that was limited to facial structures and was associated with elevated acute phase reactants, a polyclonal activation of the humoral arm of the adaptive immunity, and atopic manifestations, we favored the diagnosis of pxaoa. the patient was treated with 6 monthly infusions of cyc 750 mg / m body surface area and mp 0.8 mg / kg body weight per os with gradual tapering. after completion of all six cyc infusions the periorbital swelling had significantly regressed (figures 4a, b), complement levels had normalized and a repeat mri of the orbits showed regression of the swelling and of the abnormal signal of the eyelids and the masticatory and oculomotor muscles (figures 4c, d). mp was further tapered, while azathioprine was added to maintain the effect of the cyc / mp combination and allow for further corticosteroid tapering. nonmalignant non - langerhans histiocytoses are classified on the basis of the cell types that predominate in the histopathologic specimens and on whether the lesions are solitary, multiple or systemic.2 diseases with predominant xanthomatized histiocytes, such as juvenile xanthogranuloma, papular xanthoma, xanthoma disseminatum, etc, form a major part of this group. the age of disease onset, the specific skin changes and the potential involvement of other organs and tissues, including the orbit and ocular adnexa, varies according to the particular disease subtype.3,4 nxg displays histopathologic features similar to xanthogranulomas, ie, foamy cells and touton giant cells in relation with areas of collagen degeneration, lymphoid aggregates or follicles occasionally with germinal centers, plasma cell foci and cholesterol clefts.5 it affects equally men and women in their sixth decade and usually involves the periorbital areas, trunk, and proximal parts of the extremities. lesions initially resemble xanthelasmas, but they gradually progress to indurate yellow - brownish plaques with a tendency to ulcerate and even cause destructive skin changes. it is often associated with immune dysfunction, namely monoclonal paraproteinemia (80%), hypocomplementemia and cryoglobulinemia, whereas it occasionally is associated with multiple myeloma, non - hodgkin lymphoma (nhl) or other hematologic dyscrasias.6 association with bronchial asthma is weak, if any.4 the cause of the disease remains elusive, although it is presumed that special functional properties of the paraprotein are responsible for an aberrant immune response.6 ecd is characterized by abnormal and aggressive proliferation of non - langerhans foamy histiocytes. the main manifestations are osteosclerosis of the diaphyses and metaphyses of the long bones and diabetes insipidus due to pituitary involvement. ocular manifestations include xanthelasma - like lesions and exophthalmos and respiratory involvement includes pulmonary infiltrates, fibrosis and pleural effusion.4,7 recently, jakobiec and colleagues4 described six patients with periocular xanthogranuloma and concomitant asthma. one of the patients, on whom a necrobiotic lesion was observed later in the disease course, developed paraproteinemia as well. whether they form different aspects of the same disease spectrum or are different diseases is not clearly evident. on clinical grounds, relating pxaoa and nxg might have therapeutic and prognostic implications, particularly regarding the risk for hematologic dyscrasias. sivak - callcott and colleagues1 reviewed 137 cases of adult xanthogranulomatous disease of the orbit and ocular adnexa, among them 21 cases of pxaoa, 72 cases of nxg and 36 cases of ecd. patients with pxaoa often had signs of immune dysfunction, such as polyclonal gammapathy and reactive lymphadenopathy, while one patient developed burkitt s lymphoma and another nhl. histopathologically nxg more often showed pallisading epithelioid histiocytes together with necrobiosis, while large lymphocytic aggregates were more common in pxaoa. our patient had periorbital disease with concurrent immunological abnormalities, namely mediastinal lymphadenopathy, hypocomplementemia, and diffuse hypergammaglobulinemia. furthermore, atopic phenomena (asthma, rhinosinusitis, eosinophilia) were present. ecd diagnosis was rejected because there was no bone involvement, while lung manifestations in ecd do not include asthma.4 juvenile xanthogranuloma can be differentiated on clinical grounds (lesions are usually solitary, but, when multifocal, other parts besides the eyelids are commonly affected).3 xanthoma disseminatum can be excluded, since the patient exhibited no axillary or inguinal lesions and no mucosal involvement.3 papular xanthoma histologically rarely exhibits inflammatory infiltrates.3 regarding nxg, whereas no sites of necrobiosis were evident in two histopathological examinations, the presence of such lesions elsewhere in the pathological tissue can not be excluded. furthermore, foci of fibrosis were observed that might represent scarring of a previously necrobiotic focus. however, ulceration or ocular inflammation had never occurred in 10 years and no monoclonal b - cell abnormality could be observed. for those reasons we classified the patient as suffering from pxaoa. surgical excision is often followed by disease recurrence and scarring.4,6 orbital radiotherapy and topical corticosteroids have been used with variable results in some cases of nxg.5,6,8 patients with pxaoa often admit improvement of periorbital swelling, whenever treated with systemic corticosteroids for their asthma.4,9 there have also been various reports of pxaoa responding to systemic corticosteroids, but the disease often recurs after corticosteroid withdrawal, as was the case for our own patient as well.10 there are also several reports of effective treatment of nxg with chemotherapeutic agents particularly alkylating agents, such as chrorambucil, melphalan and cyc alone or in combination with corticosteroids.5,6,8,11,12 jakobiec suggests that patients without systemic disease receive a course of high - dose systemic steroids and periorbital radiotherapy ; if the disease does not respond, then light chemotherapy with corticosteroids could be administered.4 long - term outcomes of pxaoa following various therapeutic modalities are rarely reported. two of three patients reported by jakobiec relapsed after surgery and corticosteroids were only temporarily effective in one of them. one patient with concomitant monoclonal gammapathy responded to combined chemotherapy.4 in the series by sivak - callcott, surgery was successful in six out of eight and corticosteroids in two out of two cases of pxaoa.1 a 46-year - old patient worsened after topical radiotherapy and later, following treatment with a combination of corticosteroids and cyclosporine a, he developed a lymphoma. overall, no deaths due to pxaoa were reported.1 in rheumatology considerable experience exists concerning the use of cyc in inflammatory diseases owing to b - cell dysregulation, such as lupus nephritis, or granulomatous autoimmune diseases, such as wegener s granulomatosis and churg strauss syndrome. we thus treated the patient with 6 monthly cyc infusions and mp aiming at a rapid suppression of the inflammatory process and a disease regression that would sustain after corticosteroid withdrawal. as regards prognosis, the potential overlap between pxaoa and nxg and the known association of nxg with hematologic malignancies require close follow - up of the patient in order to timely uncover a potential emerging malignancy. thus, regular clinical evaluation, full blood count, inflammatory markers, and serum protein electrophoresis should be performed.	periocular xanthogranulomatous diseases are a rare group of disorders which are characterized by a predilection to affect the orbit and ocular adnexa and special histopathological features, in particular infiltrates comprising non - langerhans - derived foamy histiocytes and touton giant cells. the differential diagnosis is difficult and occasionally definite diagnosis can not be established even after clinical and histopathological findings are taken together. we describe a case of a middle - aged man who presented with a 10-year history of voluminous eyelid swelling with concomitant late - onset atopic manifestations, namely bronchial asthma and allergic rhinitis with nasal polyps. after thorough clinical and laboratory investigation, including a biopsy of the eyelid, we classified the patient s disease to a rare entity that has been relatively recently described : periocular xanthogranuloma associated with adult - onset asthma. in a review of the literature, no prospective trials concerning the treatment of this disease were found. the literature mainly contained case reports and case series in which corticosteroids and chemotherapy with alkylating agents have been reported to be beneficial. we treated our patient with a combination of oral corticosteroids and cyclophosphamide pulses and we observed substantial regression of the eyelid masses together with a normalization of systemic immunologic abnormalities.
venous thromboembolism (vte), which includes deep vein thrombosis (dvt) and pulmonary embolus (pe), is a common postoperative complication. a high incidence of vte has been well demonstrated in hip and knee arthroplasty.1 the incidence of dvt has been as high as 40% in some studies.1 2 less is known about the incidence of vte in spinal surgery.3 4 however, recent studies have shown that the incidence of vte following spinal surgery may be higher than previously reported.4 although the overall prevalence of symptomatic vte is considerably lower when compared with major lower extremity surgery, fatal pe is a well - known cause of postoperative mortality following spine surgery, as high as 2% in certain populations.5 for this reason, it is critical to determine the incidence of vte in patients who undergo spine surgery as well as risk factors. several studies have examined the incidence of vte in thoracolumbar procedures including various subgroups such as thoracolumbar trauma, deformity correction, and oncologic procedures.6 7 however, the number of studies looking specifically at vte in cervical spine surgery is limited. the goal of this study was to examine the 30-day incidence of and risk factors for vte including pe and dvt at a single institution between 1995 and 2012. we hypothesize that multiple risk factors will be identified including various patient comorbidities and procedure - related risk factors. after institutional review board approval, an institutional database at a level i trauma center was queried to create a list of 5,405 patients 18 years or older who underwent cervical spine procedures based on current procedural terminology (cpt) codes for cervical diskectomy, corpectomy, laminectomy, laminoplasty, and anterior or posterior arthrodesis. this group of patients was cross - referenced for lower extremity dvt or acute pe based on international statistical classification of disease and related health problems 9th revision (icd-9-cm) codes. the charts of the patients identified were reviewed to determine whether the vte event occurred within 30 days postoperatively. patients who suffered a vte outside of the 30-day postoperative period or for whom the cervical surgery was not the primary procedure were excluded from the study. the prevalence of various comorbidities were identified between the vte and non - vte patients including chronic venous insufficiency, obesity (body mass index of 30 or greater), atrial fibrillation, ischemic heart disease, malignancy, hypertension, peripheral vascular disease, diabetes, and tobacco use based on icd-9-cm codes. given the size of the patient sample, a matched cohort was created to analyze other potential risk factors for vte. multiple potential risk factors were reviewed in the patient records including demographics, length of stay, surgical indications (elective, traumatic, oncologic, or infectious), surgical approach (anterior, posterior, or both), number of surgical levels, comorbid traumatic injuries (head, abdominal, or lower extremity trauma), estimated blood loss, use of iliac crest autograft, paralysis (diagnosis of complete or incomplete quadriplegia), and staged surgery (defined as two planned cervical surgeries within 7 days). the medical record was also reviewed for use of perioperative vte prophylaxis including inferior vena cava (ivc) filters, warfarin, and low - molecular - weight heparin. the data was summarized and reported as mean (standard deviation) for continuous variables and count (percentage) for categorical data. the analysis focused on comparing patients who experienced a vte within 30 days following surgery (cases) to a set of matched patients who did not experience a vte within 30 days of surgery (controls). the controls were matched to cases in a 2:1 ratio on age (5 years), sex, and year of surgery (2 years). the association of potential risk factors with the outcome of vte was evaluated using conditional logistic regression. all statistical tests were two - sided, and p values less than 0.05 were considered significant. all analysis was conducted using sas version 9.2 (sas institute inc., cary, north carolina, united states). of the 5,405 patients who underwent cervical spine surgery in our cohort, 85 (1.57%) suffered a vte event. of those, 55 (1.02%) suffered a dvt and 51 (0.94%) suffered a pe. patients were not routinely screened for vte and all diagnostic tests were performed based on clinical suspicion. the frequency of medical comorbidities between the vte and non - vte groups is demonstrated in fig. 1. univariate analysis of patient comorbidities demonstrated chronic venous insufficiency (odds ratio [or ] 3.40), atrial fibrillation (or 2.69), obesity (or 2.67), and ischemic heart disease (or 2.21) as significant risk factors for vte (table 1). of the 85 cases of vte identified, 3 were excluded from the matched cohort analysis secondary to insufficient data in the medical record. thus 82 cases of vte were matched 2:1 based on age and sex to a control group of 164 cases without vte. abbreviations : afib, atrial fibrillation ; cvi, chronic venous insufficiency ; dm, diabetes mellitus ; ihd, ischemic heart disease ; pvd, peripheral vascular disease ; vte, venous thromboembolism. the average age of the cohort was 58.6 16.9 years with a range from 21 to 89 years old. the most common surgical indication was elective in both the vte patients (39.0%) and controls (79.3%). however, a high percentage (34.1%) of the vte cases were found in patients treated for cervical trauma. a posterior surgical approach was the most common (70.3%) in the cohort with an increased percentage of combined approach in the vte cohort (23.2%) relative to the controls (3%). the overall incidence of comorbid traumatic injuries was low (5.3%) but higher in the vte cohort (13.4%). the vte cases had higher estimated blood loss (average > 450 ml). they also more frequently involved four or more levels (46.3% versus 28.7%) and use of iliac crest autograft (39% versus 14%). a higher percentage of vte patients were paraplegic at the time of surgery (23.1%) and underwent staged surgery (17.1%). per our institution 's protocol, a form of additional vte prophylaxis was started in 22 patients (25.9%) in the vte group (prior to the vte event) and 8 patients (4.9%) in the control group. decisions regarding additional prophylaxis are made on a case - by - case basis by the treating teams based on assessed vte risk. prophylactic subcutaneous heparin was started in 5 patients (6.1%) in the vte group compared with 4 patients (2.4%) in the control group. prophylactic ivc filter was placed by interventional radiology in 14 patients (16.5%) in the vte group compared with 1 patient (0.6%) in the control group. warfarin was restarted for atrial fibrillation in 3 patients (3.5%) in the vte group compared with 3 patients (1.8%) in the control group. in the vte group following the vte event, 6 patients (7.1%) were placed on subcutaneous heparin and 7 patients (8.2%) were placed on coumadin. ivc filters were placed in 2 patients (2.4%) following an acute pe. despite prophylactic ivc filter placement in 14 patients in the vte group of the 2 patients who had an ivc filter placed following a pe, 1 had a superior vena cava filter placed as well. staged surgery (or 28.0) and paralysis (or 19.0) were highly predictive of vte. nonelective surgical indications including infection (or 18.5), trauma (or 11.1), and oncologic procedures (or 5.2) were all significant risk factors as well. in addition, comorbid traumatic injuries (or > 10) and length of stay greater than 5 days (or 3.47) were correlated with vte events. with regards to surgical characteristics, combined anterior - posterior approach (or 7.46 in comparison to posterior alone), use of iliac crest autograft (or 4.16), 4 or more operative levels (or 4.84, compared with 1 operative level only), and estimated blood loss greater than 300 ml (or 1.66) were all predictive of vte. estimated blood loss and length of stay were not included in the multivariate analysis secondary to insufficient data and an overlapping time period with the 30-day outcome window. following multivariate analysis, three independent risk factors were identified for vte : nonelective surgery (or 6.29), paralysis (or 7.86), and staged surgery (or 35.7) (table 4). this study describes the 30-day vte rate in a large series of 5,405 consecutive patients who underwent cervical spine surgery at a single institution between 1995 and 2012. our vte rate of 1.57% with a 1.02% dvt rate and 0.94% pe rate are consistent with reports in the literature. a recent review of thromboembolic disease in spine surgery highlighted the limited amount of literature on the subject. glotzbecker utilized a systematic review to address the incidence of dvt or pe after spinal surgery.4 a total of 25 studies satisfied the inclusion criteria. due to the diversity of the studies however, the authors were able to determine that the overall vte risk ranged from 0.3 to 31%.4 there are significant limitations in this study secondary to the heterogeneity of included studies, some of which involved select high - risk populations, did not report pe rates, looked exclusively at thoracolumbar surgery, or utilized screening surveillance methods.7 8 9 10 11 other studies have reported dvt and pe rates of 3.7 and 2.2%, respectively.12 pe rates have been reported as high as 12%.5 with regards to cervical surgery, epstein prospectively compared dvt rates in a limited group of patients undergoing anterior corpectomy (1%) versus multilevel anterior corpectomy and posterior fusion (7%).13 a recent review of the nationwide inpatient sample (nis) demonstrated a dvt rate of 0.27 to 1.34% and a pe rate of 0.10 to 0.63% in patients undergoing surgery for degenerative conditions of the cervical spine.14 this rate is consistent with the overall vte rates reported in this study. although this study shows a significant rate of vte following cervical spine surgery, the nis database is limited in that it provides no follow - up after discharge, is based entirely on icd-9 codes, and is unable to determine specific patient- and procedure - related risk factors found in review of patient 's medical records. although the overall incidence of vte was low, several patient comorbidities were identified as risk factors for vte following cervical spine surgery. chronic venous insufficiency was identified as a significant risk factor for vte, likely secondary to global venous dysfunction, which causes increased stasis of blood in the lower extremity veins.15 16 obesity was also shown to be a risk factor for vte, which is consistent with previous studies looking at lumbar spine surgery.17 ischemic heart disease was also a predictor for vte, which is expected because these patients are likely more prone to thrombosis and have additional medical comorbidities that increase their vte risk.18 interestingly, atrial fibrillation was also identified as a risk factor despite many of these patients being on chronic anticoagulation therapy. this finding is likely reflective as well of a patient population with a higher number of medical comorbidities but may also represent a temporary procoagulant state that occurs following resumption of warfarin.19 smoking was found to be slightly protective in our study but is likely a clinically insignificant finding given previous evidence showing an increased risk for vte in this patient population.20 21 22 although these patient comorbidities identified as risk factors are probably unmodifiable, they may help guide risk stratification and identify patients who may benefit from increased surveillance or chemical prophylaxis. performing a matched cohort analysis allowed us to identify additional risk factors related to the surgical approach, surgical indication, and operative details. in general, we found the greater magnitude and morbidity of the procedure, the higher the risk for postoperative vte. thus, combined anterior - posterior surgery, use of iliac crest autograft, increased number of surgical levels, and increased estimated blood loss were all found to be significant predictors of vte in the univariate analysis. such surgeries would be anticipated to have increased operative times, longer periods of recumbency, and slower postoperative mobilization, increasing the risk of vte.6 11 similarly, comorbid traumatic injuries may lead to additional surgeries and more prolonged immobilization, increasing the risk of vte.23 increased length of stay was also identified as a risk factor for vte. certainly, caution should be applied in interpreting this result because increased length of stay may often be secondary to vte rather than causative. regardless, increased length of stay in the vte cohort has important financial implications as it may raise hospital costs as much as twofold.14 three independent risk factors for vte were identified in this study. of the 15 cases in this cohort that underwent staged surgery, 6 operations were for traumatic injury, 4 were for oncologic resection, 2 were for infection, and 1 each for the treatment of cervical myelopathy, pseudarthrosis, and deformity. of these 15 cases, all but 3 were done with a combined anterior - posterior surgical approach. this finding is interesting because it suggests that although a combined anterior - posterior approach is correlated with postoperative vte, the added morbidity of staging the surgery is the significant independent risk factor. previous studies have demonstrated higher vte rates with combined approaches and staged surgery.6 7 8 9 although only 1/15 patients who underwent staged surgery received prophylactic ivc filter placement, previous evidence suggests these patients may benefit from routine prophylaxis.7 24 paralysis (or 7.86) was also found to be an independent risk factor. previous studies have demonstrated an increased rate of vte in spinal cord injury patients with dvt rates as high as 38.6%.25 26 nonelective surgery (or 6.29) including traumatic, oncologic, and infectious indications was a predictor of vte. higher rates of vte have been described for patients undergoing surgery for spinal trauma, even after excluding spinal cord injury.4 malignancy is associated with a prothrombotic state that leads to an increase in vte events.27 although this study identifies several risk factors for vte in patients undergoing cervical spine surgery, further studies are needed to determine which patients would benefit from screening surveillance, chemical vte prophylaxis, or ivc filters. the benefits of intermittent pneumatic compression (ipc) devices, which were used in all patients in this cohort, have been well demonstrated in the literature. the effectiveness of ipc has been shown following both lumbar and cervical spine surgery for vte prophylaxis.2 13 epstein demonstrated that the 1 to 2% pe rate following anterior cervical surgery (with ipc prophylaxis) in their cohort was similar to rates reported following low - dose heparin prophylaxis regimens without the associated hemorrhage risk.13 although spinal epidural hematoma is always a concern following the initiation of chemical vte prophylaxis,28 29 30 there may be a role for more aggressive initiation of chemoprophylaxis in certain patient populations with close neurologic monitoring.31 despite chemoprophylaxis in one series, no difference in vte rate was seen, although these results may be subject to selection bias given the study 's retrospective nature.31 a randomized trial by kurtoglu showed no difference in vte rates or mortality with postoperative chemical prophylaxis although other prospective studies have demonstrated a reduced vte rate following chemical prophylaxis.32 33 the north american spine society clinical practice guidelines for antithrombotic therapies in spine surgery suggest that chemoprophylaxis may be warranted in complex cases, combined anterior - posterior approaches, spinal cord injury, or malignancy.34 ivc filters offer an alternative that may be effective in preventing pe in spinal surgery patients contraindicated for chemical prophylaxis, such as those undergoing staged surgery. although previous studies have demonstrated the effectiveness of ivc filters, there are significant risks associated with implantation and removal.5 33 in addition, this study found four patients who developed acute pe despite prophylactic ivc filter placement. this outcome has been described in previous reports and can occur in the setting of an unrecognized upper extremity dvt, collateral venous channels, or a clot forming on the proximal portion of the filter.24 35 36 regardless, there is little consensus regarding postoperative vte prophylaxis for spinal surgery as evidenced by a recent study showing that the 63% of surgeons base their decisions for vte prophylaxis on personal experience.37 this study does have several limitations. the vte rate may be subject to underreporting. given the large cohort of patients, icd-9-cm codes were used to identify patients who suffered a vte event, which may lead to errors related to miscoding. in addition, surveillance methods are not standardized, possibly leading to underdiagnosis of vte events. given the retrospective nature of the study, patients may have been lost to follow - up ; this rate is estimated to be 6% in this cohort. in addition, the follow - up was limited to 30 days, which would miss patients with more delayed presentations of vte. although this study makes several observations regarding vte prophylaxis, no clear recommendations can be made regarding prophylaxis or screening based on this data. in conclusion, in a review of 5,405 cervical spine cases at a single institution, a vte rate of 1.57% was established, with a dvt rate of 1.02% and pe rate of 0.94%. several patient comorbidities were identified as risk factors including chronic venous insufficiency, atrial fibrillation, obesity, and ischemic heart disease. after a matched cohort analysis, several additional risk factors were identified including combined anterior - posterior approach, comorbid traumatic injuries, use of iliac crest autograft, number of surgical levels, increased blood loss, and increased length of stay. three independent risk factors for vte were identified including staged surgery, nonelective surgery, and paralysis. clearly, there is a subset of patients at an increased risk for postoperative vte following cervical spine surgery. further studies are needed to determine if certain subsets may benefit from more aggressive vte screening or prophylaxis.	study design retrospective matched cohort analysis.objective the majority of the literature on venous thromboembolism (vte) after spine surgery is limited to studies of thoracolumbar surgery. less is known regarding the incidence of vte and associated risk factors following cervical spine surgery.methods a total of 5,405 patients at our institution underwent cervical diskectomy, laminectomy, corpectomy, laminoplasty, or fusion between 1995 and 2012 ; 85 of the 5,405 patients (1.57%) suffered either a dvt (55) or pulmonary embolus (51) within 30 days postoperatively. the cases were matched 1:2 to controls based on age, sex, and date of surgery. data regarding multiple perioperative factors, demographics, and comorbidities was collected.results several risk factors were identified for vte. significant medical comorbidities included chronic venous insufficiency (odds ratio [or ] = 3.40), atrial fibrillation (or = 2.69), obesity (or = 2.67), and ischemic heart disease (or = 2.18). staged surgery (or = 28.0), paralysis (or = 19.0), combined approach (or = 7.46), surgery for infection (or = 18.5), surgery for trauma (or = 11.1), comorbid traumatic injuries (or > 10), oncologic procedures (or = 5.2), use of iliac crest autograft (or = 4.16), two or more surgical levels (or = 3.48), blood loss > 300 ml (or = 1.66), and length of stay 5 days or greater (or = 3.47) were all found to be risk factors for vte (p < 0.05) in univariate analysis. multivariate analysis found staged surgery (or = 35.7), paralysis (or = 7.86), and nonelective surgery (or = 6.29) to be independent risk factors for vte.conclusions although the incidence of vte following cervical spine surgery is low, we identified several risk factors that may be predictive. more aggressive approaches to prophylaxis and surveillance in certain patient populations may be warranted.
the dioecious trematode ornithobilharzia turkestanicum (family, schistosomatidae) is a well - documented parasitic fluke which lives in mesenteric veins of ruminants and other mammals (1, 2). it was formerly named as schistosoma turkestanicum, but later assigned to the genus ornithobilharzia (3). o. turkestanicum has been reported from different parts of asia (5 - 7). the parasite is of great economic importance in iran because of the losses in sheep meat and wool production and intestine processing by its damages (8, 9). the phylum gastropoda is comprised of about 28,000 aquatic and terrestrial snails species worldwide (10). snails of the family lymnaeidae are of medical and veterinary importance since some 20 species in this family have been recognized to be potential transmitters of the schistosomatid trematodes (11 - 14). the miracidia of these trematodes infect their intermediate hosts, i.e. snails of the family lymnaeidae, and then leave the snails to look for their definitive hosts which can be ruminants (1, 15), rodents, wild ungulates such as reindeer (rangifer tarandus), red deer (cervus elaphus) or roe deer (capreolus capreolus) (16, 17). several snail species and subspecies of the genus lymnaea these include lymnaea gedrosiana, l. ovata, l. lagotis, l. tenera, l. acuminata, l. peregra and l. auricularia rufescens (18 - 20). among the seven identified species of lymnaeid snails in iran, the aquatic snail, l. gedrosiana (annandale and prashad, 1919) is the most widely distributed one throughout the country (14, 21, 22). it is a freshwater inhabitant which can be found in water bodies with diverse environmental conditions (23). in iran, l. gedrosiana has been reported to be a preferred intermediate host for o. turkestanicum (24) and trichobilharzia spp. (25). in iran, the cases of animal schistosomosis by o. turkestanicum were reported for the first time in 1963 from babolsar, northern iran (26). since then, it was also found in different parts of the country, i.e. the provinces of fars (27), khoozestan (28, 29), tehran (30), and mazandaran (31). nevertheless, not many researches have aimed to study the causative agents of cercarial dermatitis in iran. adult helminths causing cercarial dermatitis have been reported from animal schistosomes, i.e. ornithobilharzia, and bird hosts in southern and northern parts of iran, respectively (27, 32). the furcocercariae of animal schistosomes generating cercarial dermatitis or swimmer 's itch in the people working in the rice fields have been reported from northern (33) and southwestern iran (25). microscopic examination is the most frequently - used technique to detect the larval stages of trematodes in the snails (34). however, this technique has low sensitivity and/or specificity because of the difficulties in detection and differentiation of the trematodes larvae (35). for this reason, recent studies for discerning the experimental or natural infections with schistosomatid larvae in lymnaeid snails have employed molecular tools. all previous reports of the schistosomatid infections in the field - collected snails from iran have been made based on the detection of infection by cercarial shedding method (20, 29, 32). in this study, it was aimed to assess the utility of a molecular approach, polymerase chain reaction (pcr), for detecting the prevalence of the infection with o. turkestanicum larvae stages in the field - collected snails of l. gedrosiana from northwest iran. this study was carried out in the province of west azarbaijan, northwestern iran (35463958e and 4434723n), where the existence of plenty of water bodies and reservoirs with relatively appropriate environmental con - ditions provides suitable habitats for freshwater snails (14). field - collection of the lymnaeid snails was undertaken in six freshwater bodies located in both mountainous (altitudes over 1000 m above sea level) and plain areas of northern, central and southern parts of west azarbaijan over a period of eight months from may to december 2010 (fig.1). the collected snails of each site were placed individually into the plastic screw - capped containers and transferred alive to the laboratory of malacology of faculty of veterinary medicine, urmia university. of 6,759 collected lymnaeid snails, the snails of l. gedrosiana were identified using the identification keys provided by mansoorian (21) and pfleger (36). the identities were then verified by the parasitology museum of the faculty of veterinary medicine of tehran university. the soft tissues of the snails belonging to the specie l. gedrosiana were dissected, washed several times in 0.01 m phosphate - buffered saline (pbs, ph 7.2), and stored at 20 c until the dna extraction. the genomic dna was isolated by the modified phenol - chloroform method (37) using cetyltrimethylammonium bromide (ctab) at 60 c for 1 hr : 600l of 2x ctab buffer (100 mm tris - hcl, ph 8.0 ; 0.20 mm edta, ph 8 ; 1.4 m nacl ; 2% ctab ; 0.2% 2-mercaptoethanol) were added to 100 mg of the snail tissue. the mixture was incubated at 60 c for 60 min, vortex for 10min and centrifuged at 14000rpm for 15min. in continue, 300l phenol and chloroform - isoamyl alcohol (24:1) was added to the aqueous part, shaken for 2min and centrifuged at 14000rpm for 15min. the supernatant was transferred into a new tube and extracted with 600l of chloroform - isoamyl alcohol (24:1), followed by centrifugation at 14000rpm for 15min. in the next step, 0.7-fold volume of ethanol (etoh) was added to the supernatant and the dna was precipitated at -20c overnight followed by centrifugation at 14000rpm for 15min. the etoh was poured off and the dna pellet was rinsed in 70% etoh twice. the etoh was poured off by centrifugation at 14000rpm for 15min and the pellet was dried at room temperature and finally, dissolved in 50l pcr water overnight. two specific primers (ot - f : 5-ccttagtaactgcgagtcaacagg-3 and ot - r : 5-gagcaagacagcaggatctcacc-3) were used to amplify a fragment of the 28srrna gene of o. turkestanicum in the l. gedrosiana tissues (38). the pcr was carried out in 25l reaction containing 2l of the genomic dna (diluted 1:30), 2.5u of taq dna polymerase (fermentas, germany), 50m of each dntps (cinnagen, iran), 2 mm of mgcl2, 2.5l of pcr reaction buffer (10) and 0.5m of each primer. the reaction was performed in a bioer xp thermal cycler (china) and comprised. an initial denaturation step at 94c for 5min, followed by 35 cycles of 94 c for 60s, 57 c for 60s, and 72 c for 60s and finally, an extension step of 72c for 5min. a volume of 10l of each pcr product was analyzed by electrophoresis on 1% (w / v) agarose gel for about 1.5h at 90v. the snail samples showing the band patterns corresponding to the 28srrna gene of o. turkestanikum were considered as infected. this study was carried out in the province of west azarbaijan, northwestern iran (35463958e and 4434723n), where the existence of plenty of water bodies and reservoirs with relatively appropriate environmental con - ditions provides suitable habitats for freshwater snails (14). field - collection of the lymnaeid snails was undertaken in six freshwater bodies located in both mountainous (altitudes over 1000 m above sea level) and plain areas of northern, central and southern parts of west azarbaijan over a period of eight months from may to december 2010 (fig.1). the collected snails of each site were placed individually into the plastic screw - capped containers and transferred alive to the laboratory of malacology of faculty of veterinary medicine, urmia university. of 6,759 collected lymnaeid snails, the snails of l. gedrosiana were identified using the identification keys provided by mansoorian (21) and pfleger (36). the identities were then verified by the parasitology museum of the faculty of veterinary medicine of tehran university. the soft tissues of the snails belonging to the specie l. gedrosiana were dissected, washed several times in 0.01 m phosphate - buffered saline (pbs, ph 7.2), and stored at 20 c until the dna extraction. the genomic dna was isolated by the modified phenol - chloroform method (37) using cetyltrimethylammonium bromide (ctab) at 60 c for 1 hr : 600l of 2x ctab buffer (100 mm tris - hcl, ph 8.0 ; 0.20 mm edta, ph 8 ; 1.4 m nacl ; 2% ctab ; 0.2% 2-mercaptoethanol) were added to 100 mg of the snail tissue. the mixture was incubated at 60 c for 60 min, vortex for 10min and centrifuged at 14000rpm for 15min. in continue, 300l phenol and chloroform - isoamyl alcohol (24:1) was added to the aqueous part, shaken for 2min and centrifuged at 14000rpm for 15min. the supernatant was transferred into a new tube and extracted with 600l of chloroform - isoamyl alcohol (24:1), followed by centrifugation at 14000rpm for 15min. in the next step, 0.7-fold volume of ethanol (etoh) was added to the supernatant and the dna was precipitated at -20c overnight followed by centrifugation at 14000rpm for 15min. the etoh was poured off and the dna pellet was rinsed in 70% etoh twice. the etoh was poured off by centrifugation at 14000rpm for 15min and the pellet was dried at room temperature and finally, dissolved in 50l pcr water overnight. two specific primers (ot - f : 5-ccttagtaactgcgagtcaacagg-3 and ot - r : 5-gagcaagacagcaggatctcacc-3) were used to amplify a fragment of the 28srrna gene of o. turkestanicum in the l. gedrosiana tissues (38). the pcr was carried out in 25l reaction containing 2l of the genomic dna (diluted 1:30), 2.5u of taq dna polymerase (fermentas, germany), 50m of each dntps (cinnagen, iran), 2 mm of mgcl2, 2.5l of pcr reaction buffer (10) and 0.5m of each primer. the reaction was performed in a bioer xp thermal cycler (china) and comprised. an initial denaturation step at 94c for 5min, followed by 35 cycles of 94 c for 60s, 57 c for 60s, and 72 c for 60s and finally, an extension step of 72c for 5min. a volume of 10l of each pcr product was analyzed by electrophoresis on 1% (w / v) agarose gel for about 1.5h at 90v. the snail samples showing the band patterns corresponding to the 28srrna gene of o. turkestanikum were considered as infected. a total of 6,759 lymnaeidae freshwater snails were collected from the investigated water bodies. of these, the cercarial shedding and microscopic examination showed that 23.56% (86 out of 365) of the l. gedrosiana snails were infected with the larval stages of digenian trematode. the pcr patterns confirmed that 28.77% (105/365) snails of l. gedrosiana were infected with larvae stages of o. turkestanicum (fig 2). geographically, the infected snails were distributed over five out of the six study areas. the maximum infection rate was for ghargologh (66.66%, 20/30) located in northern part of west azarbaijan, while the minimum infection was recorded in qarabaagh (16.66%, 5/30), a water body in the central part of the province (fig.1). only 35.24% (37 out of 105) of the infected snails were from the plain areas ; the remaining were distributed in high altitudes (table 1). the expansion of the parasites with indirect life cycles, such as o. turkestanicum, is facilitated where an adequate number of intermediate and definite host species coexist (38). in the present study, l. gedrosiana showed to be common snail specie throughout the studied region living in perennial and typically acidic waters. according to moghaddam. (39) and mansoorian (21), l. gedrosiana is the most widely distributed lymnaeid snail throughout iran. 14) also reported a high abundance and wide distribution of this snail in west azarbaijan. this province is characterized by flat lands, numerous stagnant water catchments and irrigation canals covered by aquatic vegetation and agricultural tradition. these together with having average annual temperatures higher than 20c, adequate rainfall and humidity and intense population of domestic ruminants set the scene for the transmission of digenian trematodes by the snails. this is the first study in iran in which the infection of field - collected l. gedrosiana snails with larval stages of the digenian trematode o. turkestanicum was discovered by molecular examination. the application of molecular methods can give an accurate estimation of infection with a certain disease - causing organism. this is not only correct the underestimations made by the traditional methods, can also uncover the infections which could not be detected by the classical manners, especially with the larval stages of trematodes. the utility of molecular approaches for studying the epidemiology of o. turkestanikum was confirmed in this study. the animal schistosomes including o. turkestanicum have shown high infection rates and wide distribution over some iranian provinces, so that their prevalence rates ranged between 35% and 100% in sheep and goats (39). however, the prevalence of infection with o. turkestanicum larvae in l. gedrosiana observed in the current study was relatively low. in accordance with the results of this study, majoros. (38) reported very low infection rates with fasciolid larval stages in the lymnaeid snails of northern iran. determination of the seasonal distribution of l. gedrosiana as the intermediate host of o. turkestanicum is of great importance. according to eslami (40), there is a seasonal variation in o. turkestanicum infection in the ruminants of iran, and this is directly related to the abundance and incidence of the native snails. thus, infection with o. turkestanicum may outbreak following a seasonal variation (40). the prevalence of o. turkestanicum in small ruminant is more important in fall than in spring. furthermore, the incidence of the infection in a range of the host animals with season - dependent life cycles may play an important role in persistence of the infection in the livestock of the region. from the results of the current study, it was confirmed that the snail l. gedrosiana can be considered as a potent transmitter of o. turkestanicum to the domestic ruminants and humans. this should be taken into consideration in the development of control programs against the infection. further studies should be directed to understand the extent to which the infection rates in the snails affect the degree of infection in the domestic ruminants and human beings in each part of the region. it is also recommended that both traditional and pcr methods should be used to better understanding of the epidemiological situation of the infection in a given area.	backgroundinfection with ornithobilharzia turkestanicum has been reported in a wide range of animals worldwide. this study was undertaken to assess the utility of polymerase chain reaction (pcr), for detecting the infection with o. turkestanicum larvae stages in lymnaea gedrosiana.methodsa total of 6,759 lymnaeidae snails were collected from six aquatic habitats in west azarbaijan, northwest iran. of these, the snails of l. gedrosiana were identified. to detect infected l. gedrosiana with the larval stages of o. turkestanicum, they were subjected for cercarial shedding and molecular examinations. the genomic dna was extracted and pcr was performed to specifically amplify a fragment of the nuclear 28srrna gene of o. turkestanicum.resultsof all collected snails, 5.4% (365/6,759) were the snails of l. gedrosiana. the cercarial shedding method revealed that 23.56% (86/365) of the snails were infected. the pcr patterns confirmed that 28.77% (105/365) snails of l. gedrosiana were infected with larval stages of o. turkestanicum. the infected snails were observed in five studied sites. the highest infection rate (66.66%, 20/30) was recorded in the snails of ghargologh in the northern part. only 35.24% (37/105) of the infected snails were from the plain areas, whereas the remaining existed in high altitudes.conclusionit was concluded pcr method could be an efficient and fast method for uncovering the actual rate of infection with larval stages of o. turkestanicum in the snails of l. gedrosiana. this method can be also useful for the domestic animals and public health management programs in the country.
one health is a concept that aims to bring together human, animal, and environmental health. researchers including louis pasteur and robert koch and physicians such as william osler and rudolph virchow demonstrated the collaborative links between animal and human health. as the traditional boundaries between medical and veterinary practice continue to pervade society there is a need for the practical application of one health. one health is defined by the one health commission as the collaborative effort of multiple disciplines to obtain optimal health for people, animals, and our environment. in another definition, the one health initiative task force (ohitf) defines one health as the promotion, improvement, and defense for the health and well - being of all species by enhancing cooperation and collaboration between physicians, veterinarians, and other scientific health professionals and by promoting strengths in leadership and management to achieve these goals. the one health approach plays a significant role in the prevention and control of zoonoses. it has been noted by the world health organization (who) and graham. that approximately 75% of new emerging human infectious diseases are defined as zoonotic, meaning that they may be naturally transmitted from vertebrate animals to humans. new and reemerging zoonoses have evolved throughout the last three decades partly as a consequence of the increasing interdependence of humans on animals and their products and our close association with companion animals. zoonoses should therefore be considered the single most critical risk factor to human health and well - being, with regard to infectious diseases. of the 1,461 infectious diseases recognized to occur in humans by the national academy of sciences, institute of medicine, approximately 60% are caused by multihost pathogens, characterized by their movement across various species. this gives significant credence to the importance of examining health effects across species, in order to fully understand the public health and economic impact of such diseases and to help implement treatment and preventive programs. the one health concept is a broad term that covers a variety of subcategories identified as bioterrorism, animals as predictors for disease, and the psychological bonds that can exist between an animal and a human. zoonoses comprised the primary focus for this review with the overall objective to determine the status of the one health approach and its applications to zoonoses, using scholarly peer - reviewed literature that has been published since the global adoption of the concept in 1984 (for study purposes, january 1, 1984, until december 31, 2012). the first assessed scholarly resources on the one health approach published works between january 1, 1984, and december 31, 2012. one health scholarly resources were classified as peer - reviewed publications, professional presentations, grants or funding allocations, reports from the who, and books or book chapters. the second objective examined the preferred scope of one health published works within the period of study. scopes of one health subject categorizations were, namely, zoonoses, food safety, agriculture, environmental health and global health. the third objective analyzed the geographic distribution of scholarly one health resources, whether they were in developed nations or developing nations listed by the international monetary fund (imf). a cross - sectional study using internet resources was carried out to analyze one health applications to zoonoses in scholarly resources from 1984 to 2012, representing a 28-year review. before conducting the internet search, scholarly material was distinguished as eligible and ineligible using the following criteria which were found on google scholar and ebscohost.peer-reviewed publications were classified as scientific journals and literature reviews of the pertinent subject matter (human, animal, and environmental health) that had been published in peer - reviewed journals.professional presentations were represented by formal presentations made by organizations and other professionals on the subject matter of human, animal, and environmental health, presenting research material, policy developments, or promotional activities in support of one health.grants and funding allocations were characterized as proposals for funding research, policy development, and so forth in the collaborative subject matter of humans, animals, and the environment accessed from reviewing all professional publications available from the systematic search conducted.who-related reporting included updates from the website that involved relevant health issues, specifically reflecting the one health approach.book and book chapters were qualified as books or selections involving the subject matter. peer - reviewed publications were classified as scientific journals and literature reviews of the pertinent subject matter (human, animal, and environmental health) that had been published in peer - reviewed journals. professional presentations were represented by formal presentations made by organizations and other professionals on the subject matter of human, animal, and environmental health, presenting research material, policy developments, or promotional activities in support of one health. grants and funding allocations were characterized as proposals for funding research, policy development, and so forth in the collaborative subject matter of humans, animals, and the environment accessed from reviewing all professional publications available from the systematic search conducted. who - related reporting included updates from the website that involved relevant health issues, specifically reflecting the one health approach. the target population included all published studies that addressed the one health philosophy and which met the inclusion criteria. the documentation review included resources found on the internet through the search engines and databases identified, which fit into the criteria of a one health approach and which took place from the concept 's adoption of january 1, 1984. excluded from this study were studies that were not found on the internet databases, those that did not involve the one health concept, or fit the criteria of a one health approach, or those that were reported outside the period of study. database searches were conducted from may to july 2011. in the search fields for google scholar and ebscohost, the terms one health, health, human, animal, and zoonoses were typed in. the first result that appeared from the database was reviewed and then assessed, using the definitions, to determine whether it fits into one of the one health approach criteria. every fifth search result was examined and after reaching results on both databases numbered 130 onward, every second result was then considered. for every result that did not meet the inclusion criteria, the very next result was examined, and so forth, until a result did meet the criteria. after a result met the criteria, the fifth result from the last selected result each scholarly initiative that met the inclusion criteria was separated into its initiative category as well as into its year of publication. in addition, each resulting initiative was further categorized by the subject matter covered in the scholarly work. considering one health scopes, these were the common subject areas covered : zoonoses, agriculture, food safety, environmental health, and global health. these categories were condensed from a larger, more complex list provided by the one health initiative task force. for resources that contributed to more than one scope, such as agriculture and food safety, the final determination was made on the emphasis of one of the scopes from within the contents of the title. finally, each initiative was also categorized into being conducted in, or having an analysis on, either a developed or developing nation based on a country 's gross domestic product (gdp). all the results were then categorized by their year of publication, the initiative that was represented, scope covered within the work, and the geographic distribution of where the initiative was conducted or what area was analyzed. spss statistical software package version 18.0 was used to analyze the frequencies of the years of scholarly resources, the initiative types, scopes categories, and geographic distribution. all years for the review were represented, except for 1985, 1990, and 1991 as there were no publications that were sourced for these three years. there were a total of 8 resources in 1995 (4%) and 43 in 2009 (21.5%). the year 2006 began a continued increase in one health resources for the period of review. the years 2010 to 2012 were the most productive for publications on one health as 71% of publications occurred during this period of time. an overall increase in the number of published one health scholarly works was found for the review with a marked increase in the most recent years (figure 1). journal articles, presentations, who reports, and books or book chapters were included in the analysis. grants and funding allocations peer - reviewed journal articles took precedence (85%) of all publications, while presentations and books accounted for 8.0% and 6.5%, respectively ; only one who report was recorded. evaluation of scopes, covered in the 737 scholarly resources (figure 2), revealed that the predominant topics were global health, with 247 scopes (33.5%), and environmental health, with 232 total scopes (31.5%). in terms of geographic distribution of the scholarly resources, most of the resources focused their objectives within or towards countries that were already developed (70%) (figure 3). an assessment on how one health initiatives were distributed by country size and gdp was achieved by mapping and measuring the burden of zoonoses and its distribution across the world (table 1). events of zoonoses were found to be disproportionately distributed as a result of the poverty and emerging market interface. outbreaks or epidemics of emerging zoonoses were also noted to be sporadic in temporal and spatial distribution and appeared in developed countries where emerging zoonoses had not previously been reported but are increasing in incidence or geographical range. data on zoonoses extracted from the global burden of diseases noted that endemic zoonoses were concentrated among the developing countries of india, nigeria, democratic republic of congo, china, ethiopia, and bangladesh, whereas emerging zoonoses events were reported in the developed countries of the united states, united kingdom, australia, france, brazil, canada, germany, and japan (table 1). for data analysis, chi - square was conducted to determine if, in the resulting reviewed years, one health resources themselves, scopes, and country locale differed significantly from the averages expected. analysis revealed p values of less than 0.05 (p < 0.05), meaning that the resources, scopes, and country locale were all statistically different (table 2). further analysis employed linear regression, using each focus, year, one health resource, scope, and country as the dependent variables and comparing them against independent variables of themselves. this showed whether the relationship between the independent and dependent variables was predictive or dependent on one another. in the case of using year as the dependent variable, the regression shows that it was dependent on the initiative (p = 0.021), scope (p = 0.003) and the country locale (p = 0.021) (table 3). since all the values were < 0.05, the null hypothesis was rejected and it was concluded that the years selected for the study showed a dependent relationship on the one health approach conducted, the scope topic areas and the represented country in the scholarly work. the same linear regression was performed, this time using the initiative as a dependent variable against the other variables (table 4). for this analysis, the initiative showed it to be dependent on the year (p = 0.022), as also noted in table 3, but not dependant on scope (p = 0.643) nor on the country 's locale (p = 0.465). the null hypothesis failed to be rejected because no complete dependency relationships were formed between all the variables from the regression test that was conducted, as compared to the regression testing done with year. next, scope was selected as the dependent variable against the year, initiative, and country. the linear regression showed that the scope was dependent on the year, as seen before (p = 0.003), but not dependent on the initiative (p = 0.643) nor on the country (p = 0.481) (table 5). the country locale was used as the dependent variable against the others in the last linear regression. it was demonstrated that the country, whether developed or developing, was dependent on the year (p = 0.021) but not on the initiative (p = 0.445) nor the scope (p = 0.481) (table 6). again, the null hypothesis failed to be rejected for the whole dependency of scope on all other variables. the only rejected null was the dependency displayed between the year of the initiative and the initiative itself, its scope, and the country covered from within the initiative. many of the results of this study could be attributed to the occurrences in the world during the time period of the study. when observing the trend of the one health approach over time, there was a minimal spike in 1995, an increased output from 2006, and marked increase from 2010 to 2012. four (50%) of the eight scopes in 1995 were focused on environmental health, three involved global health (37.5%), and one scope involved food safety (12.5%). an environmental act was passed in 1995 in england by the environment agency and the scottish environment protection agency, office of public sector information. it is likely that the passage of this law contributed to a higher production of publications. the increase in resources produced since 2006 could be related to the e. coli contamination incidents in the united states in 2006 and to the h1n1 outbreaks [11, 12 ] and also to the passing of the one health initiative task force in 2007. two (4.7%) of the 43 defined resources in 2009 involved agriculture, eight involved environmental health (18.6%), 6 were on zoonoses (13.9%), 23 were on global health (53.5%), and four were on food safety (9.3%). the marked increase since 2010 may have resulted from the developments since 2006 which continued into 2009 which allowed for the one health approach to be placed on the research and scholarly agenda. 12 (5%) of the 236 recorded resources in 2012 involved agriculture, 50 (21%) involved environmental health, 56 (23.5%) were on zoonoses, 98 (41.5%) were on global health, and 21 (9%) were on food safety. the distribution of the years of the one health approach, the scholarly resources, the scopes, and the countries ' locale were not equally represented. for the one health concept to be appropriately beneficial to the global population, it would be necessary for a significant equal distribution of scholarly works to exist. the data, suggesting that the scopes of global health (33.5%) and environmental health (31.5%) dominated the others, including the zoonoses, produces an area of concern. the issues relating to one health, while in their genesis involved zoonoses and food safety, were identified as environmental and global health issues in the reporting and publications. while this shows evidence of the profound efforts to boost environmental and global knowledge about one health, it also demonstrated the limited body of knowledge of zoonoses, agriculture, and food safety. zoonoses, agriculture, and food safety are all interconnected topics in that they all directly impact the health of humans. in the last 30 years, there has been an average of one newly discovered emerging infectious disease every year. a total of 335 emerging infectious diseases were identified between 1940 and 2004. considering that more than 60% of infectious diseases agriculture, livestock production, and food safety practices are intimately linked with the prevention and control of zoonoses through the one health approach. considering the significance of agriculture and food safety, it was surprising that these scopes did not have a greater representation in the literature reviewed. developed countries, by virtue of their greater institutional facilities, trained personnel and financial resources are able to address the issues of one health approach. this is extremely beneficial as it enables developed nations to gain an awareness of one health initiatives and the added synergistic value of this approach. the one health initiative task force has reported that while the developed countries prevail in making one health discoveries, it is the developing countries that suffer the most from the effects of zoonoses. it has been estimated that 70% of the reasons for poverty in africa can be attributed to poor livestock production practices. zoonotic infections significantly impact animal production in this region further jeopardizing human and animal livelihoods. the dependency of the initiative year, initiative, scope, and country locale on one another revealed that the incidence of the scope or country location is somewhat dependent on the year. in other words, it can be argued that the scope or country locale was represented due to that particular year, namely, due to the associated events during that year. immediate action and scholarly resources are commonly implemented after a devastating event occurs, proving that the publishing of a particular one health topic may not be due to chance during that specific year. it is important to note that the general availability of one health resources is likely to be higher in the more recent years than in the 1980s, as the internet was still in its evolutionary stage and not yet a global resource, as it is today. the free availability of scholarly information on the internet is evolving rapidly which will equalize the field. it will then be a matter of trained personnel and resources to make appropriate advances. many of the classifications which determined the scope of an initiative were subjective. even though many of them clearly fit into their appropriate scope, some were hard to decipher, as some titles could have easily been included in more than one scope. as a result, one author 's classification of an initiative could differ from another 's opinion, resulting in interobserver bias. some resources truly belonged in their own category ; however, for the purposes of this study, only five scopes were included. this resulted in many resources being placed in the global health scope, as it is a category that could be applied to all one health approaches. subjectivity was also a limitation in classifying the country locale. in some cases, resources ' locations were clear from the article 's title or content, and others were not. some scholarly resources covered subject matter concerning a developing country, yet the actual work was conducted in a developed country. the one health approach, according to the one health initiative, has been utilized to accelerate biomedical research discoveries, enhance public health efficacy, expeditiously expand the scientific knowledge base, and improve medical education and clinical care. the increasing encroachment of people and livestock into wildlife habitats provided a multifaceted need to study bats and offer understanding for study at the human - wildlife interface. bats are an important reservoir and vector for spread of a number of emerging infectious diseases and they are associated with zoonoses with global public health significance such as lyssa, hendra and nipah viruses, severe acute respiratory syndrome (sars) like coronaviruses, and ebola and marburg viruses. the importance of wildlife as reservoirs of human diseases has also been widely recognized for most of the parasitic zoonoses, including american and african trypanosomiasis, leishmaniasis, giardiasis, cryptosporidiosis, balantidiasis, fascioliasis, opisthorchiasis, clonorchiasis, paragonimiasis, schistosomiasis, echinococcosis, taeniasis, diphyllobothriasis, sparganosis, dipylidiasis, trichinellosis, toxocariasis, strongyloidiasis, and ancylostoma caninum and a. braziliense infections. molecular phylogenetic methods used to examine the genetic diversity and species composition of these parasites in humans and their domestic and wild reservoir, paratenic, definitive, and intermediate host species have shown that they are in many instances identical. for example, african trypanosomes identified in wildlife in the serengeti in tanzania and the luangwa valley in zambia which harbour a wide range of trypanosomes are the same species which infect humans and livestock. the one health concept has successfully replaced the disease centered approach to zoonoses with a system based approach that aligns multiple disciplines, working locally, nationally, and globally, to attain optimal health for people, domestic, and wild animals and the environment. zoonotic diseases pose both major health threats and complex scientific and policy challenges, to which the social, cultural, and political norms and values are essential to address successful control outcomes. the need to employ one health is illustrated in the cases of h5n1 avian influenza in which control failed due to the lack of addressing the complex dynamics of zoonotic diseases. rapid response briefing produced a report on the ebola haemorrhagic fever outbreak which occurred in kibaale and kampala in uganda in 2012. the number of deaths in kibaale was at least 16 ; the outbreak was spread 40 miles away to kampala four months later. these two outbreaks demonstrated the continuing existence of ebola in uganda which recorded an earlier outbreak in 2000 and led to 425 cases ; more than half of the cases died. the one health approach, employing disease surveillance, management, and eradication through collaboration between veterinarians dealing with livestock and wild animal populations and ecologists examining ecosystem biodiversity and public health experts, may have yielded a more rapid resolution to the outbreak the application of the one health approach has been recognized as a critical need by international organizations as well as the preferred approach to address global health issues. the 2013 grand challenges in global health is based on the theme the one health concept : bringing together human and animal health for new solutions. the recent call for proposals for funding recognizes the lack of knowledge sharing and an artificial barrier that separates the fields of human and animal health. the grand challenges in global health specifically identified that advances in drug and vaccine discoveries for human diseases can be applied to provide tools and approaches for animal diseases that still plague developing countries. it is also noted that knowledge in veterinary medicine and animal nutrition and husbandry could provide insights into human nutrition and growth. one health has gained momentum and now encompasses zoonotic infections, food safety, and even health delivery systems. the one health concept has been expanded to encompass the health and sustainability of the world 's ecosystems. based on complex ecological thinking that goes beyond humans and animals, these approaches consider inextricable linkages beyond the human, animal, and environmental interface. collaboration between veterinary, medical, and public health professionals to understand the ecological interactions and reactions to flux in a system can facilitate a clearer understanding of climate change impacts on environmental, animal, and human health. climate change adds complexity and uncertainty to human health issues, such as emerging infectious diseases, food security, and national sustainability planning. evidence for expanded application of one health compared to separate sectoral thinking is growing and this integrative thinking is increasingly being considered in academic curricula in schools of medicine, veterinary medicine and public health, clinical practice, ministries of health and livestock / agriculture, and international organizations. the one health approach to zoonoses however remains an average priority for health care professionals. the impact of zoonoses on animal health has been largely neglected but the effects on public health usually drive control initiatives on zoonoses and are much better defined by the use of disability adjusted life years (dalys). the first zoonoses prioritization exercise involving health professionals in north america who had a limited knowledge of infectious diseases identified zoonoses as an area of priority. another study reported that local public health agencies in north america were not prepared and potentially unaware of their responsibility to be the initiator of the work on zoonotic disease information intelligence. the advancement of the one health approach has increased the discussion and reporting on the topic. there remains a lack of knowledge and application of the integrated approach to health care by the health care professionals. reaching the goal of control, and elimination and/or ultimate eradication of zoonoses pose a significant challenge for the future. standardized interlaboratory test validation, intersectoral collaboration and establishment of an international one health diagnostic platform are considered to be important strategies. the sharing of best practices on diagnosis of zoonoses and the further refinement of new, cheaper, multispecies tests which can be interpreted by minimally trained individuals could contribute to a greater level of intersectoral integration, control, and elimination of zoonoses. the projection from one health may eventually lead to a one system approach based on the inherent challenges to intersect disciplines that belong to different systems. one health approaches applied across international boundaries that share the same challenges are required to create sustainable and coordinated control. the one system approach focusing on the strengthening of the community model health system as a whole as well as developing effective and novel tools to be applied across all aspects of health, is fundamental of a one world one health approach. the future of one health is a one world approach with the continued effort towards integration of the contributing parts that form the whole which is health. the one health approach continues to be a highly investigated concept, via the pursuit of scholarly resources involving the health of humans, animals, and the environment. there is a need to increase research on zoonoses, food safety, and agriculture and to improve the understanding of the one health concept. this could be achieved by introducing more scholarly resources in developing countries by the further development of the internet and the free availability of online information on one health. the use of massive open online courses (mooc) available to developing countries is now being offered to deliver courses on the approach and applications of one health. this is critical because most of the public health and economic impacts that occur within the concept of one health occur in developing nations. the lack of basic health infrastructure in developing countries means that everything else suffers as a result, namely, the environment, human, and animal health and well - being. the future of one health is at a crossroad ; there is a need to more clearly define its boundaries and demonstrate its benefits. the greatest acceptance of one health is seen where it is having significant impacts on control of infectious diseases. there is also a continuing need for further efforts towards integration with the global community serving as the unit of a one system approach.	background. one health is a concept that was officially adopted by international organizations and scholarly bodies in 1984. it is the notion of combining human, animal, and environmental components to address global health challenges that have an ecological interconnectedness. methods. a cross - sectional study of the available literature cited was conducted from january 1984 when the one health concept was adopted till december 2012 to examine the role of the one health approach towards zoonoses. inclusion criteria included publications, professional presentations, funding allocations, official documentation books, and book chapters, and exclusion criteria included those citations written outside the period of review. results. a total of 737 resources met the inclusion criteria and were considered in this review. resources showed a continuous upward trend for the years from 2006 to 2012. the predominant resources were journal publications with environmental health as the significant scope focus for one health. there was also an emphasis on the distribution of the work from developed countries. all categories of years, resources, scopes, and country locale differed from the means (p = 0.000). year of initiative, scope, and country locale showed a dependent relationship (p = 0.022, p = 0.003, and p = 0.021, resp.). conclusion. our findings demonstrate the rapid growth in embracing the concept of one health, particularly in developed countries over the past six years. the advantages and benefits of this approach in tackling zoonoses are manifold, yet they are still not seemingly being embraced in developing countries where zoonoses have the greatest impact.
patients without fractures recovered much earlier than those with fractures.among the patients with fractures, there were no significant differences in terms of consolidation time after surgery.different surgical options of curettage only or combined with different filling materials had no effects on the follow - up outcomes.of all the patients, different surgical options of curettage only or combined with different filling materials had no effects on the follow - up outcomes (all p >.05). healing was noted to be progressive and complete in all cases and no functional restriction was observed. patients without fractures recovered much earlier than those with fractures. among the patients with fractures different surgical options of curettage only or combined with different filling materials had no effects on the follow - up outcomes. of all the patients, different surgical options of curettage only or combined with different filling materials had no effects on the follow - up outcomes (all p >.05). healing was noted to be progressive and complete in all cases and no functional restriction was observed. enchondroma is the most common bone tumor of the hand and is a benign, intramedullary, cartilaginous tumor. it originates from cartilage and is commonly located in the proximal metaphysis of the proximal phalanx. it often presents in the first through fourth decades of life and has a predilection for the ulnar sided tubular bones of the hand, and arises most frequently in the phalanges and metacarpals. furthermore, enchondromas with pathological fractures occur because the bone in question has been weakened by the disease process. often, these injuries result from minor trauma, which might not otherwise cause a fracture in healthy bone. the timing of treatment when there is a pathologic fracture is not clearly defined and there is no standardized algorithm for surgical treatment of this tumor. recent studies showed that simple curettage with or without bone grafting is an effective and safe treatment for most patients with simple solitary enchondromas. yalcinkaya reported that primary treatment of an enchondroma in the presence of a pathologic fracture did not change the outcome compared with lesions treated after fracture union. naturally, patients treated after fracture union had additional periods of immobilization. but it should be noted that traditionally, surgeons have preferred to wait for pathological fractures to heal before proceeding with operative treatment secondary to the belief that complication rates would be higher with immediate curettage. however, there are few studies to investigate the impact of pathological fractures on the final treatment outcomes in patients with fractures due to enchondromas. in this retrospective study, we reviewed our 10-year experience with a subset of patients who had solitary enchondromas. we attempted to investigate the optimal surgical intervention timing for the patients with fractures due to enchondromas and evaluate the impact of pathological fractures on the therapeutic outcome in such patients. the retrospective analysis was performed on the clinical data of patients with enchondromas treated from february 2005 to august 2015 in our hospital and approved by the institutional review board (irb). the inclusion criteria were : solitary enchondroma of the hand ; with or without pathologic bone fracture ; treated surgically ; and a complete follow - up of more than 6 months. the exclusion criteria were : the merging of other bone and soft tissue injuries ; multiple enchondromas ; a follow - up of less than 6 months ; missing follow - up data, and medical records or x - ray images. the operative details and postoperative clinical and radiological outcomes were reviewed by an independent reviewer. the disability of the arm, shoulder, and hand (dash), upper limb function scale was used to evaluate the function of the affected hand. the time to complete recovery, complications as well as the recurrence rate and in - hospital costs were recorded. bone remodeling was defined as a uniform appearance with no gap between the cancellous bone and the bone substitute. chi - square test was used for the statistical analysis of demographic data and complications after surgery between groups. one - way anova was selected to compare the differences between groups in the follow - up outcomes with spss v. 19.0. chi - square test was used for the statistical analysis of demographic data and complications after surgery between groups. one - way anova was selected to compare the differences between groups in the follow - up outcomes with spss v. 19.0. in total, 227 patients with enchondroma were identified, of which 92 cases were eventually included according to the inclusion and exclusion criteria of this study. there were 52 males (56.5%) and 40 females (43.5%) in this series, aged from 2 to 69 years (mean, 29.4 years). based on the clinical data, patients with pathological fractures were assigned into the group with fractures (n = 27) and those without fractures, who were diagnosed accidentally during routine physical examinations were assigned into the group without fractures (n = 65). patients in the fracture group were divided into 2 subgroups : primary (4 weeks, mean 16 days) surgery group and delayed (> 4 weeks, mean 87 days) surgery group according to the time of surgical intervention. the operative methods were simple curettage or combined with bone grafting with autologous or bioactive materials. patient 's demographic data (enchondroma associated with or without pathological fractures). patient 's demographic data and different treatment options. there were no significant differences in terms of consolidation time after surgery, recurrence rate, and dash scores between the groups with and without fractures (all p >.05) ; the in - hospital costs were higher in the group with fractures (bioactive and osteoconductive materials or the iliac bone graft cost) than those in the group without fractures (p =.007) ; however, patients without fractures recovered much earlier than those with fractures (p <.001). among the patients with fractures, there were no significant differences in terms of consolidation time after surgery, recurrence rate, dash scores as well as the occurrence rate of complications between the primary surgery group and the delayed surgery group (all p =.534) ; however, recovery time was statistically longer in the delayed surgery group, with an average of 76 days than that of the primary surgery group, with an average of 36 days (p <.001). the outcomes of primary and delayed surgery groups are listed in tables 4 and 5. of all the patients, different surgical options of curettage only or curettage combined with different filling materials had no effects on the follow - up outcomes (all p =.692). healing was noted to be progressive and complete in all cases and no functional restrictions were observed. enchondroma, a benign slowly growing tumor composed of hyaline cartilage cells that persists throughout development, is the most common primary bone tumor of the hand, the exact incidence varies and the exact incidence of enchondroma is still unknown. up to 70% of enchondromas occur in the hand, and the proximal phalanges are the most frequent site involved. in fact, fractures associated with these benign lesions may be allowed to heal before definitive treatment of the tumor, however, surgical intervention is suggested to minimize complications and allow early motion. traditionally, enchondromas are treated with curettage and grafting with allogeneic bone or autogenous or synthetic bone substitutes. however, there is no standardized algorithm for surgical treatment of this kind of tumor. it is not clear whether grafting after curettage is necessary or whether the type of graft used affects healing, recurrence, complications, and malignant transformation. our study shows that the choice of graft either in patients with or without pathological fractures has no effect on time required to heal, range of motion, recurrence, complications, or malignant transformation. nonetheless, considering the bone will be further weakened by curettage alone, we believe that replacement with an osteogenic or osteoconductive substance will facilitate bone healing and remodeling so that this fracture - prone period may be shortened. although autologous bone graft, which will not cause immune rejection, is the most suitable choice, it may be associated with some donor site morbidities such as infection, hematoma, and chronic pelvic pain. as an alternative the application of bioactive and osteoconductive materials which is available in various shapes and sizes has obvious advantages of reduced donor site morbidity and reduces operating time and convenience of local anesthesia. however, in the present study, we found the cost of treatment with such materials is much higher than with other options. on the other hand, we found that the recovery time was shorter in the curettage only group than in the other 2 groups with grafts. we speculated this finding might be attributed to the differences in the affected site ratios of metacarpals and phalanges. in the curettage only group, the ratio was much higher (62.5%) than the other 2 groups (12.1% and 11.5%, respectively). usually postoperative defects of digits in children who sustained no fractures heal faster when compared to patients with fractured lesions. therefore, the choice of dealing with such patients should be assessed comprehensively and individualized based on patients affordability and requirement. in literature, the timing of treatment when there is a pathologic fracture in these patients has not been clearly defined. traditionally, the mainstay of surgical intervention for such patients is carried out with a delayed procedure until the fracture site completely or partially heals so that a simple curettage may be applicable with no needs of internal fixation. early treatment was associated with a shorter period of disability, but also with significantly more complications (67% vs 10%), including stiffness and rotational deformity in the study of jacobson and ruff. but in our study, no significant differences were found between the primary surgery group and delayed surgery group in terms of consolidation time after surgery, recurrence rate, dash scores as well as the occurrence rate of complications. primarily, treatment of enchondroma in the presence of a pathologic fracture did not change the outcome compared with lesions treated after fracture union. naturally, patients treated after fracture union had additional periods of immobilization. in another study, the outcome was similar to our results. the strut bone graft served both mechanical and biologic functions. although the in - hospital expense was a bit higher in the primary surgery group than in the delayed surgery group, the time to return to work was significantly shorter in the primary surgery group actually, a long - term interval before a delayed operation will not only prolong the treatment time, but also may lead to finger dysfunction ; in addition, the potential fracture displacement may also cause a deformity of the finger. in our study therefore, the primary surgery surely has a substantial advantage in the management of patients with pathological fracture due to enchondroma. enchondroma is usually identified after an incidental finding on radiographs or as a pathologic fracture. according to the results of our investigation, 27 patients suffered from pathologic fractures. a recurrence rate of up to 13.3% after curettage and bone grafting was reported. however, there are no such events in our series. patients with a diagnosis of enchondroma with fractures had no higher rate of complications. at follow - up, no differences in outcomes in terms of consolidation time after surgery and dash scores were observed between patients with or without fractures. however, our findings showed that patients suffering from osteochondromas without fractures could resume work much earlier than those with fractures. in this series, we noted good functional recovery in all groups of patients, while complications were uncommon. only 1 patient treated with an autograft in the delayed surgery group suffered from infection and 1 case in this group required reoperation because of a secondary fracture. although there were no significant differences in the occurrence rate of persistent pain between the group with fractures and the group without fractures, a higher occurrence rate of 25.9% (7/27, ci : 11.940.1%) was observed in the former group in comparison with an occurrence rate of 15.3% (10/65, ci : 7.1423.4%) in nonfracture patients. despite soft tissue complications were not analyzed in this study, we speculate that local trauma leading to postoperative functional restrictions could be a major source of patient distress following enchondroma surgery. therefore, meticulous curettage and minimally invasive manipulation of the surrounding soft tissues is the key to achieve good results and avoid complications. pathological fractures associated with enchondromas have no significant impact on the treatment outcomes compared to those with nonfractured enchondromas. although the hospital costs were higher for patients treated primarily with pathological fractures due to enchondromas, these patients could resume to their original work much earlier than those treated by delayed surgery.	abstractenchondroma, reportedly the most common primary tumor of the long bones of the hand, usually develops during the first till fourth decades of life. however, there has no consensus been reached regarding the surgical intervention timing for these patients. we aim to evaluate the optimal surgical intervention timing for the patients with fractures due to enchondromas, investigate the impact of pathological fractures on the treatment and outcomes in these patients.medical records and x - rays of patients treated for enchondroma of the hand from 2005 to 2015 were retrospectively reviewed. we collected 148 cases in total and 92 of them had complete information including x - rays, medical records, and files of follow up.there were no significant differences in terms of consolidation time after surgery, recurrence rate, and dash scores between the groups with and without fractures ; the treatment costs were higher in the group with fractures than those without fractures ; however, patients without fractures were able to resume work earlier than those with fractures.the pathological fractures associated with enchondromas have no significant impact on the treatment outcomes compared to those with simple nonfractured enchondromas. although the cost was more expensive for patients treated primarily with pathological fractures due to enchondromas, these patients could resume their work normally much earlier than those treated by delayed surgery. early surgical intervention is recommended for better results and no increased risks for patients with pathological fractures caused by enchondromas.
an important goal of personalized cancer therapy is to tailor specific therapies to the mutational profile of the individual patient 's cancer. a major issue with this strategy is that while the aim of systemic therapy is to treat metastatic disease, typically the primary tumour is used as the source of information on the mutational profile of a patient 's cancer. for example, shah. used whole genome sequencing to compare the mutation profiles of the primary tumor and a metastatic tumor that occurred many years after the primary and showed that there were multiple additional mutations present in the metastasis. in theory, these differences could arise as a consequence of selection pressures due to therapy or selection pressures for metastatic potential ; alternatively, they may arise randomly due to heterogeneity in the primary tumour and/or the high mutation rate in cancer cells. a recent whole genome sequencing study of matched primary and metastatic tumors in pancreatic cancer indicates that both primary tumor heterogeneity and further acquired mutations contribute to differences in mutational profiles between primary and metastatic sites. the product of the pik3ca gene is a catalytic subunit for class ia phosphoinositide 3-kinases (pi 3-kinases). class ia pi 3-kinases are lipid kinases whose aberrant activation plays a key role in the pathogenesis of many types of cancers. their aberrant activation occurs by multiple mechanisms including increased activation of upstream tyrosine kinases ; loss of the tumour suppressor pten, which antagonizes pi 3-kinase activity ; mutational activation of pi 3-kinase itself. there are three genes encoding different isoforms of the regulatory subunit (pik3r1, pik3r2, and pik3r3) and three genes encoding different isoforms of the catalytic subunit (pik3ca, pik3cb, and pik3cd). in breast cancer, activating point mutations in pi 3-kinase have only been found in the pik3ca gene, although amplification of the pik3cb gene has also been reported in some cases. within the pik3ca gene, the most common helical domain mutations are e542 k and e545 k, while the most common kinase domain mutation is h1047r. biochemical, tissue culture, and animal studies have confirmed that these mutations are in fact driver mutations. in breast cancer, the opposite is true in colorectal cancer ; this may relate to the different mechanisms by which the two types of mutations activate pi 3-kinase. pik3ca mutations occur in about 2530% of breast cancers, with numbers varying depending on the specific patient population and also the types of mutations that are included in the analysis. several studies have evaluated pik3ca mutation status in breast cancer metastases, although none to date have looked specifically in bone metastases [810 ]. metastatic bone disease is not only incurable, but can also cause significant medical complications including pain, fractures and hypercalcemia. we find that these mutations occur frequently in breast cancer bone metastases, suggesting that these patients may be good candidates for treatment with the selective pi 3-kinase inhibitors that are currently under development. samples were obtained from 14 patients at the ottawa hospital with histologically confirmed breast cancer and radiological evidence of at least one bone metastasis that was amenable to ct - guided biopsy. patients underwent an outpatient posterior iliac crest bone marrow aspirate and bone marrow trephine biopsy. for all cases, biopsy samples were formalin - fixed and paraffin - embedded. for a subset of cases where additional biopsy material was obtained, samples were also flash frozen. sections were cut from formalin - fixed and paraffin - embedded samples after a brief decalcification in which a surface of the block was dipped in decalcification solution. the presence of cancer cells in the biopsies was assessed by a pathologist (m. nabavi) using hematoxylin and eosin - stained sections. isolation of cancer cells from samples was performed with an arcturus xt instrument (applied biosystems canada, streetsville, on, canada) with both uv laser cutting and laser capture microdissection capabilities. for laser capture microdissection, 5 m unstained sections from paraffin - embedded samples were mounted on regular glass slides and deparaffinized. in which the presence of bone fragments in the biopsy would have interfered with laser capture microdissection, sections were mounted on pen membrane glass slides (applied biosystems canada, streetsville, on, canada) and laser cutting was used to remove bone fragments prior to laser capture. dna was extracted using quickextract ffpe dna extraction kits (epicentre biotechnologies, madison, wi, usa). membranes were removed from the laser capture caps and immersed in 25 l of extraction solution. samples were then heated for 60 min at 56c and 2 min at 98c, as recommended by the manufacturer. whole genome amplification was performed by the multiple displacement amplification method using dna repli - g mini kits (qiagen, toronto, on, canada). amplification was performed for 16 h at 30c and terminated by heating at 65c for 3 min. dna was assayed using quant - it picogreen dsdna reagent from molecular probes (eugene, or, usa). mcf7 human breast cancer cells (heterozygous exon 9 e545 k mutation) and t47d cells (heterozygous exon 20 h1047r mutation) were from the american type culture collection. cells were grown in dmem (mcf7) or rpmi (t47d) media supplemented with 15% fetal calf serum, were passaged for less than six months continuously, and were routinely checked for absence of mycoplasma. after 25 min of incubation, cells were scraped into the nbf and transferred to a 50 ml tube. cells were then washed twice in 80% ethanol and then pelleted in a 1.5 ml microfuge tube with 200 l of paraffin at the bottom. pelleted cells were transferred to the cap of a microfuge tube with a spatula and resuspended in 100 l of 3% low - melting point - agarose. after freezing at 20c, cells in agarose discs the mutation status of exons 9 and 20 in the pik3ca gene was assessed using a nested pcr procedure. the first round of pcr was performed in a multiplexed fashion with outside primers for both exon 9 (forward primer 5-ctg tga atc cag agg gga aa-3 ; reverse primer, 5-gca ctt acc tgt gac tcc ata gaa-3) and exon 20 (forward primer 5-tga gca aga ggc ttt gga gt-3 ; reverse primer, 5-cct atg caa tcg gtc ttt gc-3) combined in the same reaction. pcrs were done in a final volume of 60 l containing 0.1 ng whole genome - amplified dna, 1.5 mm mgcl2, 200 m dntps, 400 nm each primer, and 0.025 units/l hotstart taq polymerase (qiagen), using an eppendorf mastercycler thermal cycler. 35 cycles of 95c for 20 sec, 57c for 45 sec, and 72c for 45 sec were run, with an initial denaturation step of 15 min at 95c and a final extension step of 10 min at 72c. the second round of the nested pcr and high - resolution melt analysis was performed separately for exon 9 (inside forward primer, 5-aag gga aaa tga caa aga aca g-3 ; inside reverse primer, 5-cac tta cct gtg act cca tag aa-3) and exon 20 (inside forward primer, 5-gca aga ggc ttt gga ttt c-3 ; inside reverse primer, 5-ttt tca gtt caa tgc atg ctg-3). pcrs and high resolution melting were done in a final volume of 20 l containing 0.5 ng first pcr product, 2.5 mm mgcl2, 200 m dntps, 200 nm each primer, 5 m syto9 dye, and 0.025 units/l hotstart taq polymerase, using a corbett rotorgene 6000. 30 cycles of 95c for 20 sec, 57c for 20 sec and 72c for 20 sec were run, with an initial denaturation step of 15 min at 95c. high - resolution melting analysis was performed with an initial hold at 95c for 1 sec and a melting profile from 7285c rising by 0.1 degree each step. control samples of mcf7 and t47d dna, prepared as described above, were included in each pcr run. template from the initial multiplex pcr (see above) was amplified with m13-tagged primers. pcr products were sequenced directly at stemcore laboratories, ottawa hospital research institute, using big dye terminator v 3.1 chemistry and and an applied biosystems 3730 dna analyzer. the success rate for performing the three different types of biopsies on the fourteen patients in the study (ct - guided biopsy, trephine bone marrow biopsy, and bone marrow aspiration) is shown in table 1. bone marrow aspiration and bone marrow trephine biopsies were performed on 13 of 14 patients, while ct - guided bone biopsies were performed on 9 of 14 patients. a total of 5 of 13 bone marrow trephine biopsies were positive for cancer cells, while 4 of 9 ct - guided bone marrow biopsies were positive and only 2 of 13 bone marrow aspirates were positive. overall bone metastasis samples positive for cancer cells were obtained from 6 of 14 patients. a combination of laser cutting and laser capture microdissection was used to isolate > 90% pure populations of cancer cells for genomic analyses. as the amount of starting pilot experiments performed in our lab using dna from formalin - fixed paraffin - embedded pellets of cancer cell lines showed that mutation detection was not affected by the whole genome amplification procedure. dna suitable for mutation detection was obtained from all samples that were positive for cancer cells, regardless of the type of sampling method used. pik3ca mutations were detected using a nested pcr approach and a high - resolution melting screen as described in materials and methods. we restricted our analysis to the three most common mutations, e542 k, e545 k, and h1047r, as these have been well characterized for their ability to constitutively activate pik3ca and function as driver mutations. dilution with dna from cultured cells showed that high - resolution melting could detect exon nine e545 k mutations in mcf7 dna present at 10% of total dna (a 1 in 20 allele frequency as mcf7 is heterozygous for this mutation). the limit of detection was the same for exon 20 h1047r mutations, assayed using dna from t47d cells. as the number of samples was small, all were analyzed by sanger sequencing, regardless of whether or not they showed abnormal melting curves. of the six cases where biopsies were positive for cancer cells, three showed pik3ca mutations (one heterozygous e545 k mutation, one heterozygous h1047r heterozygous mutation, and one h1047r apparent heterozygous mutation ; see table 1). there was complete concordance in pik3ca mutation status between samples from the same patient taken using the different biopsy methods (table 1). figure 1 shows an example of the detection of an e545 k mutation in a ct - guided biopsy sample ; figure 2 shows an example of detection of wild - type (i.e., nonmutated) pik3ca in a ct - guided biopsy ; figure 3 shows an example of the detection of an h1047r mutation in a trephine bone marrow biopsy sample. primary tumor samples were obtained from four of the six cases analyzed for pik3ca bone metastasis mutations. dna was isolated from these and analyzed as above, except that the whole genome amplification step was omitted. pik3ca mutation analyses in the primary tumor samples were performed blinded to the pik3ca mutation status in the bone metastasis samples. for the four cases, the pik3ca mutation status was the same between the primary and the bone metastasis sample (table 1). table 2 shows the estrogen receptor, progesterone receptor, and her2 status of the six patients for which pik3ca mutation status was determined. the elapsed time between patient diagnosis (both cancer diagnosis and metastatic cancer diagnosis) and the bone metastasis biopsy is also shown in table 2. in this pilot study, the overall success rate for determining patient pik3ca status in bone metastases was 6/14 (47%). the success rate for patients in which biopsy material contained tumor cells was 100% ; thus, the limiting factor in success is the biopsy procedure itself. pik3ca status was assessable from all types of biopsies ; future studies should focus on the use of trephine bone marrow biopsies as this procedure had the highest success rate, when both patient willingness to undergo the procedure and the likelihood of obtaining positive samples are taken into consideration. all dna samples in the study were isolated using laser capture microdissection to avoid loss of sensitivity in mutation detection due to contamination with normal cell dna. an issue with laser capture microdissection on these samples is that some contain bone fragments that can interfere with the procedure, which relies on close contact between the cap membrane and the tissue section. we found that this problem could be overcome by first using laser cutting to remove bone fragments prior to laser capture microdissection. hotspot pik3ca mutations were found in three of the six assessable samples. while these numbers are clearly too low to draw strong conclusions, this is similar to the expected frequency for a population of er+ breast cancer patients (all six cases assessed here were er+). for example, the frequency of pik3ca mutations in a previous study of 366 er+ breast cancer patients was 38.5%. although pik3ca mutations occur at a significantly lower frequency in patients with her2 amplification, they do occur in this patient population. both e545 k helical domain and h1047r kinase domain mutations were detected. with the qualification that the numbers are very small, these data suggest that the overall frequency, types of mutations, and association with other breast cancer biomarkers are similar between breast cancer bone metastases and primary breast cancer tumors. recent work has shown that there can be substantial differences in biomarker status between primary breast cancer samples and samples of metastases from the same patient. of the six patients assessed for bone metastasis pik3ca status in this study, primary tumor material was available for four. there was complete concordance between the pik3ca status in the primary tumor and the bone metastases in these four patients. other studies have reported conflicting results with regard to concordance in pik3ca mutation status between paired primary and metastatic breast cancer. jensen. reported that in a study of 104 patients, about one - third exhibited differences in their pik3ca mutation status between primary and metastasis samples ; it was proposed that this was due to heterogeneity in the primary tumor and selection for the mutation during metastasis. in contrast, kalinsky. found > 90% concordance between primary tumor pik3ca status and either lymph node or distant metastasis ; in addition, they did not find any evidence for heterogeneity in pik3ca status in primary tumors and suggested that the apparent lack of concordance might be due to technical issues. our data indicate that primary tumor pik3ca mutation status (either normal or mutant) is maintained in bone metastases, although a larger number of samples would need to be evaluated in order to determine how invariant this is. times from initial diagnosis (these correspond to the times when the primary tumor sample was obtained) to the biopsies for this study were performed ranged from < 1 to 14 years (table 2). for three of the four patients in which paired samples were assessed, a period of greater than 10 years elapsed between obtaining the primary tumor sample and the bone metastasis sample. this suggests that pik3ca status does not change substantially over time or in response to standard treatment regimens. currently, there are a large number of therapeutic agents under development that target pi 3-kinase. these include dual mtor / pi 3-kinase inhibitors such as bez235 and gsk2126458 [15, 16 ] ; selective class i pi 3-kinase inhibitors such as bkm120 and xl147 [17, 18 ] ; the pi 3-kinase isoform (encoded by pik3ca) selective inhibitor byl719 (novartis international ag). in many of the trials with these inhibitors our data suggest that these drugs may be active against breast cancer bone metastases and that the pik3ca mutation status in bone metastases can be predicted from readily available primary tumor samples.	background. an important goal of personalized cancer therapy is to tailor specific therapies to the mutational profile of individual patients. however, whole genome sequencing studies have shown that the mutational profiles of cancers evolve over time and often differ between primary and metastatic sites. activating point mutations in the pik3ca gene are common in primary breast cancer tumors, but their presence in breast cancer bone metastases has not been assessed previously. results. fourteen patients with breast cancer bone metastases were biopsied by three methods : ct - guided bone biopsies ; bone marrow trephine biopsies ; and bone marrow aspiration. samples that were positive for cancer cells were obtained from six patients. three of these patients had detectable pik3ca mutations in bone marrow cancer cells. primary tumor samples were available for four of the six patients assessed for pik3ca status in their bone metastases. for each of these, the pik3ca mutation status was the same in the primary and metastatic sites. conclusions. pik3ca mutations occur frequently in breast cancer bone metastases. the pik3ca mutation status in bone metastases samples appears to reflect the pik3ca mutation status in the primary tumour. breast cancer patients with bone metastases may be candidates for treatment with selective pik3ca inhibitors.
vision and the somatosensory and vestibular sensory systems play an important role in posture control1,2,3,4,5. vision, based on a change in the information projected on the retina, guides the relationship between the environment and the body. plantar sensation provides information on the base of support and position of the center of gravity. vestibular sensation detects gravity and acceleration to provide information about the position and movement of the head. if these sensory systems are damaged due to brain diseases, failures in standing posture control are elicited, even without motor paralysis. pusher syndrome, wallenberg s syndrome, thalamic astasia, unilateral spatial neglect, etc. although there are previous studies about the change in standing posture due to vibration stimulation and/or vestibular stimulation10,11,12, there are few studies about the role of visual intervention in standing posture. prism glasses can bias a view on sagittal and/or horizontal planes, but can not shift it on coronal plane13, 14. although there is a method of tilting seat surface during sitting, this method can not be an intervention of only vision because it influences on somatosensory and vestibular sensory15. a tool to cause a lateral tilt of a view on the coronal plane was only a large screen16, 17. however, recently, an inexpensive immersive head - mounted display (hmd) has been developed and applied in rehabilitation18. in the present study, we developed a visual inclination system by using an hmd for investigating a visual intervention (tilted view) on standing posture control. eleven healthy male university students (mean standard deviation : age, 21.5 1.5 years ; height, 170.9 6.1 cm ; weight, 64.4 8.0 kg) participated in this experiment. we excluded participants who wore glasses on a daily basis and those with any orthopedic, neurological, ophthalmic or otolaryngological disease. the ethics committee of the international university of health and welfare approved all study procedures (no. 15-io-58), which were consistent with the principles of the declaration of helsinki. the authors obtained written informed consent from all the subjects prior to their participation in the study. ca, usa), a small stereo camera (ovrvision 1, shinobiya inc., osaka, japan), and a laptop pc (15x8550-i7-vsb, unitcom, osaka, japan) were employed to constitute a visual inclination system for the experiment (fig. (c) an hmd shows the tilted visual information to the wearer.). the subjects were asked to maintain the standing posture twice for 5 s while wearing the experimental system. they were instructed to perform the experiment under two visual conditions : normal view and 20 leftward inclined view conditions, in that order. two force plates were used to measure the vertical component of the floor reaction force of each leg. a three - dimensional motion analysis system was used to quantify the subjects body movements (i.e. inclination angles of the head (h), trunk (t), and pelvis (p) in absolute coordinates). we adopted several angle definitions, h, t, and p, which are described in fig 2fig. 2.definition of the h, t, and p. the black dots are reflective markers on top of the head, bilateral acromions, anterior superior iliac spine, and posterior superior iliac spine. point b is the midpoint of points c and d. points c and d are iliac crests estimated from the position of the markers on the pelvis. points e and f are midpoints of the anterior superior iliac spine and posterior superior iliac spine. h is the angle between the axis connecting from the top of the head to point a and the vertical axis. t is the angle between the axis connecting from point a to b and the vertical axis. p is the angle between the axis connecting from point e to f and the horizontal plane.. furthermore, we defined the relative head and trunk bending angles as the difference between the inclination angles of the head and trunk, and those of the trunk and pelvis, respectively. we defined a leftward inclination angle as positive and a rightward inclination angle as negative. visual inclination system. definition of the h, t, and p. the black dots are reflective markers on top of the head, bilateral acromions, anterior superior iliac spine, and posterior superior iliac spine. point b is the midpoint of points c and d. points c and d are iliac crests estimated from the position of the markers on the pelvis. points e and f are midpoints of the anterior superior iliac spine and posterior superior iliac spine. h is the angle between the axis connecting from the top of the head to point a and the vertical axis. t is the angle between the axis connecting from point a to b and the vertical axis. p is the angle between the axis connecting from point e to f and the horizontal plane. a three - dimensional motion analysis system consisting of 10 infrared cameras (vicon mx, vicon, oxford, uk) and two force plates (amti, watertown, ma, usa) was used to record three - dimensional marker displacements and floor reaction force data at a sampling frequency of 100 hz. thirty - three reflective markers (helen - hayes marker set) were attached to each subject. in the analysis, we used seven markers, which were attached to the top of the head, the bilateral acromions, anterior superior iliac spine, and posterior superior iliac spine of the participants. a two - tailed paired t - test was used to assess individual differences between the inclined view and normal view conditions. the statistical analysis was conducted using the software package spss version 20 (ibm inc. the result of the paired t - test demonstrated that the vertical component of floor reaction forces in both legs significantly changed in the inclined view condition. the vertical component of floor reaction forces decreased in the right leg (324.4 38.6 vs. 303.4 51.3 n, p=0.03), whereas it increased in the left leg (322.3 53.3 vs. 345.9 52.2 n, p=0.02). table 1table 1.the mean inclination angles of the head, trunk, and pelvis, and the relative bending angle of the head and trunknormal view conditioninclined view conditionhead leftward inclination angle ()0.2 2.31.7 3.7trunk leftward inclination angle ()0.8 0.82.0 0.9pelvis leftward inclination angle ()1.2 2.00.9 1.8neck leftward bending angle ()1.0 2.70.3 3.3trunk leftward bending angle ()0.4 1.91.1 2.1values are expressed as a mean standard deviation. significant difference (p<0.05) between the normal view condition and the inclined view condition. represents the mean inclination angles of the head, trunk, and pelvis, and the relative bending angle of the head and trunk. in the comparison between the normal view and inclined view conditions, the paired t - test indicated that there was a significant increase in the inclination angle of the head and trunk and the relative bending angle of the trunk, but not in the inclination angle of the pelvis and the relative bending angle of the head. significant difference (p<0.05) between the normal view condition and the inclined view condition. in this study, we created a novel hmd system to alter inclined standing posture in a group of male university students. by using this system, the head and trunk of participants inclined leftward and the vertical component of the floor reaction force of the lower extremities inclined leftward due to the view presented on the display. these results are identical to those of a previous experiment with a large - sized screen16, 17. the present study proves that it is possible to elicit a change of standing posture due to a visual stimulus using an immersive hmd, and a large - scale apparatus is no longer necessary. as for the inclination angle of each body segment, even though the head and trunk angles inclined toward the tilted direction, the angle of the pelvis did not incline. the reason why inclination in the pelvis did not occur is that it could not physically occur when the participant stood with both legs extended. therefore, inclination of the pelvis might occur in dynamic movements such as walking. as for the relative head and trunk bending angles, the effect of the tilted view was confirmed only in the trunk. the reason why the neck did not bend to the side is that a very large number of muscle spindles were distributed in the neck muscles19 ; therefore, it seemed that these muscle spindles compensated for the proprioceptive sensation. the previous research about siting posture using an electric balance board also showed lateral bending of trunk and no lateral bending of the neck during tilting the seat surface15. these experimental results suggest that lateral bending of the trunk is more available than the neck in the postural control using vision. the inclination angle of the standing posture observed in the present study was smaller than the tilt angle of view. it seemed that the reason was compensation by the somatosensory or the vestibular sensory systems. somatosensory function decreases with age, and therefore the elderly tend to rely on vision for postural control20, 21. thus, it seems that visual inclination has a large effect in the elderly. in addition, if the presented tilted view is combined with a vibration stimulus or vestibular stimulation, compensation due to the somatosensory and the vestibular sensory systems will be difficult. therefore, the body inclination effect might be enhanced. when the tilt angle is too small, the effect of inclining the body is low. on the other hand, even if the tilt angle is too large, the effect of inclining the body becomes low too, because the subject is no longer trust the view. there is a previous study examining the effect of the standing posture when changing the tilt angle of the view projected on a large screen17. the study compared three tilt angle conditions (5.1, 9.1 and 20.1) and reported that 20.1 is the most effective. it suggests that the tilt angle of view in the present study is appropriate. since the immersive hmd is wearable, unlike large screens or mirrors, its advantage is that it continues to provide visual information to the wearer even when moving forward or changing direction. therefore, it is possible to use this system to reveal the effects of the tilted view during walking or turning movements. furthermore, we may be able to use the visual inclination system for balance exercises to treat vertical misperception in brain disease patients. the balance exercises should be adjusted to the degree of difficulty for maintaining balance for individuals22. if patients who can not maintain an upright posture because of severe impairment of vertical perception, it may be easier for them to hold the upright posture by seeing a view that is inclined opposite to the inclination of the vertical axis that the patient is aware of. on the other hand, there are patients with unstable gait due to the mild impairment of vertical perception. for such patients, the balance exercises in normal visual conditions are less effective because the degree of difficulty is low. thus, presenting an inclined view that emphasizes the inclination of the vertical axis that the patients are aware of might be able to increase the degree of difficulty of the balance exercises to an appropriate level. moreover, if the presented tilted view is combined with vibration and vestibular stimuli, the training effect may be increased further10,11,12. the first is the possibility that the weight of the hmd affected somatosensory perception, although it was truly a lightweight device. use of a lighter hmd may increase the effect of inclination in the standing posture. the second is that we did not strictly define the visual environment in the experimental room. however, we wanted to ensure that our results would not be affected by the presence or absence of vertical products. the third is that what we measured is only the effect of the presentation of the leftward tilted view in order to avoid the burdens of the subjects. inclination of standing posture due to brain disease is likely to occur more toward the left than right6. therefore, the effect of the presentation of the rightward tilted view may be less than that of the leftward tilted view. the fourth is that we did not measure the sensory modality that the subject was focused upon. a method to determine the sensory modality that the subject focused on should be developed. the fifth limitation is that we did not consider the effects of aging, since our study group consisted of young male participants. however, the current study revealed that presenting a tilted view using immersive hmd can shift the relative trunk bending angle and center of gravity toward the same direction of the tilted view. the developed visual inclination system seems useful and it can be applicable to various psychophysical experiments in future research.	[purpose ] the purpose of the present study is to clarify whether tilted scenery presented through an immersive head - mounted display (hmd) causes the inclination of standing posture. [subjects and methods ] eleven healthy young adult males who provided informed consent participated in the experiment. an immersive hmd and a stereo camera were employed to develop a visual inclination system. the subjects maintained a standing posture twice for 5s each while wearing the visual inclination system. they performed this task under two conditions : normal view and 20 leftward tilted view. a three - dimensional motion analysis system was used to measure the subjects postures, and two force plates were used to measure the vertical component of the floor reaction force of each leg. [results ] in the 20 leftward tilted view, the head and trunk angles in the frontal plane were similarly inclined toward the left, and the vertical component of the floor reaction force increased in the left leg, whereas it decreased in the right leg. [conclusion ] when the view in the immersive hmd was tilted, the participants trunk side bent toward the same side as that of the view. this visual inclination system seems to be a simple intervention for changing standing posture.
benign duodenocolic fistula is a fistula between the duodenum and colon with no malignancy of either section. however, the duodenocolic fistula complication of peptic ulcer has not been reported in the middle east. diagnosis of duodenocolic fistula is always established with upper and lower gastrointestinal tract contrast studies. regardless of the cause of duodenocolic fistula the best treatment is surgical modality. here, the case of a patient who was diagnosed with a duodenocolic fistula secondary to duodenal peptic ulcer underwent extended right hemicolectomy and duodenal wall en bloc resection is reported. a 44-year - old caucasian male with a history of chronic upper abdominal pain, diarrhea and 25 kg weight loss in three months was admitted. the patient had a history of dyspepsia and upper gastrointestinal (gi) bleeding 4 months before admission. gastroduodenoscopy demonstrated a small ulcer in the lesser curvature and a large ulcer in duodenal bulb. physical examination revealed cachexia, pale conjunctiva and scaphoid abdomen, with no palpable mass. laboratory evaluations were in normal ranges except 10.1 gr / dl hb and 2.6 gr / dl albumin. no obvious mass was observed in the entrance of scope from first part of the duodenum (d1) into the colon and biopsies revealed no malignancy. upper gi series with barium study revealed a duodenocolic fistula (figure 1). abdominal ct scan with oral contrast showed duodenal deformity without evidence of malignancy. intraoperative findings were duodenocolic fistula between d1 and transverse colon with no mass in stomach, duodenum or colon. the patient had complete relief from symptoms after surgery with a six months follow - up. he found a fistula between duodenum and the mid - transverse colon, which was due to duodenal diverticulum. since then about 169 benign duodenocolic fistula cases have been reported in western countries. mcpeak reported the first case of duodenal ulcer with fistula between duodenum and transverse colon. he described two cases with d1 and d2 fistula and managed them with excision of fistula (3). since then, about 40 cases of duodenocolic fistula as a complication of peptic ulcer have been reported. further cases were due to previous typhoid ulceration, ulcerative colitis, tuberculous mesenteric adenitis, duodenal diverticulum, iatrogenic trauma, spontaneous fistula, colon diverticulum, foreign body penetration, acute pancreatitis, acute appendicitis, hydatid cyst and acute cholecystitis. patients usually suffer from chronic colicky abdominal pain, which is located in the epigastric region or the right hypochondrium. patients usually have weight loss due to bacterial overgrowth, steatorrhoea, malnutrition, malabsorption and electrolyte disturbances. almost always patients experience predominant motion of colonic contents through the fistula to the duodenum. this is probably due to pyloric act as a barrier of retrograde flow of feces from duodenum to the stomach (6). although the patient in the studied case had chronic abdominal pain, severe diarrhea with food particles and overt weight loss during 3 months, the important role of colonoscopy and gastroduodenoscopy is obtaining tissue sample for suspicious malignancy in the colon or duodenum. barium enema is the most reliable with a sensitivity of over 90% than approximately 25% accuracy in upper gi studies (7). upper and lower endoscopies were performed because of patient 's previous history and alarm signs. it was confirmed with upper gi barium study. due to endoscopic findings, barium enema was excluded. duodenocolic fistula patients are usually malnourished and they usually need parenteral nutrition before surgery. before introduction of parenteral nutrition, outcome of such patients were not satisfactory (5). successful palliation of diarrhea with octreotide has been reported in malignant duodenocolic fistula (8) but the treatment of choice in benign duodenocolic fistula is surgery. the duodenal defect is either closed primarily or repaired with serosal patch, depending on the defect size. primary closure of the duodenum defect is the preferred treatment in the literature (9). other procedures include roux - en - y duodenojejunostomy, partial duodenal excision with gastrojejunostomy (10). in malignant cases more extensive resection is usually needed. in some cases of crohn 's disease and tuberculosis, fistula closed spontaneously after medical treatment (11, 12). primary closure of duodenum was possible and reconstruction with gastrojejunostomy was performed. although peptic ulcer disease is not uncommon, it 's a rare complication ; duodenocolic fistula has not been reported in iran previously. due to identical clinical presentation of duodenocolic fistula and malignancy, variant radiology modalities and endoscopy with biopsies are necessary before surgery. surgery is the only curative treatment, after resection, reconstruction method is dependent on patient 's situation. partial colectomy with en bloc excision of fistula and partial duodenectomy is the preferred treatment in most of these patients.	a 44-year - old man with upper abdominal pain, diarrhea and 25 kg weight loss since 3 months ago was admitted. he had a history of dyspepsia and peptic ulcer disease 4 months before admission.gastroduodenal endoscopy and upper gastrointestinal series with barium study were done. biopsies and ct - scan ruled out malignancies. endoscopy and radiology studies revealed a duodenocolic fistula. he underwent right hemicolectomy, fistula en bloc excision, and distal gastrectomy surgery with gastrojejunostomy and ileocolic anastomosis. radiologic modalities are necessary before surgery. surgery is the only curative treatment in benign cases and reconstruction method is dependent on patient 's situation.
however, the complexity of ill - health, which is socially constructed by individuals, health personnel and health authorities have motivated the search for other forms to approach knowledge. to discuss the complementarities of qualitative and quantitative research methods in the generation of knowledge. the purpose of quantitative research is to measure the magnitude of an event, to make predictions, develop causal explanations. to achieve this it uses a pre - established design based on hypothesis and theories, conducts extensive data collection to a statistical sample and develops statistical data analysis. quantitative research objectives can be to establish the incidence or prevalence of a health problem ; the health personnel degree of adherence to a new intervention ; or, the users level of satisfaction with a service. its design is open, flexible and circular, data collection is intensive and based on a purposive sample and results will be achieved through inductive analysis. qualitative research allows to explore aspects thought as known, understands differences in personnel opinions and practice in front of new interventions or users opinion on services utilization. quantitative and qualitative methods are different research approaches, that not only provide complementary knowledge that contributes to gaining better understanding of a problem or situation, but that can be used in a combined way, to approach a new research area, to develop instruments and to interpret results.	introductionresearch in the area of health has been traditionally dominated by quantitative research. however, the complexity of ill - health, which is socially constructed by individuals, health personnel and health authorities have motivated the search for other forms to approach knowledge.aimto discuss the complementarities of qualitative and quantitative research methods in the generation of knowledge.contentsthe purpose of quantitative research is to measure the magnitude of an event, to make predictions, develop causal explanations. to achieve this it uses a pre - established design based on hypothesis and theories, conducts extensive data collection to a statistical sample and develops statistical data analysis. quantitative research objectives can be to establish the incidence or prevalence of a health problem ; the health personnel degree of adherence to a new intervention ; or, the users level of satisfaction with a service. qualitative research aims at understanding what exists from social actors perspectives. its design is open, flexible and circular, data collection is intensive and based on a purposive sample and results will be achieved through inductive analysis. qualitative research allows to explore aspects thought as known, understands differences in personnel opinions and practice in front of new interventions or users opinion on services utilization.conclusionquantitative and qualitative methods are different research approaches, that not only provide complementary knowledge that contributes to gaining better understanding of a problem or situation, but that can be used in a combined way, to approach a new research area, to develop instruments and to interpret results.
immune cells utilize toll - like receptors (tlrs) to sense pathogen associated molecular patterns (pamps), which represents the starting point of the innate immune response. plasmacytoid dendritic cells (pdcs) are at the nexus between innate and adaptive immunity and are thereby key contributors to the host 's response to various pathogens. furthermore, pdcs are the major source of type 1 interferon (ifn) in humans and are, thus, of particular importance for antiviral immunity and autoimmune diseases. in human beings, pdcs and, to a lesser extent, b cells constitutively express tlr7 and tlr9, both of which sense nucleic acids [35 ]. tlr7, originally known to recognize imidazoquinoline derivatives (e.g., imiquimod and resiquimod / r 848) and guanine analogs (e.g., loxoribine), recognizes single - stranded (ss) rna derived from rna viruses such as influenza a virus, vesicular stomatitis virus, and human immunodeficiency virus. in pdcs, tlr7 recognizes these ligands in specialized endolysosomes, resulting in the subsequent activation of nf-b and interferon regulatory factor (irf) 7 via myd88, and induces production of inflammatory cytokines and type 1 ifn, respectively. preclinical studies utilizing tlr7 ligation revealed promising results in the treatment of cancer, allergy, and infectious diseases [6, 7 ]. however, clinical studies using tlr7 ligands systemically are problematic, as they have been associated with severe side effects such as hematological toxicity., we hypothesized that the simultaneous use of two structurally and chemically different tlr7 ligands unfolds synergistic effects on human pdcs. to test this hypothesis, we coadministered the adenine analog cl264, a potent and highly specific tlr7 ligand, together with the natural ssrna 9.2s rna to the human pdc line cal-1. our data may broaden our understanding of tlr7 activation and could impact future strategies optimizing tlr7-targeted drug design. pbs (ph 7.4) and rpmi 1640 were purchased from life technologies (ca, usa). the latter was supplemented with 10% heat inactivated fetal bovine serum (fbs superior, biochrom ag, berlin, germany) and 2 mmol / l glutamine (life technologies). cell culture flasks and 24- and 96-well flat - bottom plates were obtained from greiner bio - one (frickenhausen, germany). cl264 (tlrl - c264e) and fitc - labeled cl264 (tlrl - fc264) were purchased from invivogen (ca, usa) and dissolved in sterile, rnase - free water (sigma - aldrich, mo, usa) containing 10% dmso (sigma - aldrich) and further diluted in culture medium. cl264 (formula c47h73n13o7s, molecular weight 413.43) is a 9-benzyl-8-hydroxyadenine derivative containing a glycine on the benzyl group (in para). according to the manufacturer specification, the substance we used is highly pure (endotoxin level 10 g / ml). the immunostimulatory properties of the single - stranded rna 9.2s were first described in 2005, when hornung. originally intended to design different sirnas (named 9.1 to 9.4) downregulating the human tlr9 to avoid effects of non - target - related ifn induction in human pdcs. however, when the authors of that study transfected human pdcs using these sirnas, they surprisingly noted a consistent pattern of type 1 ifn production, with sirna 9.2 being the strongest inducer of ifn production. additional analyses revealed that a specific sequence of nine bases at the 3 end (5-guccuucaa-3) of the sense strand of sirna9.2 (termed rna 9.2s, in contrast to 9.2a, which stands for the antisense strand) was responsible for the immunostimulatory activity. injection of 9.2 rna into tlr7-deficient mice elicited, in contrast to the injection into wild - type mice, no detectable ifn serum levels. microarrays facilitate the evaluation of global changes in gene expression induced by immune stimuli [2325 ]. this has led others to use microarray approaches to monitor global changes of gene expression induced by the combination of several inflammatory stimuli [2629 ]. in general, almost all studies observed synergistic upregulation of immune - related genes when combining ligands for different tlrs [27, 29 ]. rationale is always the simultaneous activation of different pathways inducing a supra - additive immune activation. however, we opted for a slightly different approach by creating synergistic effects using structurally different ligands targeting the same receptor. we observed a more than 3-fold increase in the number of genes upregulated by the combination of cl264 and 9.2s rna compared to cl264 alone (figure 2(a)). additionally, the overall magnitude of gene upregulation was increased by more than 2.5-fold when comparing the top 50 genes induced upon combination of both ligands versus cl264 alone (figure 2(b)). studies of protein production by elisa confirmed the synergistic activation of regulatory members such as il-6, tnf, and ifn- (figure 1). these findings suggest that simultaneous exposure to both ligands magnified and accelerated the response elicited by each individual ligand. importantly, the vast majority of genes upregulated by stimulating with either ligand alone were also induced in the costimulatory setting (92%). investigating the genes upregulated in the combined cl264/9.2s rna group, nearly 54% of them were also upregulated in the monostimulatory settings (although at a lower p value). taken together, this indicates a stronger stimulation of genes yet upregulated upon stimulation by either ligand. on the other hand, this finding also delineates the upregulation of new or rather so far unregulated genes by the combination of cl264/9.2s rna (46% of the genes). these synergistic effects primarily broaden the existing immune response, rather than creating a new one. this observation is supported by the fact that the functional groups (analyzed by ipa) induced by the combinatory versus single stimulation did not alter qualitatively (innate immune function such as irf signaling or dc maturation (table 2)). finally, exclusive ipa analysis of the 62 synergistically upregulated genes (defined as upregulated 1.5-fold of the sum induced by the ligands 9.2s rna and cl264 alone ; see table 3) revealed that 65% were immune - related. indeed, more than 35% of all synergistically upregulated genes, including the most highly upregulated, coded for cytokines, chemokines, or transcription regulators (such as il-1b, il-6, il-18r, il-23a, ccl1, ccl2, rel, and nfkbiz). these findings support the hypothesis that stimulation via different tlr7 ligands synergistically broadens and increases the magnitude of the host 's protective immune response., who showed that the combination of small phosphothioate odns with the tlr7/8 agonist cl075 (a chemical base analog comparable to cl264) induced a synergistic response in murine glial cells with substantially higher levels of proinflammatory cytokines and chemokines compared to cl075., we showed increased cell surface binding (> 2-fold) of cl264 upon combination with 9.2s rna compared to cl264 alone (figure 4). by stimulating the cells sequentially, 9.2s rna could be identified as being accountable for this phenomenon (figure 1(f)). although the mechanistic background remains to be elucidated, this finding suggests an optimized surface binding and subsequent cellular uptake into the endolysosomal compartment as one explanation for the observed synergistic effects of cl264 and 9.2s rna. of note, this effect is not due to the use of the cationic polypeptide plarg together with 9.2s rna, since synergy was not observed when cl264 was administered with plarg alone. further investigations are necessary to clarify whether binding is limited at the cell surface or leads to enhanced localization of cl264 within the endosomes in the presence of 9.2s rna, maybe using confocal microscopy. we can not rule out further additive effects on the receptor level using both types of ligands. with regard to the abovementioned properties of 9.2s rna, our microarray data do not show any evidence for a specific influence of 9.2s rna on the closely related tlr9 pathway (data not shown). since cal-1 cells do not express the tlr8, costimulatory effects of tlr7 and tlr8 by the use of ssrna and cl264 are highly improbable. however, tanji. very recently showed two distinct binding sites of ssrna fragments and chemical ligands at tlr8 leading to synergistic effects when being simultaneously activated. since tlr7 and tlr8 are closely related and recognize the same ligands, it is conceivable that the same phenomenon also accounts for tlr7. thus, optimal stimulation of tlr7 at different binding sites by ssrna and base analogs could also contribute to the observed synergistic effects of this study. upcoming experiments are designed to gain more mechanistic insights into the intracellular level underlying these effects by combining different ssrna motifs and cl264. although various synthetic tlr7/8 agonists have been used as adjuvants during preclinical trials, not many of them are approved for use in humans. the tlr7/8 agonist r837 is used for the topical treatment of genital warts, basal cell carcinoma, and bladder cancer. however, systemic application of imidazoquinolines causes adverse side effects, and the development of other tlr7 agonists suitable for nontopical use as adjuvants is desirable [6, 33 ]. our study has some limitations. despite the fact that cal-1 cells share many of the phenotypic and functional properties of human pdcs, maeda. have described some significant differences between this cell line and freshly isolated primary human pdcs including little ifn- secretion in response to tlr stimulation. it is well accepted that working with purified human pdcs is challenging, since they are extremely sensible to any kind of physical manipulation (such as purifying them using different sorting techniques), resulting in significant cytokine release acting in a paracrine and autocrine fashion [20, 34 ]. kim. recently demonstrated that low levels of background ifn- (so - called primed pdcs) resulted in a strong feedforward regulation of their tlr - triggered type 1 ifn production in vitro and concluded that the in vitro activity of a tlr ligand on primary pdcs does not necessarily correspond to its in vivo activity. however, we and many other groups have meanwhile shown that cal-1 cells are suitable surrogates to study specific aspects of human pdc physiology and tlr7 and tlr9 [9, 12, 3638 ]. for the herein presented study, we chose to focus on ifn- (instead of ifn-) since we studied early time point effects up to 12 hours to minimize the mentioned auto- and paracrine cytokine effects, which is essential when studying synergism of different tlr ligands. we previously demonstrated that, in contrast to ifn-, ifn- expression peaks rather late (> 24 hours) in response to tlr stimulation, which is likewise influenced by a type 1 ifn feedback loop. a number of studies analyzed synergistic effects activating different tlrs [2629 ]. however, to our knowledge, the present study is the first to provide fundamental new insights into the simultaneous activation of a single toll - like receptor. using the human pdc - like cell line cal-1, we demonstrated that structurally different tlr7 ligands act synergistically on gene expression patterns of the inflammatory response. this phenomenon may be explained in part by an enhanced binding of cl264 in the presence of 9.2s rna. however, future studies will need to further elucidate intracellular mechanisms, as suggested by recent studies. taken together, our data could impact future strategies optimizing tlr7-targeted drug design and provide new insights into the synergistic interactions of structurally different tlr7 ligands.	objective. tlr7 ligation in plasmacytoid dendritic cells is promising for the treatment of cancer, allergy, and infectious diseases ; however, high doses of ligands are required. we hypothesized that the combination of structurally different tlr7 ligands exponentiates the resulting immune response. methods. cal-1 (human pdc line) cells were incubated with the tlr7-specific adenine analog cl264 and single - stranded 9.2s rna. protein secretion was measured by elisa. microarray technique was used to detect modified gene expression patterns upon synergistic stimulation, revealing underlying functional groups and networks. cell surface binding properties were studied using facs analysis. results. cl264 in combination with 9.2s rna significantly enhanced cytokine and interferon secretion to supra - additive levels. this effect was due to a stronger stimulation of already regulated genes (by monostimulation) as well as to recruitment of thus far unregulated genes. top scoring canonical pathways referred to immune - related processes. network analysis revealed il-1, il-6, tnf, and ifn- as major regulatory nodes, while several minor regulatory nodes were also identified. binding of cl264 to the cell surface was enhanced by 9.2s rna. conclusion. structurally different tlr7 ligands act synergistically on gene expression patterns and on the resulting inflammatory response. these data could impact future strategies optimizing tlr7-targeted drug design.
obstructive sleep apnea syndrome (osa) is a common problem among children and especially boys, with estimated prevalence of 2 - 3%, with most children being diagnosed at the age of 26 years. at night osa is characterized by interrupted sleep, snoring, and disrupted breathing. at daytime, children present hyperactive behavior, cognitive deficits, and attention problems. adenotonsillar hypertrophy is considered the leading cause of pediatric osa and tonsillectomy and adenoidectomy (t&a) is still considered and clinically used as a first line treatment for children with osa. there is growing evidence on osa 's associated neurobehavioral, cognitive, cardiovascular, and metabolic morbidities. furthermore, new studies found an improvement in cognitive and cardiovascular functions after t&a. in the last 30 years there were several studies, as well as case studies, suggesting osa as a major risk factor for growth failure [9, 10 ]. these studies were conducted mostly among kindergarten and school children and showed an improvement of somatic growth accompanied with an increased level of igf-1 and the ratio growth hormone (gh)/igf-1 after t&a [11, 12 ]. the very few studies that followed somatic growth in toddlers have also shown an improvement of somatic growth, but none of them have ever tried to estimate the role of growth hormone in the process [11, 12 ]. there are several possible mechanisms for growth retardation among children with osa, including increased energy expenditure during sleep due to labored breathing, decreased appetite, and enlarged tonsils that serve as a mechanical barrier limiting the amount of food swallowed. another possible explanation is interruption in the axis of growth hormone (gh) secretion. gh is secreted from the pituitary gland mainly during slow - wave sleep in a pulsatile fashion. gh secretion positively correlates with igf-1 and igfbp-3 levels which triggers bone growth. notwithstanding the direct effects of gh on growth plate chondrocytes, it is now accepted that the indirect local and systemic effects of gh function in a highly coordinated manner regulate growth plate activities and linear bone growth. in children with osa there is a decrease in the slow - wave sleep component, and an increase in its percentage of the total sleep time is noted following t&a. this phenomenon was revealed by others and serves as a reasonable explanation to growth retardation before t&a and the improvement in growth after surgery. linear bone growth is adversely affected in children with chronic kidney disease (ckd) and other chronic inflammatory disorders. ckd patients have been reported to have elevated circulating levels of il-6 and tnf, similar to patients with osa [16, 17 ]. the (gh)/(igf-1) pathway is anabolic to the skeleton and inflammatory cytokines compromise bone growth through several mechanisms, which include interference with the systemic as well as the tissue - level gh / igf-1 axis. recent studies have shown that, in children, osa is accompanied by systemic inflammation, reflected by increased levels of markers like circulating c - reactive protein (crp), that decrease after t&a. interestingly, the role of systemic inflammation, in regard to growth, in such children was not studied so far. therefore, we assessed growth in osa young children before and after t&a and evaluated potential mechanisms that may influence growth in these children. the study was approved by soroka university medical center human irb committee, and informed consent was obtained from the legal caretaker of each participant. young children with psg proven osa were enrolled and followed prospectively before t&a surgery and 46 months afterwards. inclusion criteria were children older than 6 and younger than 36 months of age who were diagnosed with an obstructive apnea - hypopnea index (ahi) > 5 events / hour of sleep in an overnight polysomnographic evaluation and informed consent from the legal caregiver who agreed was obtained. exclusion criteria were craniofacial, neuromuscular, syndromatic, or defined genetic abnormalities, any known previous allergies, no upper respiratory infection use of any corticosteroids or antibiotics within 4 weeks preceding the initial sleep study, and any children that had had t&a or adenoidectomy in the past. children were studied in a dedicated quiet, darkened room with an ambient temperature of 24c in the company of one of their parents. polysomnography was performed with a computerized, commercially available, sleep monitoring system (sensormedics inc. sleep staging was performed with the standard criteria published by the aasm in 2007 and not by the latest revision of 2012 since we recruited our last child in february 2012. the ahi was defined as the airflow with continued chest wall and abdominal movement over at least two breaths. hypopneas were defined as a 50% decrease in nasal flow with a corresponding 3% decrease in spo2 and/or arousal or awakening. arousals were defined as recommended by the revised aasm rules. in each visit the child anthropometric data was analyzed by the endocrinologist (n.l.) for z score using the software z score represents standard deviations gap from the mean to child 's age. a short food frequency questionnaire (sffq) for energy assessment was completed for each child on both encounters. this sffq is based on a list of items reported by 160 mothers of infants at the age of 6 to 24 months describing the previous day 's food items offered or consumed. all sffqs were assessed for caloric intake and for the differential percent of protein, fat, and carbohydrates in diet. circulating hscrp was measured by means of particle - enhanced immunonephelometry using the bn prospec system (newark, de). blood draws were performed at the morning of t&a and 46 months following surgery also at the morning hours at the pediatric sleep clinic. igf-1 levels were measured using an immunoradiometric assay after extraction (dsl, webster, tx, usa). interassay coefficient of variation (cv) was < 14% ; data are presented in nmol / l. the markers were measured at the endocrinology and immunology laboratories at the soroka university medical center. all parents filled a validated questionnaire given on the day of surgery and at the postsurgical follow - up visit in our pediatric sleep clinic. in brief, it includes eight items scored by the parent in a 04 scale (0 = never ; 4 = most of the time). questions included are being hard to awake, witnessed apnea, breathing difficulties, snoring, sweating, mouth breathing, awakening, and restless sleep. all numeric data were subjected to statistical analyses with either t tests or 2-way analysis of variance procedures for repeated measures, as described by neuman - keuls. four children were lost to followup and were not different than the remaining participants. all 16 children (10 boys and 6 girls) their mean age at the first encounter was 23 6 months (range 733 months), and the mean time of follow - up was 5 2 months (range 3.86.2 months). most children showed significant improvements in their height (4.81 cm) and weight (1.88 kg) following adenotonsillectomy (average : p < 0.001 for both). thus bmi also increased by 0.85 0.42 kg / m (p = 0.037). at diagnosis, mean z scores for height, weight, and bmi were 1.18 0.32, 1.29 0.35, and 0.51 0.15, respectively. after treatment, there were significant improvements in weight z scores (p = 0.002) and in bmi z scores (p = 0.007) as shown in figure 1. the decrease in systemic inflammation was reflected by decrements of 0.51 0.28 mg% in crp levels after t&a (p = 0.131), while small increases in igf-1 levels also emerged (2.37 1.41 nmol / l ; p = 0.104). sffq were filled by the parents and compared the caloric intake (table 1). on average, the children added 377.6 292.4 calories to their daily diet after surgery (p < 0.001). there were a significant increase in protein intake (2.0 1.4% ; p = 0.045 ; figure 2) and decrease in fat intake (4.49 2.36% ; p = 0.016). there was also an increase in consumption of carbohydrates of 2% that failed to reach statistical significance (p = 0.44). a multivariate analysis identified a significant negative correlation between systemic inflammation and weight in boys, such that, with more prominent decreases in circulating crp levels, weight gain following surgery was magnified (r = 0.775 ; p = 0.041 ; figure 3). this study found a major improvement in anthropometric parameters accompanied by a decrease in systemic inflammation and an increase in caloric intake and endocrinologic markers following t&a. furthermore, for the first time it has been proved that even young children with osa show improvement in somatic growth that is associated in boys with a reduction in systemic inflammation. this study also identified, for the first time, a significant change in diet composition in children with osa after surgery. interestingly, the improvement in somatic growth correlated with an improvement in systemic inflammation but did not correlate with the changes in caloric intake. we demonstrated that children aged 636 months who suffer from sleep disordered breathing are indeed smaller, for height and weight, than their peers before surgery. this gap was minimized following t&a, as shown in the rise in the z scores. this gap narrowing can be referred to as the catch - up growth. it has been long known that children with osa improve their somatic growth after surgery, but so far a comprehensive assessment of children 636 months was not done. previous studies that were held in this age group evaluated children for anthropometric parameters before and after t&a and found an improvement of somatic growth [11, 12 ]. none of them tried to measure circulating levels of endocrine factors or the presence of systemic inflammation and their effect on growth. furthermore, no dietary assessment has ever been performed in this age group and no one tried to estimate how surgical intervention affects the diet composition. several mechanisms have been proposed during the years to try and explain growth retardation in children with osa. among them are the presence of systemic inflammation that is well documented in children with osa [8, 14, 25 ] and dysphagia from hypertrophied tonsils that serve as a mechanical barrier and obstruct food entry, thus reducing caloric intake. one of the day symptoms of osa is hyperactive behavior which results in increased motor activity that increases energy expenditure, thus contributing to growth failure. this study tried to find the factor that imposes the biggest influence on catch - up growth in these children. circulating crp and igf-1 levels were measured to estimate level of systemic inflammation and growth hormone - insulin - like growth factor 1 (gh - igf-1) axis, respectively. in order to assess caloric intake, parents completed a short food frequency questionnaire (sffq) for energy assessment for each child on both encounters. this sffq is based on a list of items reported by 160 mothers of infants at the age of 6 to 24 months describing the previous day 's food items offered or consumed and was validated in previous studies [22, 23 ]. other studies that assessed the relationship between nutrition and t&a surgery did show a rise in carbohydrates consumption after t&a, but these studies used 24-hour dietary - recalls and were performed in preschool children. this is the first study that performed dietary assessment in toddlers with osa before and after t&a using a validated questionnaire. furthermore, this sffq evaluates meal compound during the month prior to each encounter and is less prone to recall bias. analyzing the sffqs showed that all children but one increased their caloric intake after surgical intervention. moreover, looking at their dietary compound, a rise of 2% in protein and a decline of 4.49% in fat consumption were significantly shown. a nonsignificant rise of 2% percent in carbohydrates was also noticed. although these changes were found to be significant, their actual effect on growth pattern is not clear, and there is certainly a need to repeat the sffq in a larger group in order to draw conclusions. in parallel to the rise in caloric intake, the children narrowed the weight gap between them and their peers. these two findings can be linked because t&a surgery removes the hypertrophied tissue of tonsils and adenoids, thus removing a mechanical barrier that limits food swallow. removing this barrier enables the child to eat and swallow more easily and increases his caloric intake according to physiologic requirements and gaining weight accordingly. in addition, the change in diet compound not only might indicate hormonal changes that influence appetite but also may represent a rise in anabolic process in the body that is manifested by weight gain. though being statistically significant, these changes in dietary compound are not marked enough to explain the rise in somatic growth. previous studies in children with osa reported high circulating levels of inflammatory factors such as crp. those studies showed a decline of up to 50% in crp levels after t&a [14, 25 ]. we hypothesized that an improvement in systemic inflammation will be followed by an improvement in somatic growth. there was an average decrease of 0.535 mg% in crp level after surgery ; however, the change was not significant probably due to small sample and this should be studied on a larger cohort. igf-1 mediates the effects of growth hormone in tissues, especially the bone, and promotes its growth. previous studies reported on shortening of the relative part of slow - wave sleep and improvement in its relative part after t&a. a meta - analysis that tried to determine the role of t&a on gh - igf-1 axis did find an increase in igf-1 and igfbp-3 serum levels, but all the studies were conducted on older children. this may explain the improvement in their weight but it was not significant probably due to a small sample. in order to find the factor that imposes the biggest influence on catch - up growth in osa children, a multivariate analysis was performed. the more prominent the decrease in circulating crp levels was, the more weight was gained by the child.. this might support the assumption that the major factor influencing somatic growth in children with osa is systemic inflammation and not the increase in caloric intake per se. one hypothesis is that local mediators effect bone growth, similar to children with chronic kidney disease. the growth hormone (gh)/insulin - like growth factor 1 (igf-1) pathway is anabolic to the skeleton and inflammatory cytokines compromise bone growth through a number of different mechanisms, which include interference with the systemic as well as the tissue - level gh / igf-1 axis. it is known that suppressor of cytokine signaling 2 (socs2) expression is increased in inflammatory conditions including ckd and is a recognized inhibitor of gh signaling. therefore, socs2 signaling represents a critical pathway in growth plate chondrocytes through which osa 's activated proinflammatory cytokines alter both gh / igf-1 signaling and cellular function. our report is the first to note the correlation between enhanced somatic growth and decreased systemic inflammation following surgery. it corroborates previous observations regarding other osa 's related morbidities (cardiovascular and neurocognitive) that improved following surgery and correlated with decreased systemic inflammation [8, 30 ]. finally, although our findings may be of interest, there is a need to address the limitations of our study. although they were well studied, there may be bias in the outcomes due to the small sample size. the questionnaire we used shows that the overall symptom improvement was very good (all 8 questions with a scale of 15 : preop 3.7 1.3 ; postop 1.3 0.4 ; p = 0.002), but questionnaires can be limited. the sleep disordered breathing scale predicts polysomnographic results to an extent useful for research but it is not always reliable enough for individual patients. however, the sleep disordered breathing scale may predict osa - related neurobehavioral morbidity and its response to adenotonsillectomy as well as or better than polysomnography. we also need to point out the fact that the children studied represent very accurately the average child in our lab in terms of bmi but not in other countries. although we also see obese children in our sleep lab, most of the young children are normal or underweight for their age. therefore, the population recruited in our study may not be representative of current trends in the united states, for example, but still reflects what pediatric sleep specialists encounter in many other countries. in summary, this study assessed the effect of potential mechanisms on growth in young children treated for osa. we found that young children improve their anthropometric measures substantially following surgical removal of their lymphadenoid tissues. we have learned that the increase in caloric intake is accompanied by a change in the dietary composition, that is, an increase in protein and a decrease in fat consumption. the most prominent result is the degree to which t&a changes in systemic inflammation as reported by hscrp to correlate at least in boys with the degree of catch - up weight gain. this may point to the important role systemic inflammation plays in the growth processes of children with osa.	background. obstructive sleep apnea (osa) is associated with growth impairment that usually improves following effective treatment. in this study we investigated the mechanisms underlying the growth processes in young children diagnosed with osa, before and after adenotonsillectomy (t&a). methods. young children (636 months old) were enrolled and evaluated before and several months after t&a surgery for height, weight, circulating high sensitive c - reactive protein (crp), and insulin - like growth factor 1 (igf-1) levels. caloric intake was assessed by a validated short food frequency questionnaire (sffq). results. following t&a, children added 4.81 cm and 1.88 kg to their height and weight, respectively (p < 0.001 for both) and had a significant increase in bmi z score (p = 0.002). increased caloric intake of 377 kcal / day was noted (p < 0.001), with increased protein and decreased fat intake. the decrease in crp levels correlated with the increase in body weight in boys (p < 0.05, adjusted for caloric intake). conclusions. adenotonsillectomy results in enhanced somatic growth in young children that correlates with a decrease in systemic inflammation and caloric intake increment. our findings imply that systemic inflammation may have an important role in this osa - related morbidity.
earthquakes are among the most destructive natural disasters and have been the cause of some of the world 's highest death tolls in peacetime.1 they usually occur abruptly, and, therefore, the ability to utilize resources promptly and effectively to reduce casualties is the main measure of a successful response. earthquake trauma is diverse, but many victims suffer from crush injuries from building collapses.2,3 non - earthquake traumas, by contrast, usually do not include crush injuries. in light of the unique presentations of earthquake - related trauma, the features of earthquake - related injuries in abdominal, spinal, and pelvic trauma patients have been thoroughly described in previous studies.4 - 6 non - earthquake - related head injuries have contributed significantly to trauma - related deaths, accounting for up to one half of all deaths.7 similarly, in earthquake - related traumas, head injuries account for 30% of fatalities.1,8 although head trauma is common and deadly in both earthquake- and non - earthquake - related traumas, the features of the injuries may be different because of different injury circumstances. accordingly, we retrospectively reviewed computed tomography (ct) data and compared the findings of head traumas caused by the sichuan earthquake with traumas caused by other common factors in daily life with the aim of determining the distinctive features of earthquake - related head traumas. the local institutional ethical review board approved the present study, and informed consent was waived because of the retrospective nature of this study. the study subjects were patients with head traumas caused by the sichuan earthquake from the 12th to the 25th of may 2008 as well as head traumas caused by typical, non - earthquake - related traumas over a similar time frame from the 1st to the 30th of may 2009. to illustrate the features more clearly, we chose as a control group consecutive patients with non earthquake - related head traumas at the same university hospital during a similar period one year after the major earthquake to avoid bias arising from the different seasons and the confounding influence of the earthquake related situations. we applied the following inclusion criteria : 1) the patients should have had a definite history of head trauma, 2) the patients should have had no surgical treatment before undergoing a ct examination, 3) the ct scan should have covered all injured parts of the head, and 4) the ct image should have been of adequate quality for the diagnosis. among the patients with earthquake - related head traumas, any patient whose trauma resulted from falling or from a traffic accident during the earthquake finally, 221 cases with earthquake - related head traumas and 221 cases with non - earthquake - related traumas were enrolled into this study. the head ct scans of 39, 308, and 95 patients were obtained using a philips brilliance 16 slice multiple detector row ct scanner (philips medical systems, eindhoven, the netherlands), a siemens somatom sensation 16 and a siemens somatom plus 4, and a multiple detector ct scan (siemens medical systems, forchheim, germany), respectively. according to the injury site and physical examination, cranial, maxillofacial, and cranio - maxillofacial ct scans were performed in 248, 102, and 91 patients, respectively. the cranial and maxillofacial ct scans were performed from the base to the roof of the skull and from the supraorbital margin to the inferior margin of the chin, respectively. the scanning parameters were 120 kv, 200 - 440 mas, 0.5-s rotation time, pitch of 0.891, and collimation of 160.75 mm for the philips brilliance 16mdct scanner ; 120 kv, 200 - 250 mas, 0.5-s rotation time, pitch of 0.85, and collimation of 160.75 mm for the siemens somatom sensation 16-mdct scan ; and 120 kv, 180 - 260 mas, 0.5-s rotation time, pitch of 1.5, and collimation of 40.75 mm for the siemens somatom plus 4-mdct scan. the lower current (200 or 180 mas) was used for the maxillofacial ct scan. the reconstructed section thickness was 8 mm for the cranial ct and 1 mm for the maxillofacial ct. the sagittal and coronary images used to observe maxillofacial and basal fractures were reconstructed with a thickness of 1 - 3 mm. all of the mdct scans were reviewed by the authors on the syngo workflow picture archiving and communicating system workstation (siemens medical systems, forchheim, germany). the cranio - maxillofacial soft tissue, bones, and brain were reviewed, and the anatomic distributions or types of injuries were recorded. the window width and window level used in reviewing the images were as follows : soft tissue (w : 350 hu, l : 50 hu), bone (w : 3200 hu, l : 700 hu), and brain (w : 80 hu, l : 35 hu). injuries only involving the scalp and/or maxillofacial soft tissue were classified as extracranial soft tissue injuries. when the fractures were evaluated, the whole head was divided into cranial and maxillofacial regions. the anatomical sites of the cranial region included the frontal, parietal, temporal, sphenoid, and occipital bones, and those of the maxillofacial region included the nasal and ethmoid bones, zygoma, orbit, maxilla, and mandible. the diagnosis of the fractures was achieved with transverse plane and multi - planar reformation (mpr) as well as with three - dimensional (3d) reconstructions (volume rendering, vr ; surface shade display, ssd). craniocerebral injury was traumatic injury involving the scalp, cranium, or intracranial structures (i.e., brain, meninges, and other structures), whereas intracranial injury was injury that only involved the intracranial structures. intracranial injuries included extradural hematoma, subdural hematoma, subarachnoid hemorrhage, cephalophyma, pneumocephalus, cerebral edema and cerebral contusions, and lacerations. the patients ' ages and sexes and the cause of injury for the patients with non - earthquake traumas were recorded. the numbers of patients with simple head soft tissue injuries, fractures, and intracranial injuries as well as the numbers of fractures and intracranial injuries in the patients were counted. moreover, the proportions of soft tissue injuries, skull fractures, maxillofacial fractures, and cranio - maxillofacial fractures combined with intracranial injuries in both groups were calculated. the continuous variables were expressed as meansstandard deviation, and the categorical variables were expressed as numbers and percentages. an independent - sample t - test and chi - square test were used to compare the continuous variables and the categorical variables in both groups, respectively. statistical analysis was performed with the spss statistical package (version 13.0 for windows, spss inc. a two - tailed p - value of less than 0.05 was accepted as indicating a statistically significant difference. the local institutional ethical review board approved the present study, and informed consent was waived because of the retrospective nature of this study. the study subjects were patients with head traumas caused by the sichuan earthquake from the 12th to the 25th of may 2008 as well as head traumas caused by typical, non - earthquake - related traumas over a similar time frame from the 1st to the 30th of may 2009. to illustrate the features more clearly, we chose as a control group consecutive patients with non earthquake - related head traumas at the same university hospital during a similar period one year after the major earthquake to avoid bias arising from the different seasons and the confounding influence of the earthquake related situations. we applied the following inclusion criteria : 1) the patients should have had a definite history of head trauma, 2) the patients should have had no surgical treatment before undergoing a ct examination, 3) the ct scan should have covered all injured parts of the head, and 4) the ct image should have been of adequate quality for the diagnosis. among the patients with earthquake - related head traumas, any patient whose trauma resulted from falling or from a traffic accident during the earthquake finally, 221 cases with earthquake - related head traumas and 221 cases with non - earthquake - related traumas were enrolled into this study. the head ct scans of 39, 308, and 95 patients were obtained using a philips brilliance 16 slice multiple detector row ct scanner (philips medical systems, eindhoven, the netherlands), a siemens somatom sensation 16 and a siemens somatom plus 4, and a multiple detector ct scan (siemens medical systems, forchheim, germany), respectively. according to the injury site and physical examination, cranial, maxillofacial, and cranio - maxillofacial ct scans the cranial and maxillofacial ct scans were performed from the base to the roof of the skull and from the supraorbital margin to the inferior margin of the chin, respectively. the scanning parameters were 120 kv, 200 - 440 mas, 0.5-s rotation time, pitch of 0.891, and collimation of 160.75 mm for the philips brilliance 16mdct scanner ; 120 kv, 200 - 250 mas, 0.5-s rotation time, pitch of 0.85, and collimation of 160.75 mm for the siemens somatom sensation 16-mdct scan ; and 120 kv, 180 - 260 mas, 0.5-s rotation time, pitch of 1.5, and collimation of 40.75 mm for the siemens somatom plus 4-mdct scan. the lower current (200 or 180 mas) was used for the maxillofacial ct scan. the reconstructed section thickness was 8 mm for the cranial ct and 1 mm for the maxillofacial ct. the sagittal and coronary images used to observe maxillofacial and basal fractures were reconstructed with a thickness of 1 - 3 mm. all of the mdct scans were reviewed by the authors on the syngo workflow picture archiving and communicating system workstation (siemens medical systems, forchheim, germany). the cranio - maxillofacial soft tissue, bones, and brain were reviewed, and the anatomic distributions or types of injuries were recorded. discrepancies with regard to the interpretations the window width and window level used in reviewing the images were as follows : soft tissue (w : 350 hu, l : 50 hu), bone (w : 3200 hu, l : 700 hu), and brain (w : 80 hu, l : 35 hu). injuries only involving the scalp and/or maxillofacial soft tissue were classified as extracranial soft tissue injuries. when the fractures were evaluated, the whole head was divided into cranial and maxillofacial regions. the anatomical sites of the cranial region included the frontal, parietal, temporal, sphenoid, and occipital bones, and those of the maxillofacial region included the nasal and ethmoid bones, zygoma, orbit, maxilla, and mandible. the diagnosis of the fractures was achieved with transverse plane and multi - planar reformation (mpr) as well as with three - dimensional (3d) reconstructions (volume rendering, vr ; surface shade display, ssd). craniocerebral injury was traumatic injury involving the scalp, cranium, or intracranial structures (i.e., brain, meninges, and other structures), whereas intracranial injury was injury that only involved the intracranial structures. intracranial injuries included extradural hematoma, subdural hematoma, subarachnoid hemorrhage, cephalophyma, pneumocephalus, cerebral edema and cerebral contusions, and lacerations. the patients ' ages and sexes and the cause of injury for the patients with non - earthquake traumas were recorded. the numbers of patients with simple head soft tissue injuries, fractures, and intracranial injuries as well as the numbers of fractures and intracranial injuries in the patients were counted. moreover, the proportions of soft tissue injuries, skull fractures, maxillofacial fractures, and cranio - maxillofacial fractures combined with intracranial injuries in both groups were calculated. the continuous variables were expressed as meansstandard deviation, and the categorical variables were expressed as numbers and percentages. an independent - sample t - test and chi - square test were used to compare the continuous variables and the categorical variables in both groups, respectively. statistical analysis was performed with the spss statistical package (version 13.0 for windows, spss inc., a two - tailed p - value of less than 0.05 was accepted as indicating a statistically significant difference. the baseline characteristics of the patients in the earthquake and non - earthquake groups are shown in table 1. there were fewer male patients in the earthquake group than in the non - earthquake group (p0.05), but there were more cases with ages ranging from 20 to 50 years in the non - earthquake group (p<0.01). the mean time from injury to ct scan among the earthquake patients was longer than that for the non - earthquake patients. extracranial soft tissue injuries, fractures, and intracranial injuries were detected in 112 (50.7%), 97 (43.9%), and 38 (17.2%) cases in the earthquake group, respectively, and we found 58 (26.2%), 117 (52.9%), and 112 (50.7%) cases in the non - earthquake group, respectively. the comparisons of different types of injuries between the two groups are shown in figure 2. earthquake victims had more extracranial soft tissue injury cases (50.7% vs. 26.2%, rr = 1.9, p<0.001), but fewer patients had intracranial injuries (17.2% vs. 50.7%, rr = 0.3, p<0.001) than in the non - earthquake group. among the earthquake victims with fractures and/or intracranial injuries, maxillofacial fractures and craniocerebral injuries were detected in 60 (55.0%) and 66 (60.6%) cases, respectively. these same maxillofacial fractures and craniocerebral injuries were detected in 73 (44.8%) and 127 (77.9%) patients, respectively, in the non - earthquake group. craniocerebral injuries were significantly less frequent in the earthquake group than in the non - earthquake group (60.6% vs. 77.9%, rr = 0.8, p<0.01). one hundred forty - one (mean : 1.50.9 per patient, range : 18) fractures were found in 97 patients in the earthquake group, and 294 (mean : 2.51.8 per patient, range : 19) fractures were found in 127 patients in the non - earthquake group. among these patients, the cases in the earthquake group had fewer fractures than those in the non - earthquake group (p<0.001) (figure 3). the fracture anatomic distributions in the cranial and maxillofacial regions for both groups are shown in table 3. in the earthquake and non - earthquake groups, the temporal bone, orbit, and nasal and ethmoid bones were the most commonly involved sites in the cranial and maxillofacial regions. compared with the patients with non - earthquake - related fractures, patients with earthquake - related fractures had more occipital bone and mandible fractures but fewer maxillary fractures (p<0.01). fifty (mean : 1.30.5 per patient, range : 13) intracranial injuries were found in 38 patients in the earthquake group, and 242 (mean : 2.11.1 per patient, range : 15) intracranial injuries were found in 112 patients in the non - earthquake group. among these patients, the cases in the earthquake group had fewer intracranial injuries than did the patients in the non - earthquake group (p<0.001). the principal types of intracranial injuries in the earthquake and non - earthquake groups were subarachnoid hemorrhage versus cerebral contusions and lacerations, respectively. cerebral contusions and lacerations were more common in the non - earthquake group than in the earthquake group (p<0.05). moreover, contrecoup injuries (figure 4) were common in the non - earthquake group, and intracranial injuries were usually close to cranial fractures or scalp injuries (figure 5) in the earthquake group. in the earthquake group, 12 (9.8%) of 123 patients with soft tissue injuries but no fractures, 6 (12.2%) of 49 patients with maxillofacial fractures, 13 (35.1%) of 37 with cranial fractures, and 7 (58.3%) of 12 with cranio - maxillofacial fractures had intracranial injuries, respectively. in the non - earthquake group, 45 (43.7%) of 103 patients with soft tissue injuries but no fractures, 8 (18.2%) of 44 with maxillofacial fractures, 37 (82.2%) of 45 with cranial fractures and 22 (75.9%) of 29 cases with cranio - maxillofacial fractures had intracranial injuries, respectively. a comparison of intracranial injuries among the different types of extracranial injuries is shown in figure 6. compared with the non - earthquake group, the incidence of soft tissue injuries and cranial fractures combined with intracranial injuries was significantly lower in the earthquake group (9.8% vs. 43.7%, rr = 0.2 ; 35.1% vs. 82.2%, rr = 0.4, each p<0.001). in the earthquake group, 35 (15.8%) patients had a total of 48 combined injuries (mean : 1.40.5 per patient, range : 13 injuries). in the non - earthquake group, 46 (20.8%) patients had a total of 91 combined injuries (mean : 2.00.8 per patient, range : 14 injuries). extracranial soft tissue injuries, fractures, and intracranial injuries were detected in 112 (50.7%), 97 (43.9%), and 38 (17.2%) cases in the earthquake group, respectively, and we found 58 (26.2%), 117 (52.9%), and 112 (50.7%) cases in the non - earthquake group, respectively. the comparisons of different types of injuries between the two groups are shown in figure 2. earthquake victims had more extracranial soft tissue injury cases (50.7% vs. 26.2%, rr = 1.9, p<0.001), but fewer patients had intracranial injuries (17.2% vs. 50.7%, rr = 0.3, p<0.001) than in the non - earthquake group. among the earthquake victims with fractures and/or intracranial injuries, maxillofacial fractures and craniocerebral injuries were detected in 60 (55.0%) and 66 (60.6%) cases, respectively. these same maxillofacial fractures and craniocerebral injuries were detected in 73 (44.8%) and 127 (77.9%) patients, respectively, in the non - earthquake group. craniocerebral injuries were significantly less frequent in the earthquake group than in the non - earthquake group (60.6% vs. 77.9%, rr = 0.8, p<0.01). one hundred forty - one (mean : 1.50.9 per patient, range : 18) fractures were found in 97 patients in the earthquake group, and 294 (mean : 2.51.8 per patient, range : 19) fractures were found in 127 patients in the non - earthquake group. among these patients, the cases in the earthquake group had fewer fractures than those in the non - earthquake group (p<0.001) (figure 3). the fracture anatomic distributions in the cranial and maxillofacial regions for both groups are shown in table 3. in the earthquake and non - earthquake groups, the temporal bone, orbit, and nasal and ethmoid bones were the most commonly involved sites in the cranial and maxillofacial regions. compared with the patients with non - earthquake - related fractures, patients with earthquake - related fractures had more occipital bone and mandible fractures but fewer maxillary fractures (p<0.01). fifty (mean : 1.30.5 per patient, range : 13) intracranial injuries were found in 38 patients in the earthquake group, and 242 (mean : 2.11.1 per patient, range : 15) intracranial injuries were found in 112 patients in the non - earthquake group. among these patients, the cases in the earthquake group had fewer intracranial injuries than did the patients in the non - earthquake group (p<0.001). the principal types of intracranial injuries in the earthquake and non - earthquake groups were subarachnoid hemorrhage versus cerebral contusions and lacerations, respectively. cerebral contusions and lacerations were more common in the non - earthquake group than in the earthquake group (p<0.05). moreover, contrecoup injuries (figure 4) were common in the non - earthquake group, and intracranial injuries were usually close to cranial fractures or scalp injuries (figure 5) in the earthquake group. in the earthquake group, 12 (9.8%) of 123 patients with soft tissue injuries but no fractures, 6 (12.2%) of 49 patients with maxillofacial fractures, 13 (35.1%) of 37 with cranial fractures, and 7 (58.3%) of 12 with cranio - maxillofacial fractures had intracranial injuries, respectively. in the non - earthquake group, 45 (43.7%) of 103 patients with soft tissue injuries but no fractures, 8 (18.2%) of 44 with maxillofacial fractures, 37 (82.2%) of 45 with cranial fractures and 22 (75.9%) of 29 cases with cranio - maxillofacial fractures had intracranial injuries, respectively. a comparison of intracranial injuries among the different types of extracranial injuries is shown in figure 6. compared with the non - earthquake group, the incidence of soft tissue injuries and cranial fractures combined with intracranial injuries was significantly lower in the earthquake group (9.8% vs. 43.7%, rr = 0.2 ; 35.1% vs. 82.2%, rr = 0.4, each p<0.001). in the earthquake group, 35 (15.8%) patients had a total of 48 combined injuries (mean : 1.40.5 per patient, range : 13 injuries). in the non - earthquake group, 46 (20.8%) patients had a total of 91 combined injuries (mean : 2.00.8 per patient, range : 14 injuries). according to the clinical features of the patients in the earthquake and non - earthquake groups, we found that the age distributions in both groups were similar, but there were significantly more patients with ages ranging from 2050 years old in the non - earthquake group. additionally, male patients were also more common in the non - earthquake group than in the earthquake group. these differences were consistent with previous reports.9,10 this difference possibly reflects the tendency of young and middle - aged male patients to engage in riskier social behaviors. we found that head traumas caused by the earthquake were possibly different from head traumas with other causes. additionally, the earthquake group had more cases with extracranial soft tissue injuries, but fewer of these injuries coexisted with intracranial injuries. finally, the extracranial injuries, especially the soft tissue injuries and cranial fractures, had a lower incidence of combined intracranial injuries in the earthquake group. additionally, patients with head traumas in the earthquake group had fewer combined injuries. generally, the earthquake - related head injuries as depicted on ct were less serious. differences between the head traumas caused by the earthquake and other common traumas had a possibly direct relationship with the severity of the injuries. in an earthquake, head traumas commonly result from building collapses and falling objects,2, whereas non - earthquake - related traumas are frequently caused by traffic accidents and falls.4,10,15,16 the energy of impact is significantly different between these two settings, and high - mass, low - velocity injuries may be more common among earthquake - related injuries than among non - earthquake injuries. in addition, patients in an earthquake have a little time to react to falling buildings. however, patients in traffic accidents or who have fallen are even more vulnerable, because the impact is so instantaneous that they have no time to protect themselves from the trauma, which perhaps makes these traumas more severe. in the present study, the head traumas in the earthquake group mainly resulted from building collapses, and the non - earthquake - related traumas from falls and traffic collisions were more serious than the traumas in the earthquake group. similarly, injury mechanisms could also account for the different ct findings between earthquake - related and non - earthquake - related head traumas. the crush injuries caused by building collapses do not involve notable head motion after the initial collision. however, everyday traumas, such as those resulting from traffic collisions, cause deceleration injuries, which can also result in countercoup injuries from the relative motion of the brain.10, therefore, deceleration - related brain injuries are more common in non - earthquake - related head traumas and seem to cause more complicated brain injuries, such as the brain contusions and lacerations seen in this study. in addition, external conditions may also be responsible for the differences in head traumas. in the massive sichuan earthquake, traffic accidents, complicated infrastructure, and aftershocks all delayed rescue to the outlying areas, and patients with severe injuries died before they could receive emergency services. by contrast, rescue in the downtown area of chengdu city was more prompt, and, therefore, even severely injured patients could be quickly sent to hospitals for treatment. we recognized the significant difference in the time to treatment and ct scans between the earthquake and non - earthquake groups. thus, the mortality for the earthquake group was lower because some severely injured patients died before arriving at the hospital, and there were more patients with severe injuries in the non - earthquake group, although some died eventually. understanding the difference between head traumas caused by earthquake- and non - earthquake - related traumas is helpful for directing the specific diagnosis and treatment of injuries after an earthquake. as demonstrated in our study, patients in the non - earthquake group had more serious and complex head injuries. to diagnose their injuries, it was necessary to have them undergo ct examinations as quickly as possible. the patient load in a local area will suddenly increase, resulting in a sudden excess demand for ct scanning for head traumas and other injuries. under these conditions, selecting the patients who need urgent treatment will help improve the survival rate and optimize the use of limited medical resources. this study suggests that patients with suspected maxillofacial fractures have the greatest risk of intracranial injuries and, therefore, might deserve priority among head injury patients. mdct to examine the head is advantageous in an earthquake because it can save scanning time. the diagnosis is rapid, which will shorten the time required for lifesaving treatment.20 additionally, high - quality mpr images can demonstrate extra - intracranial injuries accurately and thoroughly and exclude the obvious intracranial injuries.21 - 23 furthermore, patients with head traumas present with varying levels of consciousness. however, physical examinations can not accurately depict the extent of severe trauma. in such conditions, some of the patients with serious head traumas died before rescue workers could bring these patients to our university hospital. the sichuan earthquake, the epicenter of which was 92 km (57 miles) from the hospital, damaged much of the infrastructure in the area, hampering rescue efforts directed to these outer suburban areas. the results of our study only address the patients who were stable enough to have survived the rescue effort and transport. although we could not evaluate the injuries of the victims who died in the earthquake, the study of the survivors ' injuries is more meaningful and reflects how we can make an actual difference in an earthquake 's aftermath. delayed rescue is common after a massive earthquake, and studying injuries in these conditions provides our medical teams with the practical knowledge that they will need to respond. in the present study, we determined the features of earthquake - related head traumas by comparing the ct findings of head injuries caused by the sichuan earthquake and by typical, non - earthquake - related events. compared with non - earthquake - related head traumas, the severity of earthquake - related traumas in survivors, as depicted on ct, was milder, and isolated extracranial injuries were more frequent, which may have been the result of the different injury causes, mechanisms and settings. a comparative study of the features of earthquake - related head injuries is helpful for understanding this common condition in such a special setting. this study was supported by the science foundation for distinguished young scholars of sichuan province in china (grant n 2010jq0039).	objective : the features of earthquake - related head injuries may be different from those of injuries obtained in daily life because of differences in circumstances. we aim to compare the features of head traumas caused by the sichuan earthquake with those of other common head traumas using multidetector computed tomography.methods:in total, 221 patients with earthquake - related head traumas (the earthquake group) and 221 patients with other common head traumas (the non - earthquake group) were enrolled in our study, and their computed tomographic findings were compared. we focused the differences between fractures and intracranial injuries and the relationships between extracranial and intracranial injuries.results:more earthquake - related cases had only extracranial soft tissue injuries (50.7% vs. 26.2%, rr = 1.9), and fewer cases had intracranial injuries (17.2% vs. 50.7%, rr = 0.3) compared with the non - earthquake group. for patients with fractures and intracranial injuries, there were fewer cases with craniocerebral injuries in the earthquake group (60.6% vs. 77.9%, rr = 0.8), and the earthquake - injured patients had fewer fractures and intracranial injuries overall (1.50.9 vs. 2.51.8 ; 1.30.5 vs. 2.11.1). compared with the non - earthquake group, the incidences of soft tissue injuries and cranial fractures combined with intracranial injuries in the earthquake group were significantly lower (9.8% vs. 43.7%, rr = 0.2 ; 35.1% vs. 82.2%, rr = 0.4).conclusion : as depicted with computed tomography, the severity of earthquake - related head traumas in survivors was milder, and isolated extracranial injuries were more common in earthquake - related head traumas than in non - earthquake - related injuries, which may have been the result of different injury causes, mechanisms and settings.
rheumatoid arthritis (ra) increases the risk of atherosclerotic cardiovascular disease (cvd) to a similar extent as diabetes [13 ]. atherogenesis in ra remains poorly elucidated and current recommendations on cvd risk stratification reportedly have major shortcomings [5, 6 ]. hence, there is a need for identifying novel biomarkers of enhanced cardiovascular risk in ra [5, 6 ]. numerous studies have documented that adiponectin exerts antidiabetic and vasculoprotective effects [814 ]. amongst the different adiponectin isoforms, high - molecular weight adiponectin particularly protects against the development of diabetes and cvd. in cellular studies, adiponectin was shown to increase gene expression and protein synthesis of many proinflammatory and prodestructive molecules that participate in the pathophysiology of ra [1719 ]. both the production and effects of adipokines can be altered in patients with autoimmune diseases including ra [20, 21 ]. in this regard, indeed, increased as well as reduced adiponectin concentrations in ra were reported by different groups [2224 ]. also both inverse or favorable and direct or paradoxically unfavorable associations of adiponectin concentrations with metabolic risk factors were found in ra [21, 24, 25 ]. with regard to direct effects of adiponectin on atherogenesis, we recently reported a cardiovascular risk factor independent direct relationship between adiponectin concentrations and early endothelial activation in white patients with ra. additionally, no association was found between adiponectin levels as well as carotid intima - media thickness (cimt) and coronary artery calcification scores in ra [21, 26, 27 ]. however, compared to cimt, carotid plaque is a more reliable indicator of severe atherosclerosis [2834 ]. thus, cimt reflects mostly arterial media thickening in response to aging and elevated blood pressure whereas plaque represents intimal pathology and advanced atherosclerosis that links more closely to coronary heart disease risk factors and myocardial infarction [2834 ]. in the present study, we examined the independent relationships of total and hmw adiponectin concentrations with cimt and plaque. as the production and effects of adipokines on cardiovascular risk depend on pathophysiological context [21, 25, 3539 ], we also determined whether the presence of conventional and nonconventional cardiovascular risk factors modified adiponectin concentrations and their associations with atherosclerosis. the present study was conducted according to the principles outlined in the helsinki declaration. the human research ethics committee (medical) from the university of the witwatersrand in johannesburg, south africa, approved the protocol (approval number : m06 - 07 - 33). participants gave informed, written consent. this investigation forms part of an ongoing study on cardiovascular risk in ra [25, 36, 38, 39 ]. two hundred and ten consecutive african patients (119 black and 91 white) that met the 1988 american college of rheumatology and 2010 american college of rheumatology / eular criteria for ra [40, 41 ] were enrolled. baseline characteristics and conventional metabolic risk factors were recorded using previously reported methods [25, 36, 38, 39 ]. overall adiposity, abdominal obesity, and fat distribution were estimated by the body mass index (bmi), waist circumference, and waist - hip ratio, respectively. extra - articular manifestations included the current or previously recorded (hospital record review) presence of pericarditis, pleuritis, felty 's syndrome, cutaneous vasculitis, neuropathy, scleritis or episcleritis, retinal vasculitis, glomerulonephritis, vasculitis affecting other organs, amyloidosis, keratoconjunctivitis sicca, xerostomia, sjogren 's syndrome, pulmonary fibrosis, bronchiolitis obliterans organizing pneumonia, cervical myelopathy, subcutaneous nodules, and rheumatoid nodules in other locations. standard laboratory blood tests of erythrocyte sedimentation rate, renal and liver function, hematological parameters, lipids, and glucose were performed. the glomerular filtration rate was estimated using the modification of diet in renal disease equation. recorded metabolic risk factors included systolic, diastolic and mean blood pressure, lipid concentrations and ratios, and glucose levels. hypertension was defined as an average systolic blood pressure 140 or / and diastolic blood pressure 90 mmhg or / and current use of antihypertensive medications. dyslipidemia was diagnosed when the atherogenic index, that is, the cholesterol - hdl cholesterol ratio, was > 4. diabetes was identified as the use of glucose lowering agents or a fasting plasma glucose 7 mmol / l. as reported previously, we also measured resistin concentrations. bas (see acknowledgement) and as performed the carotid artery ultrasound measurements in private and public healthcare patients, respectively. both operators obtained images of at least 1 cm length of the distal common carotid arteries for measurement of the intima - media thickness of the far wall from an optimal angle of incidence defined as the longitudinal angle of approach where both branches of the internal and external carotid artery are visualized simultaneously and with high resolution b - mode ultrasound (image point, hewlett packard, andover, ma, usa, and sonocalc imt, sonosite inc., bothell, wash, usa, used by bas and as, resp.) employing linear array 7.5 mhz probes. the equipment used by as involves the application of a unique semiautomated border detection program that was previously found to provide highly reproducible results. the intima - media thicknesses in the left and right common carotid artery were measured and the cimt was defined as the mean of of the two obtained values. carotid artery plaque was defined as a focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the surrounding intima - media thickness value or demonstrates a thickness of > 1.5 mm as measured from the media - adventitia interface to the intima - lumen interface. repeat ultrasound examinations by both operators on 23 patients revealed spearman correlations between repeat cimt measurements of 0.983 and 0.956 for bas and as, respectively, and the correlation between measurements made by bas and as was 0.926. both operators identified carotid artery bulb or / and internal carotid artery plaque in 11 of these 23 patients with full agreement. total and hmw adiponectin concentrations were measured using solid - phase sandwich enzyme - linked immunosorbent assays (elisa) (quantikine hs, r&d systems, inc., the inter- and intra - assay coefficients of variation were 6.5 and 3.5% for total and 8.5 and 3.0% for high molecular weight adiponectin, respectively. dichotomous variables are expressed as proportions or percentages and continuous variables as mean (sd) or median (interquartile range (iqr)) when nonnormally distributed. associations of baseline characteristics with total and hmw adiponectin concentrations in the present cohort were previously reported and considered in multivariable analysis in the present study, that is, upon deciding which characteristics are potential confounders or mediators. these variables comprised age, sex, race, glomerular filtration rate, and waist circumference. we assessed the associations of hmw and total adiponectin concentrations with cimt and plaque in framingham score (calculated from age, sex, and major conventional risk factors), race, glomerular filtration rate, waist circumference, and c - reactive protein level adjusted mixed linear or logistic regression models as appropriate. the framingham score was used rather than its individual components in order to avoid over - fitted models as these can produce false positive and false negative results. patients with ra that experience conventional risk factors or severe disease are reportedly at high risk of cardiovascular disease [16 ]. for these reasons together with the influence of physiological context on adipokine effects and our recent experience with adipokine metabolism in ra [21, 25, 3539 ], we assessed the impact of patient characteristics on total and hmw adiponectin concentrations and their relations with carotid cimt and plaque in subgroups with and without patient characteristics of interest in the present context. for this purpose, patients with a bmi of 30 kg / m and those that met the national cholesterol education program for metabolic syndrome (mets) waist criterion were considered to sustain overall and abdominal obesity, respectively. when appropriate, patients were categorized in subgroups based on median values. in view of the small number of patients that were rheumatoid factor negative or had extra - articular features, sensitivity analysis in subgroups based on the presence or absence of these characteristics, silverspring, maryland, usa) and sas software, version 9.1 (the sas institute, cary, nc). the demographic features, lifestyle factors, anthropometric measures, conventional metabolic risk factors, c - reactive protein concentrations, glomerular filtration rate, and use of antirheumatic and cardiovascular agents in the present cohort were previously reported. the median (range) erythrocyte sedimentation rate was 14 (0120) mm / hr. total and hmw adiponectin concentrations in all patients and relevant subgroups are given in table 1. total adiponectin concentrations were smaller in patients with compared to those without abdominal obesity and both total and hmw adiponectin concentrations larger in those with compared to those without deformed joints. the mean (sd) cimt and plaque prevalence in 208 of the patients is given in table 2., total adiponectin concentrations were related to plaque prevalence in patients with but not without abdominal obesity and in those without but not with joint deformities. as applied to total adiponectin, hmw adiponectin concentrations were not associated with atherosclerosis in all patients and related to plaque prevalence in patients with but not without abdominal obesity and in those without but not with joint deformities. the use of the framingham score in our analysis may not have fully accounted for the confounding effect of age on atherosclerosis. however, in additional analysis in which we constructed models in groups as in tables 4 and 5 but with adjustment for age, sex, race, and waist circumference as potential confounders, total and hmw adiponectin concentrations remained associated with plaque in patients with abdominal obesity and without joint deformities (p = 0.02 for each relationship). in view of these findings we evaluated whether cimt and plaque prevalence differed by abdominal obesity and joint deformity status. plaque prevalence was numerically smaller in patients with compared to those without abdominal obesity and larger in patients with compared to those without joint deformities. the latter relationship was significant with an odds ratio (95% confidence interval) of 3.31 (1.467.51) for the association of deformed joint with plaque. in this regard also, obesity protects against joint damage in ra. in the present study, the median (range) deformed joint count was smaller in patients with compared to those without abdominal obesity (5 (036) versus 10 (045), p = 0.05). when, in additional analysis, we constructed models in groups as in table 5 but with adjustment for age, sex, race, and waist circumference as potential confounders, the plaque prevalence remained higher in patients with compared to those without deformed joints (p = 0.01). in age, sex, race, glomerular filtration rate, cardiovascular drug use, and waist circumference adjusted models, total and hmw adiponectin concentrations were not significantly (p 0.2) associated with those of resistin in all patients and those with and without abdominal obesity or deformed joints. also, upon further adjustment for resistin concentrations in tables 3 and 4, total and hmw adiponectin concentrations remained associated with plaque (p = 0.02 to 0.002) in patients with abdominal obesity and without joint deformities. when we repeated the analyses using log transformed variables in the mixed regression models, the results were materially unaltered (data not shown). previous studies on the potential involvement of adiponectin in enhanced cvd risk in ra have produced contradictory results [2427 ]. herein, we show for the first time that both total and hmw adiponectin concentrations are independently associated with reduced plaque prevalence in ra patients with abdominal obesity or clinical absent joint damage. a 1-standard deviation increment in total adiponectin concentration was independently associated with a 13 and 6% reduction in plaque prevalence amongst patients with abdominal obesity and clinical absent joint damage, respectively. carotid artery plaque independently predicts incident cardiovascular event rates in non - ra subjects and patients with ra. first, as applies to osteoprotegerin and retinol binding protein 4 (rbp4), which is an adipokine that enhances atherogenesis, adiponectin is a promising cardiovascular risk biomarker that could improve cardiovascular risk stratification in ra [46 ]. second, considering adiponectin concentrations only may be inadequate in estimating the impact of this adipokine in cardiovascular risk in ra. indeed, despite the presence of reduced total and hmw adiponectin concentrations in patients with abdominal obesity or absent joint damage, it was in these groups that we observed inverse - adiponectin atherosclerosis relations. thus, small rather large adiponectin concentrations concurred with a favorable association with cardiovascular risk in the present investigation. taken together, increased adiponectin concentrations in ra do not necessarily translate into adiponectin mediated cardioprotective effects. third, targeting adiponectin in an attempt to reduce ra severity [1719 ] could be expected to additionally increase cardiovascular risk, at least in patients with abdominal obesity or less severe ra. fourth, as previously documented by us [3639 ], stratified analysis should be considered upon elucidating of the potential role of adipokines in cvd risk in ra. obesity is paradoxically associated with decreased incident cardiovascular event rates and overall mortality in ra. in this study congruently, we recently also found an independent inverse relation between abdominal adiposity and endothelial activation in ra. the present investigation suggests that altered and favorable effects of adiponectin may constitute a mechanism that links abdominal obesity to reduced cardiovascular risk in ra. this relationship was explained by adiponectin concentrations. in the present investigation, we also found an inverse association between abdominal obesity and joint damage. joint deformity counts as well as radiographic damage in ra are associated with increased atherosclerosis. our current results further indicate that altered effects of adiponectin may additionally contribute to the link between mild ra and reduced cardiovascular risk. we recently reported a paradoxically positive relation between adiponectin concentrations and endothelial activation amongst white patients with ra. as also indicated by other investigators, we suggested that this finding represents a compensatory increase in adiponectin production aimed at reducing cardiovascular risk rather than a paradoxically altered adverse impact on atherogenesis. our current results support this possibility since total adiponectin concentrations exhibited a borderline inverse association with plaque prevalence in white patients (or = 0.89 per 1-standard deviation increment, p = 0.07). interestingly in this regard, we recently documented that rbp4 concentrations are paradoxically associated with reduced endothelial activation and, at the same time, also with enhanced atherosclerosis in ra. high grade inflammation is associated with atherosclerosis and cardiovascular events in ra [57, 58 ]. in this regard, adiponectin is an anti - inflammatory adipokine [1719, 21, 25 ] whereas resistin is proinflammatory [36, 59 ]. indeed, resistin concentrations are associated with those of c - reactive protein in ra [36, 59 ]. moreover, resistin levels are independently related to surrogate markers of early atherogenesis and may contribute to the link between inflammation and enhanced cardiovascular risk in ra. our analysis argues against circulating adiponectin and resistin being linked in ra and the adiponectin concentration - atherosclerosis relations were not explained by resistin levels. in a recent investigation, koskinen and colleagues showed that in men with osteoarthritis (oa), circulating adiponectin concentrations correlate positively with levels of oa biomarkers and both circulating adiponectin levels and adiponectin concentrations released by cultured cartilage are associated with oa severity. furthermore, when adiponectin was added at physiological concentrations to cultures of intact oa cartilage or primary oa chondrocytes, this adipokine enhanced the production of inflammatory and/or destructive factors nitric oxide, interleukin-6, and matrix metalloproteinases 1 and 3 in a mitogen - activated protein kinase - dependent manner. hence, reported evidence together with the findings in the current study indicates that adiponectin can exert a dual role in arthritis comprising catabolic and proinflammatory effects on cartilage and protection against atherosclerosis. we evaluated relationships of both total and hmw adiponectin concentrations with atherosclerosis in a relatively large and well characterized ra cohort. a substantial number of potential confounders or / and mediators were included in mixed regression models. however, we previously found that radiographic scores and joint deformities are strongly correlated in our setting (r = 0.808, p < 0.0001)., the present study indicates that consideration of adiponectin can improve cardiovascular risk stratification amongst ra patients with abdominal obesity or mild disease.	in the present study, we examined the potential impact of adiponectin on carotid ultrasound determined atherosclerosis in 210 (119 black and 91 white) ra patients in mixed regression models. total adiponectin concentrations were smaller in patients with compared to those without the metabolic syndrome (mets) defined waist criterion (median (range) = 6.47 (1.2334.54) versus 8.38 (0.8285.30) ng / ml, p = 0.02, resp.) ; both total and high molecular weight (hmw) adiponectin concentrations were larger in patients with compared to those without joint deformities (7.97 (0.8285.30) and 3.51 (0.0135.40) versus 5.36 (1.2919.49) and 2.34 (0.0119.49) ng / ml, p = 0.003 and 0.02, resp.). total and hmw adiponectin concentrations were associated with carotid artery plaque in patients with mets waist (odds ratio (95% ci) = 0.87 (0.760.99) and 0.92 (0.850.99) per 1-standard deviation increment, p = 0.02 for both) and those without joint deformities (odds ratio (95% ci) = 0.94 (0.880.99) and 0.94 (0.890.99), p = 0.03 for both). plaque prevalence was lower in patients without compared to those with joint deformities (23.4% versus 42.6, p = 0.004 in multivariable analysis). in ra patients with abdominal obesity or no clinically evident joint damage, adiponectin concentrations are reduced but nevertheless associated with decreased carotid atherosclerosis.
multilevel cervical canal stenosis causesd by the ossification of posterior longitudinal ligament (opll) or cervical spondylotic myelopathy (csm) is common42326). most patients are asymptomatic, but those with severe spinal cord compression are predisposed to symptoms of cervical radiculopathy or myelopathy 1626). surgical treatment is recommended when neurologic symptoms are severe. the technique enlarges of the canal which indirectly decompresses the spinal cord by allowing the dural sac to drift away from the spondylotic bars223). several technical variations exist, such as the z - plasty14) or the open door technique6). the french - door laminoplasty consists of a median lamina split followed by a lateral thinning and opening of both hemilaminae. the technique seems to provide satisfying and reliable long - term results in patients with opll and myelopathy due to cervical spondylosis and posterior thickening of the ligamentum flavum225). cervical expansive laminoplasty was originally carried out as a modified french - door laminoplasty using the spinous processes as spacers15). since the design of classic open - door laminoplasty with the use of sutures, the procedure has been modified to reduce complications such as restenosis, axial symptoms, and segmental motor paralysis315). with the development of surgical implants, various kinds of lamina spacers, such as the spinous processes, hydroxyapatite spacers, and centerpiece each methods had its own advantages and disadvantages, but the recently developed laminoplasty fixation peek system with a biocompatible polymer securing the locking hinge of the plate without radiological artifacts has not been studied for its efficacy yet. the authors therefore began to use maxpacer (seohancare, gyeonggi - do, korea, fig. 2) as a lamina spacer with the french - door method, and compared the canal expansion rate among these methods. between june and december 2012, combined surgery for multilevel cervical stenosis was performed by one neurosurgeon in a single university hospital. we performed a retrospective study of the 15 patients, analyzing the clinical and radiological results, including the difference in spinal canal areas between pre- and postoperative imaging. the surgical procedure in the cases discussed here consisted of french - door laminoplasty with and without maxpacer-l type (fig. horizontal amputation of the spinous processes was performed and bilateral laminar exposure was carried out. the upper half of spinous process was removed and applied to the gutter side after maxpacer was used for only bone fusion. midline laminotomy was then performed with a drill, and lateral outer cortical bone drilling was done to facilitate elevation. during performing lateral outer cortical bone drilling, the surgeon found the lamina - facet junction, which was a landmark for drilling. it was important to drill just medial of the facet joint in order to have enough spinal canal area. since the narrow drilling space can induce the lamina fracture when the lamina is elevated, adequate space was important to avoid fracturing the lamina. after drilling, the ligament flavum was split centrally and each lamina and ligamentum flavum was opened bilaterally until the lamina stood straight. after proper positioning of the laminae, maxpacers were applied to the space between both laminae and secured by 8mm - sized titanium screws (fig. the decision of where to apply the maxpacer was determined when the canal expansion was no longer sustained by the conventional french - door laminoplasty without the support of medical devices. the authors used imaging programs (pacsplus ppw, medical standard, korea) to measure the spinal canal area (fig. 4). in all vertebrae, anatomical and radiological (magnetic resonance image, mri) anterior - posterior diameter was the maximum distance between midpoint of the posterior border of vertebral body shadow and shadow of the spinolaminal junction of same vertebra12). the lamina open - door angle was equal to the angle of two lines, one through both sides of the vertebral body crossing the transverse foramen posteriorly and the other connecting with the inside edge of the hinge side of the tangent of the lamina31). the c2 - 7 angle was measured by formal cobb methods that verified the angle between the horizontal line of the c2 lower end plate and the horizontal line of the c7 lower end plate30). the area of the preoperative spinal canal was defined as the region surrounded by the posterior border of the vertebral body and the inner border of the lamina, while that of the postoperative spinal canal was defined as the region surrounded by the posterior border of the vertebral body, the inner border of the lamina, and the inner border of a spacer. in cases of opll, opll was not excluded in the spinal canal area to prevent overestimation of the expansion rate. we adjusted the images to be the same size among the same sections and compared the number of pixels in the spinal canal. the canal expansion rate was calculated by the increase in pixels from the preoperative to the postoperative images. the severity of clinical symptoms was assessed using the various clinical score systems such as the visual analog scale (vas), neck disability index (ndi), short - form 12 (sf-12), japanese orthopedic association (joa) score, and odom 's score. the preoperative and the one - year clinical scores were evaluated to determine whether there were significant differences. persisting nuchal pain distributed over the posterior neck and shoulder pain in the area of the suspensory muscles were defined as axial symptoms. preoperative neck and shoulder pain and subjective outcomes regarding axial symptoms were assessed using a vas questionnaire, on a scale from ten points (extremely severe pain) to one point (almost no pain) at discharge, at 6 months and at the one - year follow - up. pre- and post - operative vas scores were evaluated to determine whether there were significant differences between the two groups. the results are expressed as the meanstandard deviation. paired student t - test was used to assess the statistical differences of the demographic, clinical, and radiological data at each time point between the groups using sas software for windows (sas institute inc., among the 15 patients included in this retrospectively analyzed study, five patients had opll, nine had csm, and one had a combined csm and opll. a patient had a previous operative lesion with anterior cervical discectomy and fusion (acdf). the mean age of the patients was 50.0 years (range 35 - 72). one patient underwent a laminoplasty c6 with maxpacer with a subtotal laminectomy c5 and c7. another patient had a laminoplasty c2 - 6 with maxpacer. eight patients underwent laminoplasties of c3 - 6 with maxpacer. four patients had laminoplasties of c4 - 6 with maxpacer one patient underwent a laminoplasty c5 - 7 with maxpacer. cervical french - door laminoplasties with and without maxpacer were applied in all patients with 53 levels. maxpacers were used in 25 levels, and an average 1.67 levels of maxpacer were applied. the most commonly maxpacer applied cervical level was c6 (n=13) followed by c3 (n=5), c4 (n=3), c5 (n=3), and c2 (n=1). although this data included only a limited number of cases (n=15), the most of radiological outcomes after applying maxpacer were significantly increased compared to the preoperative baseline. anterior - posterior diameter (mm) was grossly increased from 9.42.2 in preoperative patients to 16.21.1 in the postoperative status (p 160 mm achieve a better outcome5). it was believed that the optimal enlargement of the stenotic canal by laminoplasty is greater than 4 - 5 mm in the sagittal diameter7). however, although the spinal canal area can be greatly increased during laminoplasty, excessive opening of the lamina may cause problems. the kinking of the nerve root induced by maximal decompression might be related to the occurrence of postoperative c5 nerve root palsy and radiculopathy29). excessive opening also creates epidural space and can lead to the formation of more epidural scar tissue than expected81015). device - related complications were very low in using the maxpacer, as our study showed only two cases with screw malposition (4% among total 50 screws in maxpacer). the screw displacements did not disturb the clinical outcomes in this retrospective study. in summary of this study and the literatures, maxpacer showed several advantage compared to conventional laminoplasty such as excellent surgically accessibility with easy handling27), sufficient increase of spinal canal volume1527), reasonable clinical outcome27), very low chance of intraoperative device failure with the device stability after even though device mal - positioned, and the consistency of device after plate implanted. therefore, combined french - door laminopalsty using maxpacer is a useful and safe surgical technique, as long as the surgeon assures that the screws are properly positioned in the posterior laminaes. despite our findings, this study had several limitations. in particular, the number of patients was small and the follow - up period was too short to allow a generalization of our results. indeed, we only performed an observational study and did not compare our results with those of alternative techniques. so, additional study is required to compare the french - door laminoplasty with the medical spacer in cases with identical operative indications. combined cervical expansive laminoplasty using a maxpacer with french - door laminoplasty is an effective treatment for multi - level cervical stenosis. despite the small cohort and short follow - up duration in our study, future studies will be able to further confirm our findings.	objectivethe purpose of this study was to evaluate the safety and efficacy of cervical midline - splitting french - door laminoplasty with a polyether ether ketone (peek) plate. the authors retrospectively analyzed the results of patients with cervical laminoplasty miniplate (maxpacer) without bone grafts in multilevel cervical stenosis.methodsfifteen patients (13 males and 2 females, mean age 50.0 years (range 35 - 72)) with multilevel cervical stenosis (ossification of the posterior longitudinal ligament and cervical spondylotic myelopathy) underwent a combined surgery of midline - splitting french - door laminoplasty with or without mini plate. all 15 patients were followed for at least 12 months (mean follow - up 13.3 months) after surgery, and a retrospective review of the clinical, radiological and surgical data was conducted.resultsthe radiographic results showed a significant increase over the postoperative period in anterior - posterior diameter (9.42.2 cm to 16.21.1 cm), open angles in cervical lamina (46.516.0 to 77.213.1), and sectional volume of cervical central canal (100.50.7 cm2 to 146.54.9 cm2) (p<0.001). the sagittal alignment of the cervical spine was well preserved (31.710.0 to 31.27.6, p=0.877) during the follow - up period. the clinical results were successful, and there were no significant intraoperative complications except for screw displacement in two cases. the mini plate constructs did not fail during the 12 month follow - up period, and the decompression was maintained.conclusiondespite the small cohort and short follow - up duration, the present study demonstrated that combined cervical expansive laminoplasty using the mini plate is an effective treatment for multilevel cervical stenosis.
the l - tyrosine is a substance that generates a neurotransmitter such as adrenaline, norepinephrine (noradrenaline), or dopamine and becomes the thyroid hormone that controls the metabolism and is a precursor of melanin [1, 2 ]. therefore, the development of a method to determine small amounts of l - tyrosine is important in the medical and pharmacology fields. moreover, the intake of l - tyrosine is also necessary for the preservation of health and is contained in nutrition supplements. recently, the guarantee of its quality and analysis has become especially important problems in the nutritional science field. for example, ultraviolet absorptiometry at the wavelength of 280 nm and the lowry method using the phenol reagent are known as a general determination analysis methods, but both have too low a sensitivity. they are as follows : spectrophotometry using the phenylalanine ammonia - lyase enzyme reaction, fluorimetry using the 1,5-bis(4,6-dichloro-1,3,5-triazinyl - amino) synthesis reaction or using the mo(vi)-phenyl - fluorone quenching reaction, chemiluminescence (cl) using the oxidation reaction of k3fe(cn)6 and kmno4 [79 ], cyclic voltammetry using the multiwalled carbon nanotube/4-amino - benzenesulfonic acid film - coated glassy carbon electrode oxidation reaction, uv detection of microcolumn electrophoresis, and hplc - fl. however, only a few rapid, simple, and highly sensitive analyses of l - tyrosine are known. the setup is simple and has the advantage that the measurement time is short. the cl analysis of various samples is performed in many fields such as biochemistry, clinical chemistry, and environmental chemistry for this reason. however, the absolute number of high sensitive and high functional cl substance is a few although the cl system using luminol or peroxyoxalate has been many reported [13, 14 ]. therefore, the appearance of a high cl substance is strongly desired. up to now, the authors have reported about the chlorophyll cl and the iron - chlorophyllin complex cl. in these cls, each compound itself has multifunctions such as a luminescence substrate, a catalyst, and an energy donor. moreover, in the iron - chlorophyllin complex cl, the luminescence was quenched when l - ascorbic acid was made to coexist in an oxidation reaction with hydrogen peroxide. the quenching - cl determination of 10 m for l - ascorbic acid was developed. on the other hand, the cl phenomenon with respect to the iron - phthalocyanine tetrasulfonic acid (fe - pts) was reported [18, 19 ]. the fe - pts complex had multi - cl functions similar to the iron - chlorophyllin complex, because the fe - pts as a luminescence substrate is the analog of an iron - chlorophyllin complex. in this paper, as a result of examining the fe - pts cl system, the coexistence of l - tyrosine significantly influenced the cl signal intensity. the quenching - cl method of l - tyrosine with fe - pts was then developed, especially, the determination of tyrosine in soft drink and dietary supplement were examined. iron - phthalocyanine tetrasulfonic acid was obtained from aldrich (milwaukee, wi, usa). sodium tetraborate (borax) was obtained from the kanto chemical co. (tokyo, japan). the l - tyrosine was obtained from the tokyo chemical industry co. (tokyo, japan). the practical samples were obtained from a soft drink (amino supli, kirin beverage co., tokyo, japan) and a dietary supplement (amino body, orihiro, gunma, japan). a genelight200s (the spectra range of pmt : 400570 nm ; microtec niti - on, chiba, japan) was used for the cl measurements. the distilled water was obtained using a swac-500 (shimadzu co., kyoto, japan). a 300 l sample of water containing l - tyrosine was injected into a glass cell using a microsyringe. a 100 l ph buffer solution and 50 l iron - phthalocyanine complex solution next, the cell was then placed into the cell holder of the cl detection equipment. a 50 l aliquot of hydrogen peroxide was then injected using a microsyringe and the cl signal was measured. the cl = cl0cls, where cl is the difference in the cl signal intensity, cl0 is the intensity of the l - tyrosine additive - free sample, and cls is the intensity of a certain amount of l - tyrosine contained in the sample. a 1.0 ml soft drink was diluted to the marked line with distilled water in a 50 ml volumetric flask. next, 5 ml of a 6.7 10 m l - tyrosine solution was added to 5 ml of this soft drink. the measurement was done using the standard procedure concerning the determination of l - tyrosine. a 0.25 g sample of the tablet (the content of l - tyrosine is 6.00 mg) was dissolved in 15 ml of a 1.0 m sodium hydroxide solution and then was diluted to the marked line with distilled water in a 250 ml volumetric flask. a 1.4 ml of this solution was diluted to the marked line with distilled water in a 10 ml of volumetric flask, and the cl signal was measured using the standard procedure concerning the determination of l - tyrosine. l - tyrosine is a reducing agent, naturally, authors think that it may work as h2o2 scavenger. in a previous paper, it is because the similar quenching - cl phenomenon by l - ascorbic acid was observed in iron - chlorophyllin / h2o2/l - ascorbic acid. it was examined whether l - tyrosine works as a ligand of fe - pts. the absorbance of fe - pts was decreased with increasing l - tyrosine concentration. from this result, it is thought that l - tyrosine works as a ligand of fe - pts. it was examined whether l - tyrosine might work as a quencher (energy transfer type). stern - volmer plot from 2.0 10 to 2.0 10 m in the range of concentrations of l - tyrosine became linear. therefore, stern - volmer plot showed that l - tyrosine worked as a quencher. in conclusion, the function of l - tyrosine in the cl phenomenon is presumed form the above to work compositely and to happen. based on the difference between the cl intensity of the blank reaction and the cl intensity for the 2.0 10 m l - tyrosine concentration that was the highest, each condition was optimized. the concentration of the fe - pts was varied in the concentration range of 1.0 10 m to 5.0 10 m. the cl intensity increased with the increasing concentration of fe - pts. the optimal concentration of fe - pts was 4.0 10 m according to the procedure optimization. the concentration of the hydrogen peroxide was varied in the range of 1.6 10 m to 8.0 10 m. as a result, the maximum cl0 intensity could be measured at 6.4 10 m hydrogen peroxide. however, the maximum cl could be obtained at 3.2 10 m hydrogen peroxide according to the procedure optimization. the ph was varied within the range of 9 to 12. the maximum cl intensity according to the ph change was observed at ph 10. the cl intensity quantitatively decreased when the concentration of the coexisting l - tyrosine increased. the cl signal for various l - tyrosine concentrations is shown in figure 3. in the calibration curve of l - tyrosine, the relationship obtained between the concentration range of 2.0 10 m to 2.0 10 m of l - tyrosine, and the difference in the cl intensity (cl) was y = 2544 ln(x) + 39864, where y is cl and x is the l - tyrosine concentration [m ]. the correlation coefficient was 0.981, the rsd was 1.63% (n = 5) at a 2.0 10 m l - tyrosine concentration, and the detection limit (3) was 1.81 10 m. moreover, the result of determination of tyrosine of enantiomer is shown in table 1. as for the allowable limit, the change in time was within 5% based on the difference in the cl intensity with the blank when no foreign substances were added., l - ascorbic acid could be present up to 5-fold without affecting the results of the test. also, although ni and co could be present up to equivalent without affecting the results of the test and cu could be present up to 0.5-fold without affecting the results of the test, they could be masked up to 10-fold with the addition of edta as a masking reagent. also, l - cysteine could be present up to 0.5-fold without affecting the results of the test. it is reported that l - cysteine and zn form a complex under neutral and alkaline conditions. as a result of the experiment, l - cysteine the proposed method can then be comparatively said to be selective as an analytical method for l - tyrosine. the proposed method and other methods were compared. the proposed method (procedure time : approximately 3 min) was proved to be rapid and simple, but the detection limits (detection limits : 1.81 10 m) are poor for fluorophotometry by the synthesis reaction with 1,5-bis(4,6-dichloro-1,3,5-triazinyl - amino) (detection limits : 6.8 10 m, procedure time : 30 min). also, the proposed method was rapid, simple, and highly sensitive compared to spectrophotometry using the phenylalanine ammonia - lyase enzyme reaction (detection limits : 5.0 10 m, procedure time : 2 h) and quenching - fluorophotometry using mo(vi)-phenyl - fluorone (detection limits : 5.2 10 m, procedure time : 5 min). the determined concentration of l - tyrosine was 1.96 10 m for 2.00 10 m, and the recovery was 98%. the indicated value of the components in the supplement was measured by the japan food research laboratories, and the various amino acids were measured by the amino acid analytical method using ninhydrin reaction. the experimental value that had been obtained by the measurement was 2.04 10 m for the tabulated one of 2.00 10 m l - tyrosine. in this study, rapid, simple, and highly sensitive analysis method of trace amounts of l - tyrosine based on the cl reaction of fe - pts was developed. in the future, a new fia cl - detection will be developed for application to multisamples containing trace amounts of l - tyrosine.	the chemiluminescence (cl) signal immediately appeared when a hydrogen peroxide solution was injected into an iron - phthalocyanine tetrasulfonic acid (fe - pts) aqueous solution. moreover, the cl intensity of fe - pts decreased by adding l - tyrosine. based on these results, the determination of trace amounts of l - tyrosine was developed using the quenching - chemiluminescence. the calibration curve of l - tyrosine was obtained in the concentration range of 2.0 107 m to 2.0 105 m. moreover, the relative standard deviation (rsd) was 1.63 % (n = 5) for 2.0 106 m l - tyrosine, and its detection limits (3) were 1.81 107 m. the spike and recovery experiments for l - tyrosine were performed using a soft drink. furthermore, the determination of l - tyrosine was applied to supplements containing various kinds of amino acids. each satisfactory relative recovery was obtained at 98 to 102%.
mucormycosis (mcm) is a devastating infection with high mortality rates despite recent advances in its diagnosis and treatment. it is caused by the filamentous fungi of the mucorales order of the class of zygomycetes. although it is classically defined as an opportunistic infection, preferentially affecting patients with diabetes mellitus (dm), neutropenia, malignancy, chronic renal failure, and acquired immunodeficiency syndrome and those who have received organ or hematopoietic stem cell transplants, it can affect immunocompetent hosts as well (such as trauma patients) [1, 2 ]. the incidence of mcm worldwide appears to be increasing, particularly in oncological patients and those with dm. along with aspergillus, it is one of the most common invasive fungal infections affecting immunosuppressed individuals. despite aggressive surgical and polyene antifungal therapy, overall mortality for mcm infection remains high, with figures ranging from 20 to 50% [46 ]. depending on patient characteristics (such as critically ill or immunocompromised patients) and site of infection, mortality rises markedly, nearing 7090% for cases of disseminated mucormycosis [46 ]. inhalation of sporangiospores is the most common route of transmission, although ingestion of spores, direct implantation into injured skin (burns), trauma with contaminated soil, or intravenous (drug users) transmission have also been described. after nasal inoculation it takes a rapidly progressive course extending to neighboring tissues, including the orbit, and sometimes to the brain. lipid formulations of amphotericin b are the mainstay of treatment, along with aggressive surgical therapy. however, such drug formulations are not available worldwide due to their elevated costs. most of the information on the epidemiology and clinical characteristics of mcm comes from case series and small studies on specific populations, such as those in oncology centers. considering the high mortality associated with mcm and the increasing recognition of the importance of this disease in latin american countries, we set out to describe the demographic and clinical characteristics of mcm patients in a tertiary - care teaching hospital in mexico. we received approval from the ethics committee of our institution to carry out this study. from 2007 to 2012 jose eleuterio gonzalez, and their records were obtained to analyze the cases retrospectively. demographic characteristics, presentation, signs, and symptoms as well as treatment and outcomes were analyzed. cases were sought through manual and electronic searches in hospital records (using discharge diagnoses). we used the 2008 european organization for research and treatment of cancer / mycoses study group (eortc / msg) criteria for the diagnosis of mcm, but only proven cases were retained. descriptive statistical methods were used to analyze demographic characteristics, and differences between groups were calculated using student 's t - test for independent samples or fisher 's exact test, where appropriate. statistical significance was established at p < 0.05. data were analyzed using an spss statistical package (spss statistics 15.0, spss inc. the mean age of the patients was 39.9 + / 20.3 years (range 565 years), with men presenting the majority (64.3%). the most common underlying disease was dm (71.4%), followed by hematological malignancy (42.8%) and chronic renal insufficiency on hemodialysis (21.4%). five patients with hematological malignancy had all and only one had acute myelocytic leukemia ; they had all received chemotherapy, including steroids, and two were neutropenic on diagnosis. patients also complained of headache, ocular pain, facial edema, and when ocular involvement was present, visual abnormalities. all patients had rhinocerebral involvement (figure 1). on presentation, 4 patients had overt orbital involvement. all patients received medical treatment with conventional (deoxycholate) amphotericin b at 11.5 amphotericin b was administered through a central line, with a median duration of treatment of 11.5 days. medical treatment was initiated empirically in 10 patients and after microbiological identification in the remaining cases. hypokalemia and creatinine elevations appeared in 6 patients after treatment, but these alterations did not ultimately require switching or modifying therapy. all of the patients that presented with diabetic ketoacidosis died, and all nonsurvivors underwent surgery. some routine laboratory tests on admission were also analyzed. no differences were found in hemoglobin, white blood count, or serum electrolytes (data not shown) between survivors and nonsurvivors. mean creatinine was 3.68 + / 3.15 mg / dl in nonsurvivors and 1.69 + / 1.15 mg / dl in survivors, with no statistical difference between groups. serum albumin levels were significantly lower on admission in nonsurvivors (2.3 + / 0.23 g / dl) in comparison with survivors (3.15 + / 0.46 g / dl), with a p = 0.03. there was no difference in the time of initiation of treatment between survivors and nonsurvivors (data not shown). the majority of the patients in our series were male, a trend that has been consistently reported in case series from different countries [1012 ]. dm is associated with impaired neutrophil function, microvascular insufficiency, and in the case of ketoacidosis, other metabolic abnormalities that promote fungal growth [1113 ]. rhizopus species have an active ketone reductase system and thrive in high glucose and acidotic conditions. notably, in our series, 4 patients presented with diabetic ketoacidosis. in a series of 28 mcm cases, 64% cases had dm and 55.6% of those cases had diabetic ketoacidosis, a proportion similar to our findings. however, in other series, complications associated with dm accounted for only 17% of cases of mcm. chronic renal insufficiency is another condition that predisposes to mcm infection. in our series, patients with renal disease all had concomitant dm and were on hemodialysis. in a 1997 case series with patients from our institution, rhinocerebral mcm was diagnosed in 22 patients over 15 years. in contrast with our study, hematologic malignancy was uncommon, with only one patient presenting with myelodysplastic syndrome. this could indicate increasing number of patients with hematological malignancies admitted to our institution, the use of more aggressive immunosuppressive regimens in these patients, and improved control of dm complications in our population. these trends have been recognized in other studies as well [12, 16 ]. in the largest case series, patients with hematological malignancies (mainly acute leukemia) represent the group with the highest prevalence and with rapidly increasing rates of mcm [12, 16 ]. in our series 6 of our patients had hematological malignancies, and in all cases these were acute leukemia (all and aml). although neutropenia has also been hailed as an important factor in the development of mcm, only 2 of our patients with all presented with neutropenia. mcm infection may have a rhinocerebral, rhinoorbital, pulmonary and soft - tissue extension, among others. recent registries have reported conflicting data about the most common site of infection. in a global registry, the lung (58.5%) was the main site of infection, followed by rhinocerebral or rhinoorbital involvement (19.5%). in an italian series, rhino - orbital - cerebral involvement was the most common site of infection (35%), followed by the lung (25%). interestingly, we found no evidence of lung involvement or of disseminated disease in our series. the most common signs and symptoms were fever, rhinorrhea, and headache, while the most ominous symptom was vision loss. despite aggressive medical and surgical treatment, in - hospital mortality in our patients was 50%, which is similar to rates reported in other case series. although it is higher than the rate reported in a recent population - wide study (22%), our series is quite different in terms of infection site (mainly rhinocerebral) and treatment (conventional amphotericin b). all of our patients received conventional (as opposed to liposomal) amphotericin b at doses that ranged from 1 to 1.5 mg / kg / day. although surgical therapy has been associated with improved outcome in some studies [5, 6 ], all of the nonsurvivors in our study underwent surgical treatment. this could be explained by clinically milder forms of mcm infection in those patients that did not have to undergo surgery. notwithstanding our small sample, we could identify factors that were significantly different in nonsurvivors when compared with survivors : these were older age, dm, and ketoacidosis at presentation. ketoacidosis represents a severe complication of dm in its own right and indicates poor glycemic control, so along with old age it is not surprising that it would be associated with worse outcomes. of note, this accounts for the higher creatinine levels (although not significant) on admission in nonsurvivors. serum albumin on admission was lower in nonsurvivors as well, which could indicate malnutrition, another predisposing factor for immunosuppression. in conclusion, mcm is a life - threatening infection that most commonly affects immunocompromised individuals and that despite aggressive multimodal treatment carries a significant risk of mortality. a high index of suspicion is required in order to begin the appropriate diagnostic workup and treatment. our cases most commonly involved the rhino - orbital - cerebral cavities, and the main underlying disease was dm. unfortunately, due to economic limitations, the use of liposomal amphotericin b in third world countries is often prohibitive, and our patients were instead treated with conventional amphotericin b. fortunately, there were no cases in our series where side effects (such as renal injury or hypokalemia) forced a change in therapy. in light of evidence suggesting that early and aggressive use of liposomal amphotericin b could improve outcomes,	mucormycosis (mcm) is a life - threatening infection that carries high mortality rates despite recent advances in its diagnosis and treatment. the objective was to report 14 cases of mucormycosis infection and review the relevant literature. we retrospectively analyzed the demographic and clinical data of 14 consecutive patients that presented with mcm in a tertiary - care teaching hospital in northern mexico. the mean age of the patients was 39.9 (range 565). nine of the patients were male. ten patients had diabetes mellitus as the underlying disease, and 6 patients had a hematological malignancy (acute leukemia). of the diabetic patients, 3 had chronic renal failure and 4 presented with diabetic ketoacidosis. all patients had rhinocerebral involvement. in - hospital mortality was 50%. all patients received medical therapy with polyene antifungals and 11 patients underwent surgical therapy. survivors were significantly younger and less likely to have diabetes than nonsurvivors, and had higher levels of serum albumin on admission. the clinical outcome of patients with mcm is poor. uncontrolled diabetes and age are negative prognostic factors.
physicians have shown great concern on the relationship between lymphoma and inflammatory bowel disease (ibd) over the years. in 1928, bargen described initial report of lymphosarcoma in a patient with ulcerative colitis. since then, numerous case reports and meta - analyses have been presented, but composite lymphoma in ibd patients had not previously been reported. composite lymphoma is a rare malignancy and defined as the presence of two or more distinct cytological and histological variant of lymphoma in the same tissue. this combination consists of hodgkin lymphoma (hl) with non - hodgkin lymphoma (nhl) or b - cell nhl with t -cell nhl, or two distinct subtype of b - cell or t - cell nhl. in this study, we present a case of a composite diffuse large b - cell lymphoma and nodular sclerosing hl in para - aortic lymph node and spleen in a 45-year- old female patient diagnosed with ulcerative colitis. the patient was a 45-year - old female with a 2-year history of left - sided ulcerative colitis. only mesalamine was responsible for keeping the disease under control and no other immunosuppressive agents such as azathioprine and anti tnf were administered. the patient was referred to hematologic ward with a one - month history of malaise, weakness and fatigue. upon physical examination, hematologic findings consisted of pancytopenia, elevated erythrocyte sedimentation rate (esr) and lactate dehydrogenase (ldh). results of renal and liver function tests, electrolytes, urine analysis and hiv test were within the normal range. contrast - enhanced computed tomography scan of the chest and abdominopelvic regions showed lymphadenopathy of middle and posterior mediastinum, para - aortic lymphadenopathy and splenomegaly with heterogeneous density (fig.1). to achieve a definite diagnosis, splenectomy, excisional biopsy of para - aortic lymph node, aspiration and biopsy of marrow histomorphological evaluation of the spleen and para aortic lymph node (fig : 2) show focal involvement by classical hodgkin s lymphoma and nodular sclerosis variant, wherein typical nodules are seen in which the lacunar cells and classic red - sternberg (r - s) cells are surrounded by small mature lymphocytes with a few eosinophils and plasma cells. however, in other areas, diffuse sheets of centroblastic and (less) immunoblastic cells are also evident, which is in keeping with diffuse large b - cell lymphoma (dlbcl). 3), the lacunar cells and r - s cells in the hl component show negative leukocyte common antigen (lca), cd20 and positive ki - a10 (one of the hl associated markers) although cd30 and cd15 are negative. accordingly, there is strong positivity of lca and cd20 in the dlbcl component. treatment of composite lymphoma depends on histological grading of components and is towards the high grade histology. the patient was treated with r - chop- based chemotherapy (rituximab, cyclophosphamide, adriamycin, vincristine and prednisolone). after eight courses of chemotherapy with r - chop and one course of high - dose methotrexate, complete remission was achieved. the patient was a 45-year - old female with a 2-year history of left - sided ulcerative colitis. only mesalamine was responsible for keeping the disease under control and no other immunosuppressive agents such as azathioprine and anti tnf were administered. the patient was referred to hematologic ward with a one - month history of malaise, weakness and fatigue. upon physical examination, hematologic findings consisted of pancytopenia, elevated erythrocyte sedimentation rate (esr) and lactate dehydrogenase (ldh). results of renal and liver function tests, electrolytes, urine analysis and hiv test were within the normal range. contrast - enhanced computed tomography scan of the chest and abdominopelvic regions showed lymphadenopathy of middle and posterior mediastinum, para - aortic lymphadenopathy and splenomegaly with heterogeneous density (fig.1). to achieve a definite diagnosis, splenectomy, excisional biopsy of para - aortic lymph node, aspiration and biopsy of marrow histomorphological evaluation of the spleen and para aortic lymph node (fig : 2) show focal involvement by classical hodgkin s lymphoma and nodular sclerosis variant, wherein typical nodules are seen in which the lacunar cells and classic red - sternberg (r - s) cells are surrounded by small mature lymphocytes with a few eosinophils and plasma cells. however, in other areas, diffuse sheets of centroblastic and (less) immunoblastic cells are also evident, which is in keeping with diffuse large b - cell lymphoma (dlbcl). 3), the lacunar cells and r - s cells in the hl component show negative leukocyte common antigen (lca), cd20 and positive ki - a10 (one of the hl associated markers) although cd30 and cd15 are negative. accordingly, there is strong positivity of lca and cd20 in the dlbcl component. treatment of composite lymphoma depends on histological grading of components and is towards the high grade histology. the patient was treated with r - chop- based chemotherapy (rituximab, cyclophosphamide, adriamycin, vincristine and prednisolone). after eight courses of chemotherapy with r - chop and one course of high - dose methotrexate, complete remission was achieved. inflammatory bowel disease is a chronic disorder of gastrointestinal tract including two main diseases : ulcerative colitis and crohn s disease. the etiology of these diseases is unknown, but evidence suggests that genetically susceptible patients immune dysfunction triggered by environmental factors is responsible for disease. the risk of lymphoma in ibd patients has been a great concern in the medicine for many years. environmental, genetic, infectious and iatrogenic factors in the patients with inflammatory bowel disease can predispose them to increase the risk of lymphoma. overall risk of lymphoma in ibd patients is not higher than general population, but the basic question is whether treatment with immunomedulatory agents can lead to an increased risk of lymphoma. the use of azathioprine /6-mercaptopurine increases the incidence of lymphoma in patients with inflammatory bowel disease although usefulness of azathioprine therapy outweigh the risk of lymphoma has been suggested in decision analysis model. ebv in the patients with ibd who received azathioprine/6-mercaptopurine associated with increased risk of lymphoma. methotrexate is another immunomedulatory drug which is used as alternative treatment in moderate to severe crohn s disease. unlike azathioprine, there is no repeated and reliable data for the risk of lymphoma in ibd patients treated with methotrexate. in the cesame study conducted by beaugerie., (2009) some studies like those conducted on rheumatoid arthritis provide some information on the effect of methotrexate. in these studies, there was no increase in the risk of lymphoma among patients treated with methotrexate. at that time, anti - tnf agents such as infliximab were approved for the treatment of crohn s disease. there is a great concern over safety of drugs ; moreover, the assessment of specific risk of these agents on lymphoma development is difficult because most patients had a previous exposure to methotrexate and azathioprine. a comprehensive meta - analysis that was conducted by siegel., (2009) showed that the rate of nhl in patients with crohn s disease who treated with anti - tnf agents in combination with immunomedulators was not increased. non - hodgkin s lymphoma, particularly diffuse large b - cell lymphoma, appears to be more common than other types of lymphoma in the patients with inflammatory bowel disease. she had no exposure to azathioprine, methotrexate or anti tnf agents. despite the large number of ibd patients with lymphoma, the incidence rate of composite lymphoma, known as rare malignancy, is estimated to be 1 to 4.7% of all other types of lymphoma. in contrast, the combination of non - hodgkin s lymphoma and hodgkin s disease rarely occurs. nodular lymphocyte - predominant hodgkin s lymphoma and diffuse b - cell lymphoma are more frequently found among these combinations.	a 45-year - old female patient with a diagnosis of ulcerative colitis complicated with composite lymphoma in the spleen and para - aortic lymph node presented with a one - month history of malaise, weakness and fatigue. only mesalamine kept ulcerative colitis under control. in physical examination, splenomegaly was revealed and pancytopenia was obtained from laboratory data. computed tomography scan revealed para - aortic mediastinal lymphadenopathy with splenomegaly. splenectomy and excisional biopsy of abdominal lymph node were performed and disease was diagnosed as composite lymphoma, consisting of diffuse large b - cell lymphoma and nodular sclerosing hodgkin lymphoma.
the clinical usefulness of light was first recognized some 3,000 years ago when it was used to treat rickets, psoriasis, and other common ailments.1 more recently, the term photodynamic therapy (pdt) surfaced and has been used to describe the use of light to treat a variety of diseases, including cancer.25 specifically, over the last 100 years, great strides have been made in developing photodynamic therapies for use in the clinical setting. the first component is the photosensitizer, the photosensitive material that targets diseased tissue, akin to chemotherapeutic agents. the second component is the irradiation of the target area with light of a specific wavelength. excitation of the photosensitive material to a triplet state in the presence of oxygen results in triplet triplet annihilation, producing cytotoxic reactive oxygen species (ros) that destroy cellular components, thus triggering cell death. therefore, the success of photodynamic therapeutics depends largely on the photosensitizer concentration throughout the tissue, adequate intracellular oxygen concentration, and proper source of excitation. the discovery of this process was extremely promising in providing a more targeted, regulated method of cancer treatment. however, one of the main drawbacks of this method of pdt is that it is oxygen dependent. although cancer cells have abnormally high vascularization, intracellular oxygen concentration is low due to poor morphology of the vessels and abnormally high metabolic and oxygen consumption rates, which are characteristics of cancerous tissues.2 without oxygen available to interact with the photosensitizer, ros can not be formed and the therapeutic method becomes relatively ineffective or far less effective than intended. in addressing the aforementioned issue, we recently took an alternative approach in attempting to trigger cell death by employing a photoresponsive agent with low dark cytotoxicity. rather than producing ros as the main damaging component, we used a non - toxic sulfonium - based photoacid generator (pag) to cause intracellular ph imbalance.6 upon proper light excitation, the pag lowers the ph in the moderately ph - sensitive intracellular environment ; the acidic environment thus created leads to the malfunction and destruction of many cellular components, likely including key enzymes. this process is considered an oxygen - independent pdt (oi - pdt) and, essentially, relies on two main components, the sulfonium salt distribution in the affected area and a proper source of excitation, rather than the three components mentioned earlier. in essence, reducing the number of variables in the therapeutic process development improves the chances of success in material design and subsequent treatment. given the hydrophobic nature of pags prepared in our preliminary study,6 we opted to introduce a polyethylene glycol (peg) unit that can confer hydrophilicity to the pdt agent while maintaining its photo - induced cytotoxicity. along the same line, we prepared highly monodispersed silica nanoparticles (sinps) functionalized with amines that served as handles to covalently bond pag molecules via an amide linkage. the incorporation of the pag into a nanomaterial delivery formulation is also intended to improve delivery properties of the proposed pdt agent as nanomaterials have emerged as a promising solution to the issues of cellular specificity, time - controlled delivery, and aqueous solubility.7,8 herein, we report the design, synthesis (scheme 1 and supplementary material), and investigation of a new pag and its incorporation on sinps (sinp pag9), which offers better solubility in aqueous media, greater efficacy than water - soluble pegylated pag (peg pag9), and low dark cytotoxicity. pag9 and sinp pag9 were designed having similar aa core structure to the one reported in our initial work, where the two acceptor units (a), no2 and sph2, flank a fluorenyl - stilbene -spacer. using the c(9) of fluorene 1 to attach a handle for further functionalization, a propanoic acid unit was added as depicted in scheme 1. initially, treatment of 1 with one equivalent of n - buli at 78c generated a carbanion, stabilized by resonance through the fluorene -system ; this was followed by careful addition of ethyl bromide to afford the monoalkylated intermediate 2. bi - substituted fluorene 3 was obtained through a triton b - assisted michael addition of 2 with acrylonitrile in satisfactory yield (77%). although later stages of the synthesis that use aqueous acids have resulted in the hydrolysis of the nitrile moiety, such acidic hydrolysis was incomplete and often resulted in low yields and multiple side products. therefore, we opted to hydrolyze the nitrile group using aqueous naoh to afford the carboxylic acid derivative 4 in good yield. subsequent intermediates 59 were prepared following our reported procedure to afford pag 9 in ~61% yield over four steps. this compound was conjugated to an amine - terminated peg moiety by preparing the benzotriazole intermediate in situ, followed by the addition of the amine - terminated peg to afford peg sinps having amine appendages sinp(nh2)n (preparation detailed in the supplementary material) were treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide condensation agent, followed by the addition of an excess of pag 9 ; the good solubility of pag 9 in water allowed the dialysis of the resulting sinp surface modification of sinp(nh2)n by addition of pag 9 molecules was first assessed using a zetasizer nano system (malvern instruments, malvern, uk), where such addition resulted in particle size increase from an average of 4 nm to 9 nm (supplementary material). in addition, the nuclear magnetic resonance (nmr) spectrum of sinp pag9 in d2o (supplementary material) showed the presence of peaks in the aromatic region that corresponded to pag 9, hence verifying the successful functionalizing of the sinps with the pdt (pag) molecules. next, the photophysical properties of peg pag9 and sinp pag9 were examined to ensure that the magnitude of photoacid generation is preserved after modification. initially, the absorption profiles of peg pag9 and sinp pag9 were obtained in water, where both show a similar absorption band to previously reported pl-127-encapsulated pag, with an absorption max of 384 nm. pag9 was maintained at ~0.4 (supplementary material), suggesting that the aqueous environment has little effect on the relevant photophysical properties of peg furthermore, the ph drop inside cell lysosomes was estimated by recording the changes in the absorption intensity of rhodamine b (rhb) base. conceptually, it is safe to assume that the number of protons generated by peg pag9 upon exposure is the same as the number of rhb base molecules converted to rhb+, which is visualized as an increase in the absorption peak at ca. extrapolation of the calibration curve (figure 1b) suggests that 50 m of peg pag9 would result in an increase of [h]lysosomal by 9.110 m after a radiation dose of 10 min ; consequently, the lysosomal ph would be reduced by at least 0.3 ph units to ~4.4 (supplementary material).9 in order to evaluate the intrinsic toxicity of peg pag9 and sinp pag9, cell viability assays with celltiter 96 aqueos one solution reagent (promega corporation, fitchburg, wi, usa) were performed in the dark (dark viability) to prevent the production of acid and determine suitable concentration for effective pdt (supplementary material). in this regard, hct-116 cells (human colorectal carcinoma) were incubated with various concentrations of peg pag9 and sinp pag9 has very low dark cytotoxicity, even at 100 m dose concentration (figure 2a). pag9 loading was doubled from 10 m to 20 m (figure 2b). in order to further assess the cytotoxicity of pdt agents, post - exposure viability assays were performed using 50 m and 10 m of peg pag9 and sinp pag9, respectively. as can be seen in figure 3, exposure of hct-116 cultures incubated with peg such a drastic decrease in cell viability can be the result of pag distribution within the lysosomes and endosomes. mechanistically, photoexcitation of pag 9 molecules that are densely grafted on sinp(nh2)n generates a highly localized dose of protonated species, and, thus, a greater localized ph drop. consequently, a localized surge in the protonated species concentration creates an acidic microenvironment that could override mechanisms by which cells tend to regulate intracellular [h ], resulting in severe cytoplasmic acidification and inducing cell death.10 although this report is largely qualitative, the error bars of the cell viability in figures 2 and 3 provide some insight into the statistical significance of the results. pag9 exhibits high cytotoxicity at very low concentration and short exposure time. in order to determine the localization of peg pag9 in hct 116 cells, lysotracker green (thermo fisher scientific, waltham, ma, usa), a commercial dye that localizes in lysosomes after cell uptake, was used along with peg pag9. pag9 was collected inside cells (figure 4a c), showing a good uptake efficiency of peg overlay image (figure 4d) exhibited good colocalization between peg pag9 and lysotracker green, which indicated that peg next, we verified the reduced lysosomal ph by employing lysosensor green as a suitable intracellular ph sensor.6 since the fluorescence quantum yield of lysosensor green increases in increasingly acidic environments,11 fluorescence intensity as a function of exposure time was studied, which showed a drop in intralysosomal ph following irradiation in hct-116 cells incubated with peg pag9 (figure 5 and supplementary material). as demonstrated in our previous study,6 time - lapsed micrographs (figure 6) of hct-116 cells incubated with peg pag9 and sinp pag9 (figures 6 and 7, respectively) show significant cell swelling, loss of cell adhesion that is followed by a figure 8 shows a simplified jablonski diagram summarizing the process of photoacid generation by either one - photon or two - photon (2p) excitation. we demonstrated that sulfonium salts can be used to selectively induce cell death by photoexcitation. pags induced necrotic cell death by creating a ph imbalance in the hct-116 cells via generation of photoacid within the lysosomes. higher efficacy was achieved when these pag molecules were conjugated to silica - based nanoparticles, resulting in a higher cytotoxic effect. ultimately, near - infrared (ir) 2p excitation of the water - soluble pag should afford deep tissue excitation and pdt. thus, light - guided acid generation could be considered an attractive tool for treatment of hypoxic tumors and other diseases. currently, our efforts are focused on evaluating the prepared pags in vivo and for 2p excitation in the near - ir spectral range.	photodynamic therapy (pdt) processes involving the production of singlet oxygen face the issue of oxygen concentration dependency. despite high oxygen delivery, a variety of properties related to metabolism and vascular morphology in cancer cells result in hypoxic environments, resulting in limited effectiveness of such therapies. an alternative oxygen - independent agent whose cell cytotoxicity can be remotely controlled by light may allow access to treatment of hypoxic tumors. toward that end, we developed and tested both polyethylene glycol (peg)-functionalized and hydrophilic silica nanoparticle (sinp)-enriched photoacid generator (pag) as a nontraditional pdt agent to effectively induce necrotic cell death in hct-116 cells. already known for applications in lithography and cationic polymerization, our developed oxygen - independent pdt, whether free or highly monodispersed on sinps, generates acid when a one - photon (1p) or two - photon (2p) excitation source is used, thus potentially permitting deep tissue treatment. our study shows that when conjugated to sinps with protruding amine functionalities (sinp pag9), such atypical pdt agents can be effectively delivered into hct-116 cells and compartmentalize exclusively in lysosomes and endosomes. loss of cell adhesion and cell swelling are detected when an excitation source is applied, suggesting that sinp pag9, when excited via near - infrared 2p absorption (a subject of future investigation), can be used as a delivery system to selectively induce cell death in oxygen - deprived optically thick tissue.
menopause is associated with increased risk of metabolic diseases such as type 2 diabetes and metabolic syndrome (1, 2). dyslipidemia, obesity, and insulin resistance are of the most common features of the metabolic syndrome in menopausal woman and known to be important risk factors for cardiovascular diseases (3 - 5). menopause is also associated with a wide range of changes in the expression and secretion of proinflammatory cytokines which may explain increased incidence of osteoporosis, alzheimer, arthritis, and other inflammatory diseases seen in a postmenopausal period (6 - 9). for many years, hormone therapy was the first primary treatment modality for the management of menopause ; however, it had own limitations like increasing risk of stroke, coronary heart disease, pulmonary embolism, and ovarian cancer (1, 10). therefore, development of new agents with anti - inflammatory action and lesser side effects for management of menopause remains an attractive subject. it has been reported that a number of medicinal plants may be useful for preventing or treating some menopausal related complications such as insulin resistance, dyslipidemia, hepatic steatosis, weight gain, inflammation, and loss of bone mass (11 - 13). trigonella foenum - graecum (fenugreek), a plant of fabaceae family, has a long history for management of diabetes and dyslipidemia (14, 15). several studies also support anti - inflammatory and immunomodulatory activities of t. foenum - graecum in adult male animals (16 - 18). recently, sindhu. (19) reported that mucilage extracted from ethanolic extract (ee) of t. foenum graecum has anti - inflammatory and antioxidative properties on adjuvant - induced arthritis in female rats. further, hakimi. showed that t. foenum graecum seed decreases the number of hot flashes and vasomotor symptoms in menopausal women (20). however, to the best of our knowledge, no study has yet evaluated the effect of this plant on the level of proinflammatory cytokines in menopause. the present work was performed to investigate the effects of chronic treatment with t. foenum graecum seed extracts on serum concentration of interleukin-1 (il-1), interleukin-6 (il-6) and tumor necrosis factor- (tnf-) in ovariectomized rats. the seeds of t. foenum - graecum were purchased from local market (guilan, iran) and identified at the herbarium of agricultural research center of guilan university (iran). the ee and hexanic extract (he) of t. foenum - graecum were prepared using a maceration method as described previously (21, 22). briefly, the air - dried powder of the seeds was suspended in 96% ethanol (5 ml per g of seed) and maintained for 72 hour at 37c. then the solvent was removed and a new extraction started again with fresh ethanol. finally, the ee was filtered through whatman no.1 filter paper and dried using a rotary vacuum evaporator (glf, germany) at 50c. female wistar rats initially weighing 190 to 220 g were obtained from pasteur institute, hesarak branch, iran. the animals were maintained at 22c 4c on a light / dark cycle of 12 hour and away from stress. at all stages of the study, they had free access to enough laboratory chow and tap water ad libitum. ethical rules of working with laboratory animals were approved by guilan university of medical sciences. after two weeks acclimatization period, animals were randomly divided into the following 7 experimental groups, each group including 7 animals : group i included sham control rats ; group ii comprised ovariectomized control animals ; groups iii and iv included ovariectomized animals and administrated intraperitoneally ee of t. foenum - graecum (50 and 150 mg / kg, respectively) ; groups v and vi comprised ovariectomized animals and treated intraperitoneally he of t. foenum - graecum (50 and 150 mg / kg, respectively) ; groups vii included ovariectomized positive control rats, which administrated orally 10 g / kg of 17- estradiol valerate (aburaihan pharmaceutical co., iran). the treatments were started one day after removal of ovaries and lasted for 42 days. during this period, fasting blood glucose (fbg) and body weight of all of the rats were recorded prior to the start and at the end of treatments. after an overnight fast, each rat was anesthetized with intraperitoneally injection of 320 mg / kg chloralhydrate (arya pharmaceutical co., iran) (23). then, a small abdominal skin incision was performed and after exposing ovaries, they were removed bilaterally and tubal ends were closed using 2/0 silk string to prevent internal bleeding. then the fat pad was repositioned into the abdomen and muscle and skin the animals in the control group underwent a similar procedure without eliminating the ovaries. at the end of the 42th day, the samples were centrifuged for 5 minutes at 3000 rpm and supernatants were maintained at -20c for further analysis. the level of fbg was determined using accu - check glucometer (roche diagnostic, germany). concentrations of il-1, il-6 and tnf- were measured by e0071ra, e90079ra, and e90133ra elisa kit, respectively (life science inc, china). differences between means of different groups were analyzed using one - way analysis of variance (anova) followed by tukey s post hoc test for multiple comparisons. paired t - test was used to compare the differences between day 0 and day 42. results were exhibited as the mean standard error of the mean (sem). the seeds of t. foenum - graecum were purchased from local market (guilan, iran) and identified at the herbarium of agricultural research center of guilan university (iran). the ee and hexanic extract (he) of t. foenum - graecum were prepared using a maceration method as described previously (21, 22). briefly, the air - dried powder of the seeds was suspended in 96% ethanol (5 ml per g of seed) and maintained for 72 hour at 37c. then the solvent was removed and a new extraction started again with fresh ethanol. finally, the ee was filtered through whatman no.1 filter paper and dried using a rotary vacuum evaporator (glf, germany) at 50c. female wistar rats initially weighing 190 to 220 g were obtained from pasteur institute, hesarak branch, iran. the animals were maintained at 22c 4c on a light / dark cycle of 12 hour and away from stress. at all stages of the study, they had free access to enough laboratory chow and tap water ad libitum. ethical rules of working with laboratory animals were approved by guilan university of medical sciences. after two weeks acclimatization period, animals were randomly divided into the following 7 experimental groups, each group including 7 animals : group i included sham control rats ; group ii comprised ovariectomized control animals ; groups iii and iv included ovariectomized animals and administrated intraperitoneally ee of t. foenum - graecum (50 and 150 mg / kg, respectively) ; groups v and vi comprised ovariectomized animals and treated intraperitoneally he of t. foenum - graecum (50 and 150 mg / kg, respectively) ; groups vii included ovariectomized positive control rats, which administrated orally 10 g / kg of 17- estradiol valerate (aburaihan pharmaceutical co., iran). the treatments were started one day after removal of ovaries and lasted for 42 days. during this period, fasting blood glucose (fbg) and body weight of all of the rats were recorded prior to the start and at the end of treatments. after an overnight fast, each rat was anesthetized with intraperitoneally injection of 320 mg / kg chloralhydrate (arya pharmaceutical co., iran) (23). then, a small abdominal skin incision was performed and after exposing ovaries, they were removed bilaterally and tubal ends were closed using 2/0 silk string to prevent internal bleeding. then the fat pad was repositioned into the abdomen and muscle and skin the animal was placed in a warm place to come intellect. the animals in the control group underwent a similar procedure without eliminating the ovaries. at the end of the 42th day, blood sample was collected from tail vein of fasted animals for biochemical assays. the samples were centrifuged for 5 minutes at 3000 rpm and supernatants were maintained at -20c for further analysis. the level of fbg was determined using accu - check glucometer (roche diagnostic, germany). concentrations of il-1, il-6 and tnf- were measured by e0071ra, e90079ra, and e90133ra elisa kit, respectively (life science inc, china). differences between means of different groups were analyzed using one - way analysis of variance (anova) followed by tukey s post hoc test for multiple comparisons. paired t - test was used to compare the differences between day 0 and day 42. results were exhibited as the mean standard error of the mean (sem). in nonovariectomized sham group, the mean fbg on day 0 (5.72 0.12 mmol / l) and day 42 (5.38 0.11 on the other hand, concentration of fbg at the end of the study was significantly higher than day 0 in ovariectomized control animals (6.16 0.13 mmol / l vs. 8.77 0.47 mmol / l, p < 0.05). although the administration of estradiol or t. foenum - graecum (50 and 150 mg / dl of he and 150 mg / kg of ee) to ovariectomized animals significantly diminished (p < 0.05) the increase of fbg, in all treated groups the level of fbg was still higher than that of day 0 (table 1). abbreviations : ee, ethanolic extract ; he, hexanic extract ; and ova, ovariectomy. there was a significant difference regarding weight gain between the sham control (53 9 g) and ovariectomized control groups (111 7 g, p < 0.05). estradiol and both extracts of t. foenum - graecum significantly decreased the body - weight gain compared to the ovariectomized control group (p < 0.05). as shown in figure 1, the serum il-1 level of the saline - injected rats in the ovariectomized group was significantly higher than those of the sham animals (990 2.9 vs. 300 2.8 pg / ml, p < 0.05). administration of both he and ee of t. foenum - graecum significantly decreased serum il-1 in the ovariectomized rats compared to the saline group (p < 0.05). the level of il-1 in groups received 50 and 150 mg / kg of he was 440 2.8 pg / ml and 410 2.2 pg / ml, respectively. also, concentrations of il-1 in animals treated with 50 and 150 mg / kg of ee were 410 2.2 pg / ml and 610 2.7 pg / ml, respectively. the effect of he was comparable to that of induced by 10 g / kg of estradiol (389 2.9 pg / ml). the animals were subjected to ovariectomy surgery and then treated intraperitoneally with 10 g / kg estradiol or concentrations of 50 mg / kg and 150 mg / kg of he and ee of t. foenum - graecum seeds for 6 weeks. p < 0.05 vs. control group ; # p < 0.05 vs. saline group. similarly, serum il-6 significantly increased in ovariectomized control group when compared to the sham animals (1258 2.7 vs. 483 3 pg / ml, p < 0.05). as shown in figure 2, hexanic and ethanolic extracts of t. foenum - graecum, like estradiol, diminished the il-6 level in ovariectomized animals (p < 0.05 as compared with saline group). treatment with 50 and 150 mg / kg of he was also able to significantly decrease serum tnf- from 999 2.4 pg / ml (saline group) to 457 2.5 and 445 2.6 pg / ml, respectively, comparable to the effect of estradiol (430 2.6 pg / ml). a similar decrease in tnf- concentration was induced by both doses of ee (figure 3). the animals were subjected to ovariectomy surgery and then treated intraperitoneally with 10 g / kg estradiol or concentrations of 50 mg / kg and 150 mg / kg ofhe and ee of t. foenum - graecum seeds for 6 weeks. p < 0.05 vs. control group ; # p < 0.05 vs. saline group. the animals were subjected to ovariectomy surgery and then treated intraperitoneally with 10 g / kg estradiol or concentrations of 50 mg / kg and 150 mg / kg of he and ee of t. foenum - graecum seeds for 6 weeks. p < 0.05 vs. control group ; # p < 0.05 vs. saline group. in nonovariectomized sham group, the mean fbg on day 0 (5.72 0.12 mmol / l) and day 42 (5.38 0.11 on the other hand, concentration of fbg at the end of the study was significantly higher than day 0 in ovariectomized control animals (6.16 0.13 mmol / l vs. 8.77 0.47 mmol / l, p < 0.05). although the administration of estradiol or t. foenum - graecum (50 and 150 mg / dl of he and 150 mg / kg of ee) to ovariectomized animals significantly diminished (p < 0.05) the increase of fbg, in all treated groups the level of fbg was still higher than that of day 0 (table 1). abbreviations : ee, ethanolic extract ; he, hexanic extract ; and ova, ovariectomy. there was a significant difference regarding weight gain between the sham control (53 9 g) and ovariectomized control groups (111 7 g, p < 0.05). estradiol and both extracts of t. foenum - graecum significantly decreased the body - weight gain compared to the ovariectomized control group (p < 0.05). as shown in figure 1, the serum il-1 level of the saline - injected rats in the ovariectomized group was significantly higher than those of the sham animals (990 2.9 vs. 300 2.8 pg / ml, p < 0.05). administration of both he and ee of t. foenum - graecum significantly decreased serum il-1 in the ovariectomized rats compared to the saline group (p < 0.05). the level of il-1 in groups received 50 and 150 mg / kg of he was 440 2.8 pg / ml and 410 2.2 pg / ml, respectively. also, concentrations of il-1 in animals treated with 50 and 150 mg / kg of ee were 410 2.2 pg / ml and 610 2.7 pg / ml, respectively. the effect of he was comparable to that of induced by 10 g / kg of estradiol (389 2.9 pg / ml). the animals were subjected to ovariectomy surgery and then treated intraperitoneally with 10 g / kg estradiol or concentrations of 50 mg / kg and 150 mg / kg of he and ee of t. foenum - graecum seeds for 6 weeks. p < 0.05 vs. control group ; # p < 0.05 vs. saline group. similarly, serum il-6 significantly increased in ovariectomized control group when compared to the sham animals (1258 2.7 vs. 483 3 pg / ml, p < 0.05). as shown in figure 2, hexanic and ethanolic extracts of t. foenum - graecum, like estradiol, diminished the il-6 level in ovariectomized animals (p < 0.05 as compared with saline group). treatment with 50 and 150 mg / kg of he was also able to significantly decrease serum tnf- from 999 2.4 pg / ml (saline group) to 457 2.5 and 445 2.6 pg / ml, respectively, comparable to the effect of estradiol (430 2.6 pg / ml). a similar decrease in tnf- concentration was induced by both doses of ee (figure 3). the animals were subjected to ovariectomy surgery and then treated intraperitoneally with 10 g / kg estradiol or concentrations of 50 mg / kg and 150 mg / kg ofhe and ee of t. foenum - graecum seeds for 6 weeks. p < 0.05 vs. control group ; # p < 0.05 vs. saline group. the animals were subjected to ovariectomy surgery and then treated intraperitoneally with 10 g / kg estradiol or concentrations of 50 mg / kg and 150 mg / kg of he and ee of t. foenum - graecum seeds for 6 weeks. p < 0.05 vs. control group ; # p < 0.05 vs. saline group. the prevalence of metabolic syndrome and inflammatory diseases increases during menopause with the overproduction of proinflammatory cytokines. the present study showed that both he and ee of t. foenum - graecum seed are able to improve metabolic status and decrease level of these cytokines in ovariectomized rats. ovariectomy is a standard experimental model of menopause in rodent to investigate pharmacological management of postmenopausal symptoms (11, 12, 24, 25). in our study, in agreement with previous reports, ovariectomy led to increased body weight gain (25, 26). similarly, abbas and elsamanoudy observed that estradiol decreases fbg, fasting insulin, and homeostatis model assessment of insulin resistance (homa - ir, a predictor of insulin sensitivity) in ovariectomized rats (24). the beneficial effects of estradiol on glucose homeostasis and body weight were mimicked by t. foenum - graecum. hypoglycemic effect of t. foenum - graecum has been demonstrated previously in several experimental and clinical studies (27). this effect may be achieved by inhibition of intestinal glucose absorption, enhancing insulin actions on tissues, and activation of hepatic enzymes (28, 29). the inhibitory effect of t. foenum - graecum on weight gain of ovariectomized rats may be related to improvement of insulin resistance. this is in consistent with previous reports that t. foenum - graecum reduces the body weight gain and hyperlipidaemia induced by high - fat diet (30, 31). as reported by other researchers, in our study ovariectomy led to increased level of proinflammatory cytokines. the mechanism underlying elevated inflammation markers in postmenopausal period is not exactly clear ; however, excess production of cytokines by fat and bone marrow can be involved (4, 32). the cytokines tnf-, il-1, and il-6 have obtained the most attention due to their prominent causal role in postmenopausal inflammatory diseases (7, 33). in this work, estradiol replacement could restore the level of these cytokines to near that of control animals. the mechanisms by which estrogen modulates the level and activity of cytokines may include alterations of immune cell function, antioxidative actions, and changes in nitric oxide activity (6, 32, 33). similar mechanisms may describe an inhibitory effect of t. foenum - graecum on the level of tnf-, il-1, and il-6. it has been reported that this plant has estrogenic activities in vitro (34). the presence of phytoestrogen compounds like diosgenin in t. foenum - graecum may be responsible for this estrogenic effect (35). on the other hand, several studies have shown that t. foenum - graecum has antioxidative, anti - inflammatory, and immunomodulatory activities in male and female animals (16 - 19). for example, it was demonstrated that diosgenin induces anti - inflammatory action in adipose tissue (36). reported that ee of t. foenum graecum decreases the blood levels of il-1, il-2, il-6, and tnf- in female rat model of arthritis (19) also, treatment with hydroalcoholic extract of t. foenum graecum seed reduces oxidative stress and the proinflammatory cytokines tnf- and il-6 in diabetic rats (37). further, it has been reported that improvement in glucose metabolism with weight loss is independently associated with reduction of cytokine levels (38). in this study, we used ee and he of t. foenum - graecum to test beneficial effects of this plant in the management of metabolic and inflammatory alterations associated with menopause. for extraction of plant materials, nonpolar solvent such as hexane is used to extract lipophilic constituents (e.g. fatty acids, alkanes, sterols, coumarins, and some terpenoids). solvents with medium - polarity such as ethyl acetate are used to extract constituents with intermediate polarity (e.g. flavonoids and some alkaloids). a more polar solvent such as ethanol is used to extract more polar constituents (e.g. tannins and glycosides) (39, 40). interestingly, in our experiment, both ee and he of t. foenum - graecum could correct metabolic and inflammatory alterations induced by ovariectomy (although the he showed stronger effects). therefore, it can be concluded that both polar and nonpolar constituents of t. foenum - graecum are involved in the beneficial effects of this plant. diosgenin, a steroid saponin with anti - inflammatory, antioxidative, and estrogenic properties, may be of the candidate constituents responsible for the above - mentioned effects of t. foenum - graecum (31, 35, 36). further works on isolation and characterization of active compound of the plant would be of interest. this study described observations on the effect of t. foenum - graecum on metabolic and inflammatory alterations in a rat model of menopause, which may not mimic human menopause. another limitation of the present work is the failure to address exact compound responsible for the effects of t. foenum - graecum. also, long - term safety and potential adverse effects of t. foenum - graecum in menopause women should be well - defined. in conclusion, the present study showed that the administration of t. foenum - graecum improves metabolic features, and corrects inflammatory alterations associated with ovariectomy and thus has a potential for management of menopause.	background : several experimental and clinical studies support beneficial effects of trigonella foenum - graecum (fenugreek) in the management of metabolic diseases and inflammatory disorders.objectives:the purpose of this study was to examine the effect of t. foenum - graecum seed extract in reducing the metabolic and inflammatory alternations associated with menopause.materials and methods : in this experimental study, 49 rats were divided into seven groups : (i) sham - control, (ii) ovariectomized - control, (iii and iv) ovariectomized treated with 50 and 150 mg / kg of t. foenum - graecum seed ethanolic extract, (v and vi) ovariectomized treated with 50 and 150 mg / kg of t. foenum - graecum hexanic extract, (vii) ovariectomized - positive control treated with 10 g / kg of estradiol. the extracts were injected intraperitoneally one day after ovariectomy and the treatments were lasted for 42 days.results:fasting blood glucose and body weight gain increased significantly in the ovariectomized - control group compared with that in the sham animals (p < 0.05). administration of estradiol and t. foenum - graecum (50 and 150 mg / dl of hexanic extract and 150 mg / kg of ethanolic extract) significantly diminished the increase in glucose and body weight (p < 0.05). the serum level of interleukin-1 (il-1), interleukin-6 (il-6), and tumor necrosis factor- (tnf-) in the ovariectomized control group was significantly higher than those in the sham animals (p < 0.05). both hexanic and ethanolic extracts as well as estradiol were able to decrease level of these cytokines in the serum of ovariectomized rats (p < 0.05).conclusions : the results of the present study show that administration of t. foenum - graecum corrects metabolic and inflammatory alterations associated with ovariectomy and has a potential for the management of menopause.
the t cell immunoglobulin and mucin domain (tim) family is located on chromosome 11b1.1 in mice and consists of several members (tim-18). in humans it is located on chromosome 5q33.2 and consists of three members (tim-1, 3, and 4). individual tim family members may serve as susceptibility markers for asthma, allergies, and autoimmune diseases, as well as potential cell surface markers for t helper (th) type 1 and th2 cells [1, 2 ]. therefore, the human tim gene family is critical in the regulation of th1/th2 mediated immunological reactions. tim-1 was first identified as a hepatitis a virus cellular receptor 1 [3, 4 ] and a kidney injury molecule, kim-1 [5, 6 ]. tim-1 is expressed on cd4 t cells after activation and its expression is sustained preferentially in th2 but not th1 cells [1, 7 ]. the high - avidity anti - tim-1 antibody enhances the severity of experimental autoimmune encephalitis by increasing autopathogenic th1 and th17 responses, whereas the low - avidity antibody inhibits autopathogenic th1 and th17 responses. tim-4 is a natural ligand of tim-1 and is exclusively expressed on antigen - presenting cells, including dendritic cells (dcs) and macrophages [9, 10 ], where it mediates phagocytosis of apoptotic cells and plays an important role maintaining tolerance [11, 12 ]. tim-1 and tim-4 interact to regulate th cell responses and modulate the th1/th2 cytokine balance. dc - derived tim-4 maintains tim-1 in th2 cells in a stable status and plays a critical role sustaining th2 polarization. a higher dose of tim-4-ig consistently leads to an increase in t cell proliferation upon ligation with the t - cell receptor, whereas a lower concentration of tim-4-ig inhibits t cell proliferation. human tim-1 is also associated with other types of immune dysfunction, such as atopic dermatitis, allergy, rheumatoid arthritis, asthma, and systemic lupus erythematosus (sle) [1418 ], suggesting that tim-1 may regulate immune responses. in addition, tim-4 expression in peripheral blood mononuclear cells (pbmcs) also increases in patients with sle. behet 's disease (bd) is a th1-polarized, chronic, multisystemic inflammatory disorder with arthritis, gastrointestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. the etiology of bd is unclear ; however, viral infection has long been postulated as one of the main factors. since behet first proposed a viral etiology, his hypothesis has been verified by detecting virus in saliva, intestinal ulcers, and genital ulcers [24, 25 ] of patients with bd. subsequently, herpes simplex virus (hsv) inoculation of the earlobes of icr mice resulted in the development of bd - like symptoms. manifestations in mice inoculated with hsv include multiple symptoms such as oral ulcers, genital ulcers, skin ulcers, eye symptoms, intestinal ulcers, arthritis, and neural involvement, as well as skin crusting. the frequencies of these symptoms are similar to those of patients with bd. tim-1 and tim-4 have not been studied much in bd until now. in this study, we investigated the tim expression in a bd mouse model with bd - like symptoms. the expression tim-1 and tim-4 was analyzed in bd mice and the changes in bd - like symptoms were observed by regulating of tim-1 or tim-4 expression. furthermore, the changes in cellular phenotypes and cytokine levels on immune cells were confirmed after upregulation of tim-1 or downregulation of tim-4 in bd mice. mouse anti - cd4, anti - tim-1, anti - tim-4, anti - cd8a, anti - cd122, anti - cd11b, anti - cd11c, and anti - cd25 antibodies as well as an anti - foxp3 staining kit were purchased from ebioscience (san diego, ca, usa). icr male mice (4 - 5 weeks old) were infected with hsv type 1 (1 10 pfu / ml, f strain) grown in vero cells as described previously. we used anesthetic composed of a mixture of zoletil (virbac lab, carros, france) and rompun (bayer, seoul, korea). the ratio of zoletil and rompun was 1 : 4, and it was administered at a dose of 40 l / mouse (tiletamine 10 mg / kg, zolazepam 10 mg / kg, and xylazine hydrochloride 36 mg / kg) via intramuscular injection. virus inoculation was conducted twice at 10 day intervals, after which the animals were observed for 16 weeks. animals were handled in accordance with a protocol approved by the animal care committee of ajou university school of medicine (institutional approved number : amc-102). multiple symptoms were observed in the mice after hsv inoculation, and 12% of the hsv - injected mice developed bd - like symptoms. disappearance of symptoms or a > 20% decrease in dimension of lesion size was classified as effective. determination of the bd severity score was followed by determining the value of the bd activity index, as outlined on the bd activity form (http://www.behcet.ws/pdf/behcetsdiseaseactivityform.pdf). symptoms exhibited by patients, including mouth ulceration, genital ulceration, erythema, skin pustules, skin ulceration, joints - arthritis, diarrhea, blurred or red eye (right, left), reduced vision (right, left), loss of balance, discoloration of skin, and swelling of the face were selected and analyzed in the bd mouse model. the score of each symptom was one, and the total score was used to determine the bd severity score. briefly, the tim-1 open reading frame (excluding the start codon and signal sequence) was amplified from this clone by polymerase chain reaction and ligated into a pcdef3 expression plasmid. all plasmids used were purified by two passes through endo - free columns (qiagen, chatsworth, ca, usa) as described previously. cac auc aga uca aca gca guu -3, and antisense : 5- cug cug uug auc uga ugu gau uua guu -3. the tim-4 sirna with transfection reagent jetpei (polyplus - transfection, llkirch, france) was used to inject into mice. ten g of tim-1 vector was intraperitoneally injected four times at 2 day intervals into bd mice when the bd - like symptoms appeared, followed by 2 weeks of observations. five g of sirna tim-4 was intraperitoneally injected three times at 2 day intervals into bd mice to downregulate tim-4, followed by a 2-week observation. pbmcs and lymph node cells were isolated from mice and erythrocytes were removed from cell suspensions in ack solution, then washed with phosphate buffered saline (pbs). the cells were surface - stained with anti - mouse antibodies (cd4, cd8, cd11b, cd11c, cd25, cd122, tim-1, and tim-4) for 30 min at 4c in the dark. an anti - mouse foxp3-staining buffer set was used according to the manufacturer 's instructions to detect foxp3 intracellularly. briefly, cells were fixed using fix / perm buffer after washing with 1 permeabilization buffer and then incubated with anti - mouse foxp3 antibody. for analysis, the cells were gated and then the population of stained cells was analyzed by a flow cytometer (facs canto ii ; becton dickinson, franklin lakes, nj, usa) with 10,000 gated lymphocytes. serum was collected 14 days after the first administration of the tim-1 vector and tim-4 sirna into bd mice. serum was analyzed using commercial elisa kits for the detecting mouse interleukin (il)-6 (r&d systems, minneapolis, mn), tumor necrosis factor (tnf)- (r&d systems), il-17 (r&d systems), il-4 (r&d system), and interferon (ifn)- (r&d systems), according to the manufacturer 's instructions. the elisa reader was bio - rad model 1706850 microplate reader, and samples were read at a wavelength of 450 nm. all data are expressed as mean standard deviation. statistical differences between the experimental groups were determined using student 's t - test with a bonferroni correction. the frequencies of tim-1, cd4tim-1, and cd8tim-1 cells were analyzed in cells from lymph nodes (ln), spleen (sp), and pbmcs of normal healthy (nor) and bdn (hsv-1 was inoculated but no symptoms) mice and compared with those in bd mice by facs. in ln, the frequencies of tim-1, cd4tim-1, and cd8tim-1 cells in bd mice were significantly lower than those in bdn mice (bdn versus bd (%) : tim-1, 20.1 11.5 (n = 12) versus 11.1 6.7 (n = 12), p = 0.01 ; cd4tim-1, 3.4 1.9 (n = 9) versus 2.2 1.6 (n = 9), p = 0.09 ; cd8tim-1, 1.9 1.1 (n = 9) versus 1.1 0.6 (n = 9), p = 0.04) (figure 1(a)). in sp, the bd mice showed significantly lower frequencies than nor or bdn mice (nor versus bdn versus bd (%) : tim-1, 21.3 6.5 (n = 6) versus 18.3 8.5 (n = 10) versus 12.2 5.9 (n = 10), nor versus bd p = 0.006, bdn versus bd p = 0.04 ; cd4tim-1, 3.1 1.6 (n = 6) versus 1.9 1.0 (n = 8) versus 1.7 0.5 (n = 8), nor versus bd p = 0.02, bdn versus bd p = 0.35). cd8tim-1 cells showed similar frequencies among three groups (figure 1(b)). in pbmcs, the frequencies of bd mice were slightly lower than those of bdn, but not significantly (bdn (n = 9) versus bd (n = 9) (%) : tim-1, 27.6 17.1 versus 23.6 8.4, p = 0.54 ; cd4tim-1, 8.3 5.9 versus 5.7 4.4, p = 0.31 ; cd8tim-1, 7.6 5.0 versus 5.2 4.3, p = 0.29). bd mice had significantly higher levels than those of nor mice (figure 1(c)). these data indicate that the frequencies of tim-1 expressing cells in bd mice were downregulated compared to those in bdn mice. the frequencies of tim-4 cells were also analyzed in ln, sp, and pbmcs of nor, bdn, and bd mice. in ln, the frequencies of tim-4 cells in bd mice were significantly higher than those in nor and bdn mice (nor versus bdn, versus bd (%) : 1.3 0.8 (n = 11) versus 3.2 0.9 (n = 15) versus 5.8 2.0 (n = 15), nor versus bdn p = 0.000005, nor versus bd, p = 0.0000001, bdn versus bd p = 0.00003) (figure 2(a)). in sp, the frequencies of tim-4 cells in bd mice were also significantly higher than those in nor mice (nor versus bd (%) : 3.9 2.8 (n = 11) versus 6.7 4.3 (n = 17), p = 0.035). however, the difference between bdn and bd mice was not significant (figure 2(b)). in pbmcs, the frequencies of tim-4 cells in bd mice were higher than those in nor mice (figure 2(c)). moreover, in peritoneal macrophages, the frequencies of tim-4 cells in bd mice were significantly higher than those in nor and bdn mice (nor versus bdn versus bd (%) : 6.1 2.0 (n = 5) versus 8.6 4.0 (n = 9) versus 26.6 17.0 (n = 5), nor versus bdn, p = 0.11, nor versus bd, p = 0.02, bdn versus bd, p = 0.005). no differences between nor and bdn mice were observed (figure 2(d)). in addition, we also examined the frequencies of cd11btim-4 and cd11ctim-4 cells in ln and sp. in ln, both markers in bd mice were higher than those in nor and bdn mice (nor (n = 5) versus bdn (n = 6) versus bd (n = 5) (%) : cd11btim-4, 1.1 0.3 versus 1.7 0.7 versus 3.0 1.4, nor versus bdn, p = 0.05, nor versus bd, p = 0.01, bdn versus bd, p = 0.04 ; cd11ctim-4, 0.1 0.0 versus 0.5 0.1 versus 0.7 0.5, nor versus bdn, p = 0.00003, nor versus bd, p = 0.01, bdn versus bd, p = 0.14) (figure 2(e)). in splenocytes, expression of cd11ctim-4 cells was similar to that in ln in nor (0.3 0.1%, n = 5), bdn (0.6 0.4%, n = 6) and bd mice (1.0 0.6%, n = 8) (nor versus bdn, p = 0.07, nor versus bd, p = 0.07, bdn versus bd, p = 0.1). in contrast, the frequencies of cd11btim-4 cells in bd mice were significantly lower than those in nor, but higher than those in bdn mice (figure 2(f)). these data suggest that the frequencies of tim-4 expressing cells in bd mice were upregulated compared to those in bdn mice. we investigated the change in bd - like symptoms according to the regulation of tim-1 expression. the tim-1 vector was injected intraperitoneally at 10, 30, and 50 g / mouse into nor mice twice at 2 day intervals. the frequencies of tim-1 cells from ln were compared to those of the control vector injected group the day after the last injection. the expression of tim-1 cells in the 10 g tim-1 vector injected group (19.1 5.1%, n = 4) was significantly higher than that in the control vector injected group (10 g : 9.4 2.6%, n = 4, p = 0.01 ; 50 g : 10.4 5.4%, n however, the 30 (n = 3) and 50 g (n = 4) tim-1 vector injected groups showed lower expression than that in the 10 g injected group. ten g of tim-1 vector was injected intraperitoneally into bd mice four times at 2 day intervals, followed by observations for 2 weeks. skin and genital ulcers improved after administering the tim-1 vector when compared to those in the control vector - injected group (figure 4(a)). additionally, the severity score was 2.83 0.41 before and 2.83 0.75 at 1 week in the control vector group and 2.67 1.21 at 2 weeks after the first injection of control vector (n = 6). the severity score in the tim-1 vector group was 3.17 0.75 before injection, 1.50 1.22 at 1 week after injection (p = 0.004), and 1.33 1.51 at 2 weeks after injection in bd mice (p = 0.03, n = 6) (figure 4(b)). the severity score decreased after injecting the tim-1 vector, whereas the control vector injection did not result in a difference. two weeks after the first administration of the tim-1 vector to bd mice, isolated ln and pbmcs were analyzed for tim-1 cells by facs. in ln, the frequencies of tim-1 cells in tim-1 vector injected group were slightly higher compared to those in the control vector injected group (con versus tim-1 (%) : 10.3 1.7 (n = 4) versus 11.9 3.2 (n = 5), p = 0.19). however, cd4tim-1 and cd8tim-1 cells were similar to those in the control vector injected group (figure 4(c)). the frequencies of tim-4 cells were also not different (figure 4(d)). in pbmcs, tim-1 cells were slightly lower in the tim-1 vector injected group, but cd4tim-1, cd8tim-1, and tim-4 cells were not different (figures 4(c)-4(d)). interestingly, cd4 t cells in the tim-1 vector injected group were slightly higher than those in the control vector injected group (con versus tim-1 (%) : 21.3 10.7 (n = 8) versus 27.6 13.7 (n = 8), p = 0.36) (figure 4(c)). several cellular phenotypes in the ln and pbmcs the frequencies of cd4cd25, cd4foxp3, and cd4cd25foxp3 (regulatory t, treg) cells in ln were not significantly changed in the tim-1 vector compared to those in the control vector injected group (con (n = 5) versus tim-1 (n = 6) (%) : cd4cd25, 6.5 1.6 versus 6.1 2.3, p = 0.35 ; cd4foxp3, 5.3 1.5 versus 4.7 1.7, p = 0.29 ; treg, 5.2 1.4 versus 4.7 1.7, p = 0.3) (figure 5(a)). but, in pbmcs, cd4cd25, cd4foxp3, and treg cells in the tim-1 vector injected group were significantly higher than those in the control vector injected group (con (n = 8) versus tim-1 (n = 8) (%) : cd4cd25, 0.31 0.18 versus 0.61 0.21, p = 0.01 ; cd4foxp3, 0.10 0.10 versus 0.31 0.12, p = 0.004 ; treg, 0.04 0.08 versus 0.16 0.10, p = 0.03) (figure 5(a)). cd8cd122 t cells are newly identified, regarded as treg cells, and are involved in anti - inflammatory responses. another type of treg cells, cd8cd122 t cells, were also analyzed in ln of the tim-1 vector injected group (con (n = 5) versus tim-1 (n = 6) (%) : 1.3 0.3 versus 1.6 0.7, p = 0.25) (figure 5(b)). in pbmcs, cd122 and cd8cd122 t cells in the tim-1 vector injected group were also higher than those in the control vector injected group (con (n = 8) versus tim-1 (n = 8) (%) : cd122, 1.2 1.0 versus 9.8 7.9, p = 0.04 ; cd8cd122, 1.9 1.5 versus 2.4 2.1, p = 0.58). our study indicated that the tim-1 vector upregulated treg cells and cd122 cells in bd mice. up - regulation of these cellular phenotypes may be involved in improved bd - like symptoms after injection of the tim-1 vector. sera were analyzed by elisa at 2 weeks after tim-1 vector administration into bd mice to determine the levels of cytokines. the il-17 level in tim-1 vector injected group was significantly lower than that in the control group (con (n = 7) versus tim-1 (n = 7) (pg / ml) : 11.53 4.45 versus 6.84 2.17, p = 0.03). tnf- also decreased significantly in the tim-1 vector injected group compared to that in the control group (con (n = 11) versus tim-1 (n = 11) (pg / ml) : 16.5 13.8 versus 6.9 5.1, p = 0.04). the il-6 level in the tim-1 vector injected group decreased (con (n = 8) versus tim-1 (n = 8) (pg / ml) : 145.7 176.9 versus 72.2 38.8, p = 0.27). in contrast, il-4 increased slightly in the tim-1 vector injected group (con (n = 8) versus tim-1 (n = 9) (pg / ml) : 9.3 5.4 versus 10.8 5.0, p = 0.53). but, ifn- levels did not differ between the tim-1 and control vector injected group (figure 6). consequently, these results indicate that the tim-1 vector might downregulate proinflammatory cytokines in bd mice. in bd mice, the frequencies of tim-4 cells were higher than those in nor and bdn mice in ln cells and peritoneal macrophages (figure 2). sitim-4 was injected into nor mice intraperitoneally to downregulate tim-4 cells, and the frequencies of tim-4 cells were analyzed in peritoneal macrophages by facs. tim-4 sirna (sitim-4) at 2, 5, or 10 g / mouse or negative control (nc) scrambled sirna (2 and 5 g / mouse) was injected (nc : 5 g versus sitim-42, 5, and 10 g (n = 4) : 8.9 1.4% versus 6.6 3.4% (p = 0.42), 3.7 0.9% (p = 0.013), 2.8 2.2% (p = 0.04)) (figure 7(a)). sitim-4 downregulated tim-4 macrophages dose dependently and showed a significant difference in the 5 g and 10 g administered groups. therefore, 5 g injections were used for the following experiment. to determine the time - dependent efficacy of tim-4 sirna, 5 g of sitim-4 was injected, and the frequencies of tim-4 macrophages were analyzed by facs after 24, 48, and 72 hours. the frequencies of tim-4 at 48 hours were lowest (nc versus sitim-4 (n = 2) : 24 h, 7.9 0.6% versus 4.9 0.2%, p = 0.02 ; 48 h, 8.9 1.4% versus 2.9 0.4%, p = 0.03 ; 72 h, 15.4 0.1% versus 4.2 3.0%, p = 0.03) (figure 7(b)). five g of sitim-4 was injected intraperitoneally three times at 2 day intervals into bd mice and the mice were observed for 2 weeks (figure 8(a)). after administration of sitim-4, bd - like symptoms, such as skin and genital ulcers, were compared to the control group. in the negative control group, the severity score was 2.25 0.46 before, 2.00 1.07 at one week (p = 0.35), and 1.88 1.13 at 2 weeks after the first injection of bd mice (p = 0.28, n = 8). in the sitim-4 treated group, the score was 2.63 0.52 before, 1.25 0.81 at 1 week (p = 0.001), and 1.25 0.89 at 2 weeks after injection into bd mice (p = 0.001, n = 8) (figure 8(b)). at 1 and 2 weeks after the first administration of sitim-4 to bd mice the frequencies of tim-4 cells at 1 and 2 weeks after sitim-4 treatment tended to be lower than those in the negative control group, but the difference was not significant (1 week : 18.6 4.8% versus 15.7 3.7%, p = 0.31, 2 weeks : 20.7 6.5% versus 18.8 3.3%, p = 0.42) (figure 8(b)). however, in ln, tim-4, cd11btim-4, cd11ctim-4, tim-1, cd4tim-1, and cd8tim-1 cells in the sitim-4 treated group were similar to those in the control treated group at 1 week after the first sitim-4 injection (figures 8(c)-8(d)). even at 2 weeks after the first injection, those markers were not different (data not shown). in these results, bd - like symptoms improved with sitim-4 treatment and decreased the severity score. the frequencies of cd4cd25, cd4foxp3, and treg (cd4cd25 foxp3) cells were analyzed by facs at 1 and 2 weeks after the first treatment with sitim-4 in bd mice (figure 9). after 1 week, treg cells increased slightly in sitim-4 treated bd mice compared with those in the nc treated group (nc versus sitim-4 : cd4cd25, 5.82 2.01% versus 6.84 2.03%, p = 0.45, n = 5 ; cd4foxp3, 4.64 1.9% versus 5.82 2.4%, p = 0.41, n = 5 ; treg, 3.28 1.57% versus 4.08 1.73%, p = 0.47, n = 5). after 2 weeks, treg cells were significantly higher in sitim-4 treated bd mice than those in the nc treated group (nc versus sitim-4 : cd4cd25, 4.8 0.6% versus 5.9 1.2%, p = 0.03, n = 6 ; cd4foxp3, 4.1 0.7% versus 5.2 1.2%, p = 0.03, n = 7 ; treg, 2.7 0.5% versus 3.5 0.9%, p = 0.04, n = 7). these results suggest that the increased number of treg cells is associated with knock down of tim-4 in bd mice. after administering sitim-4 to bd mice, the serum level of il-17 was analyzed by elisa and compared with nc sirna treated bd mice. the level of il-17 tended to decrease in the sitim-4 treated group compared to that in the nc treated group, but the difference was not significant (nc versus sitim-4 (n = 8) : 19.4 11.5 pg / ml versus 15.6 8.1 pg / ml, p = 0.25) (figure 10). bd mice downregulated tim-1 levels and upregulated tim-4 levels in ln, sp, pbmcs, and peritoneal macrophages compared to those in bdn mice. administration of the tim-1 vector upregulated the frequencies of tim-1 cells, and tim-4 sirna downregulated the frequencies of tim-4 cells. administering the tim-1 vector improved bd - like symptoms, such as genital and skin ulcers, and decreased the severity score. tim-1 expression is lower in patients with active sle compared to that in inactive patients. human tim-1 is associated with immune dysfunction, such as atopic dermatitis, allergy, rheumatoid arthritis, asthma, and sle [1418 ]. our data indicate that the tim-1 related t cells phenotype did not change much after administering the tim-1 vector. interestingly, the frequencies of cd4 t cells in pbmcs increased in the tim-1 vector injected group compared to those in the control vector injected group. actually, tim-1 is expressed on cd4 t cells after activation and its expression is preferentially sustained on th2 but not th1 cells [1, 7 ]. reported that tim-1-fc triggers a significant increase in the frequencies of cd4 t cells. in our study other treg cells, cd8cd122 t cells, were also higher in pbmcs and ln of the tim-1 vector injected group compared to those in the control group. cd8cd122 t cells are newly identified and regarded as treg cells and have an effect on anti - inflammatory responses. our results suggest that the function of tim-1 influenced the bd - like symptoms and is associated with treg and cd8cd122 t cells. recent studies suggest that il-17 may play a dominant role in provoking chronic autoimmune inflammation and is considered essential for t cell - mediated colitis and promotion of inflammation [3436 ]. the association of il-17 and il-22 was also reported in patients with bd [37, 38 ]. tnf- overexpression has been implicated in acute and chronic inflammatory diseases, such as septic shock, bowel disease, crohn 's disease, rheumatoid arthritis, atopic dermatitis, psoriasis, and bd. overproduction of il-6 plays a role in rheumatoid arthritis, juvenile idiopathic arthritis, inflammatory bowel disease, and sle. the anti - tim-1 antibody (high avidity / agonistic) is a down - regulator of pro - inflammatory th-17 cells. tim-1-tim-4 interaction is involved in the regulation of th cell responses and modulation of the th1/th2 cytokine balance. our data also indicate that administering the tim-1 vector decreased proinflammatory cytokines, such as il-17, tnf-, and il-6, but the th2 type cytokine il-4 was upregulated in the tim-1 vector injected group. recently, upregulated cytokine il-22 in ocular bd patients was also downregulated by anti - il-6 and anti - tnf-. bd mice displayed markedly increased tim-4 levels in ln and peritoneal macrophages compared to those in nor and bdn mice. the frequencies of tim-4 cells were downregulated when tim-4 sirna was administered to nor mice compared to those in the nc group. sitim-4 administered bd mice displayed improved symptoms such as skin and genital ulcers and arthritis and showed a decreased severity score. tim-4 is a ligand for tim-1 and is not expressed in t cells but is exclusively expressed in antigen - presenting cells, including dcs and macrophages [9, 10 ]. two groups have reported that tim-4 deficient mice with a 129 or b6 background develop little or no autoimmunity [9, 43 ]. in conjunction with improved symptoms, treg cells in the sitim-4 treated group were significantly upregulated compared to those in the control group, and il-17 level decreased. in our previous study, in addition, the frequencies of treg cells are lower in patients with autoimmune and inflammatory diseases, such as crohn 's disease, multiple sclerosis, and sle, than those in healthy controls and inactive patients. the tim-1-tim-4 interaction is important for regulating t cell proliferation and modulating the th1/th2 cytokine balance. our data suggest that upregulated treg cells induced by sitim-4 might contribute to improve bd - like symptoms. in conclusion, the frequencies of tim-1 cells in bd mice were downregulated compared to those in bdn mice. administering the tim-1 vector to bd mice improved the bd - like symptoms and decreased the severity score by up - regulating treg cells and down - regulating pro - inflammatory cytokines. in addition, treatment with sitim-4 upregulated treg cells and improved bd - like symptoms, which were related to the tim-1 and tim-4 expression and the tim-1-tim-4 interaction. furthermore, tim-4 sirna downregulated the level of il-17, which may have been involved in improving the bd - like symptoms. consequently, regulation of tim-1 and tim-4 was effective for improving bd - like symptoms in mice.	the t cell immunoglobulin mucin (tim) proteins regulate t cell activation and tolerance. tim-1 plays an important role in the regulation of immune responses and the development of autoimmune diseases. tim-4 is a natural ligand of tim-1, and the interaction of tim-1 and tim-4 is involved in the regulation of t helper (th) cell responses and modulation of the th1/th2 cytokine balance. behet 's disease (bd) is a chronic, multisystemic inflammatory disorder with arthritic, intestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. tim-1 expression was lower in a herpes simplex virus - induced bd mouse model compared to that in asymptomatic bd normal (bdn) mice. tim-4 expression was higher in bd mice than that in bdn mice. in this study, we investigated the tim expression in a bd mouse model with bd - like symptoms. tim-1 and tim-4 expression was regulated by an expression vector or sirna injected into the bd mouse model. the tim-1 vector injected into bd mice resulted in changes in bd - like symptoms and decreased the severity score. treatment with tim-4 sirna also improved bd - like symptoms and decreased the severity score accompanied by upregulation of regulatory t cells. we showed that regulating tim-1 or tim-4 affected bd - like symptoms in mice.
propofol is a popular intravenous (iv) anesthetic agent providing fast onset and smooth anesthetic induction with rapid recovery. however, the disadvantage of pain or discomfort during iv injection may be as high as 85% in children when given into small vein on the dorsum of the hand 1 - 3. although various drugs have been used to decrease propofol injection pain such as ketamine, lidocaine, alfentanil, remifentanil, and thiopenthal 4 - 8, it still represents a clinical problem in children, especially in younger children because of the smaller size of the accessible veins 9. the two most commonly used methods are pretreatment of the vein with lidocaine and mixing propofol with lidocaine immediately before injection 3. however, prior or concomittant administration of lidocaine is not effective in all children 9 - 11. tramadol is a centrally - acting drug, which is effective in the treatment of moderate to severe pain. in addition to its systemic effect, the local anesthetic effect of tramadol has been shown in both clinically and laboratory studies 12,13. according to this action, pretreating the vein with iv tramadol has proved to be effective in preventing propofol injection pain in adults, the incidence of tramadol treated patients was 23% vs 69% in the control group 14. however, to date no published data are available for tramadol on reducing propofol injection pain in children. to the best of our knowledge this is the first study of tramadol on preventing propofol injection pain in pediatric population. as the beneficial effect of tramadol on propofol injection pain in adults is already well known, we aimed to investigate the effect of preatreatment with tramadol on reducing both the incidence and severity of propofol injection pain and how it compared with lidocaine in children. after receiving the institutional ethics committee approval (ec.071.00/2631), written informed consent was obtained from the parents of 120 asa physical status i or ii patients aged 6 - 13 years, who were scheduled for elective orthopedic and otolaryngological surgery with general anesthesia between november 2010 and june 2011 were enrolled in this study. exclusion criteria include crying children on arrival in the operating room, emergent cases, presence of hepatic or renal dysfunction, musculoskeletal disorders and known allergies to propofol, tramadol and lidocaine. patients were also excluded if it was impossible to insert a venous line into the dorsum of the hand. when the children arrived in the operating room, routine monitors for heart rate, noninvasive blood pressure (nibp) and peripheral oxygen saturation was applied. the cannula was inserted 15 min before induction without the use of local anesthetic because of the lack of availability of topical anesthetic creams. a continous of isomix 1/3 was applied at the maintenance rate according to the children ' weight. the children were randomly allocated using a table of random numbers to one of three treatment groups. children in group c (group control) received normal saline placebo 60 sec before propofol (180 mg 1% propofol with 2 ml saline) ; group t (group tramadol) received 1 mg.kg tramadol iv 60 sec before propofol (180 mg 1% propofol with 2 ml normal saline) ; group l received normal saline placebo 60 sec before propofol - lidocaine mixture (180 mg 1% propofol with 2 ml % 1 lidocaine). after the injection of the pretreatment drug, propofol (at room temperature) 3 - 4 mg. kg was delivered through the iv cannula at an approximate rate of 2 ml per 10 sec by observers until the loss of eyelash reflex. during a ten - second pause after the 25% of the calculated propofol dose had been given, another anesthesiologist who was unaware of the study groups, assessed propofol induced pain using a four point behavioral scale : 1= no pain (no reaction) ; 2= mild pain (grimace) ; 3= moderate pain (grimace+cry) ; 4= severe pain (cry+withdrawal) 9. then fentanyl iv 1 - 2 g.kg was administered only after assessment of the pain after propofol injection. induction of anesthesia was completed with the remaining dose of propofol and tracheal intubation was facilitated with 0.6 mg.kg rocuronium. anesthesia was maintained with sevoflurane and nitrous oxide 50% in oxygen, with controlled ventilation and intermittent fentanyl and rocuronium was given if required. before the skin closure 10 mg.kg paracetamol given intravenously for postoperative analgesia. within 24 h after the operation, the injection site was checked for pain, edema or allergic reactions by an anesthesiologist who was unaware which drug had been administered. statistical analyses were performed by using spss software (statistical package for the social sciences, version 16.0, spss inc, chicago, il, usa). the primary endpoint of the study was the number of patients in each group without injection pain. the sample size was determined according to a previous study which was estimated the pain was 66 % after iv 1 % propofol injection 8 with a significance level of = 0.05 and = 0.80 and 29 patients in each group was found sufficient. we decided to include 40 patients in each group for the possibility of drop out. patient demographic data were determined by analysis of variance (continous variables : age, weight, eg), as well as test (discrete variables : sex, asa physical status). pain severity scores were determined by using the mann - whitney test and the incidence of pain was determined using the test. statistical analyses were performed by using spss software (statistical package for the social sciences, version 16.0, spss inc, chicago, il, usa). the primary endpoint of the study was the number of patients in each group without injection pain. the sample size was determined according to a previous study which was estimated the pain was 66 % after iv 1 % propofol injection 8 with a significance level of = 0.05 and = 0.80 and 29 patients in each group was found sufficient. we decided to include 40 patients in each group for the possibility of drop out. patient demographic data were determined by analysis of variance (continous variables : age, weight, eg), as well as test (discrete variables : sex, asa physical status). pain severity scores were determined by using the mann - whitney test and the incidence of pain was determined using the test. one hundred and twenty patients were enrolled in the study but one patient in group c was drop out from the study because of the difficulty of insertion of iv cannula into the dorsum of the hand. there were no significant differences in age, weight, gender and asa physical status of the patients and the mean induction dose of propofol, time to loss of eyelash reflex (p>0.05) (table 1). the patients in tramadol group had less intraoperative fentanyl consumption and postoperative analgesic requirement one hr after surgery than other groups. 11 (27.5 %) patients in tramadol group had received additional fentanyl intraoperatively whereas 25 (64.1 %) in control (p=0.01). and 24 (60 %) patients (p0.05). according to mild pain there was no statistically difference found among groups (p>0.05). in respect to moderate pain 14 (35.9 %) in control group, 6 (17.5 %) in tramadol group and 4 (10 %) patients in lidocaine group had moderate pain. severe pain had been experienced by 11 (28.2 %) patients in only control group and it was found statistically significant when compared with both tramadol and lidocaine groups (p<0.001). additionally, no patient had an experience of severe pain in tramadol and lidocaine groups so there was no statistically significance between these two groups with respect to severe pain. no complications, such as pain, edema, wheal or flare response were observed at the injection site within the first 24 h after the operation. tramadol 1 mg.kg and lidocaine 20 mg premixed with propofol were equally effective in reducing especially severe pain during iv injection of propofol. injection pain which is a recognized adverse effect of propofol can be very distressing to patients 15 and still a limitation of this popular iv anesthetic agent. the mechanism of propofol injection pain is still unclear, it has been postulated to be due to either a direct irritant effect given rise to an immediate sensation of pain or an indirect effect via the release of mediators such as bradykinin leading to a delayed onset 16. the most common practices including iv lidocaine before propofol injection, after mixing with propofol and iv lidocaine given with a tourniquet were all used for preventing propofol injection pain 3,4. however, there is the possibility that lidocaine, a local anesthetic, reversibly blocks peripheral nerve pathways in the arm 17. besides, the analgesic effect of lidocaine on propofol injections not only based on its local anesthetic effect but also on the decrease in ph of the propofol when given as a lidocaine mixture. tan lh, compared the effects of propofol - lidocaine mixture and lidocaine pretreatment in adults on propofol injection pain and the dose of propofol required for the induction of anesthesia 18. they showed no statistically difference between the groups for propofol induced pain and induction dose of propofol like in our study. bilotta 19 administered 2 mg.kg 2% lidocaine or an equivalent volume of saline as a pretreatment in pediatric icu patients for different invasive procedures. they investigate the effect of lidocaine on both propofol injection pain and propofol induced motor disturbances such as spontaneous dystonic or choreiform movements. they found that lidocaine not only reduced pain and motor disturbances but also reduced propofol requirement. in contrast, in our study we did not show the difference in the required propofol dose, because we used low dose of lidocaine for propofol injection pain but in their study the dose was higher and reduced propofol dose might be related with the high dose of lidocaine. although this study was not designed to assess the neurologic and motor disturbances of propofol, in contrast to their study, we did not observe any motor disturbances in our study groups. we thought that this might be related to the pain thresholds and the children investigated. we only assessed the intensity and severity of pain with a behavioral scale of cameron which was described the withdrawal of the arm as a severe pain 9. furthermore, iv retention of lidocaine with a tourniquet was found the best method for reducing pain 3,4. the tourniquet which isolates the arm veins from the rest of circulation presents a useful model for studying the peripheral actions of a drug in the absence of central effect. it is advised that the duration of venous occlusion with a tourniquet which is applied to the forearm could be for a period of 30 - 120 sec before propofol injection 3,4. but in this study, we did not administer a tourniquet because of not being practical in the pediatric population, therefore we were not rule out the possible central role of tramadol in reducing propofol injection pain. tramadol, is a centrally acting weak -receptor agonist, inhibits noradrenaline re - uptake as well as promotes seratonin release and can be used to treat moderate and severe pain 21 - 24. in addition to its systemic effect, the local anesthetic effect of tramadol on peripheral nerves has been shown in both clinically and laboratory studies 13,24,25. more complete data have been produced the effect of tramadol on the release of monoaminergic neurotransmitters in the central nervous system and its agonist action at peripheral and central opioid receptors. desmolues and co - workers 25 have confirmed in humans that the analgesic effect of tramadol is apportioned between the opioid and monoaminergic components. pang 26 observed a local anesthetic effect with intradermal injection of tramadol and lidocaine. jou 12 suggested that tramadol affects sensory and motor nerve conduction by a similar mechanism to that of lidocaine which acts on the voltage dependent sodium channel leading to axonal blockage. opiates were shown to exert peripheral analgesic action in addition to their well known central effects, a clear cut discrimination between peripheral and central analgesics is debatable 27. the analgesia produced by both peripheral and central mechanisms may be additive or even synergistic. moreover, peripheral opioid receptors have been described and shown to mediate analgesic effect when activated by opioid agonists 28,29. it was suggested that both peripheral and central actions could be at the basis of the antiinflammatory effects of the tramadol 29. it, in fact, exerts a dual mechanism action due to binding opioid receptor and potentiation of the monoaminergic system. therefore, the antiinflammatory action of tramadol could be at least in part, related to its opioid effect. accordingly pharmacokinetics of tramadol in adults and children is reported to be similar when administered intravenously 30. it has been used as an alternative agent to traditional opioids for pain relief in pediatric population with the recommended dose of 1 - 2 mg.kg for children as we used in our study without increasing adverse events in the pediatric population 31. hiller and saarnivaara 11 compared three different doses of alfentanil one min prior to propofol with 10 mg lidocaine premixed with propofol in children. they found the incidence moderate to severe pain was 4 % in lidocaine group, 40 %, 16 % and 20 % in the groups receiving 10, 15, 20 g.kg alfentanil respectively. al - refai and coworkers 32 compared remifentanil, alfentanil, lidocaine premixture with placebo and they found that three drugs were all effective in reducing propofol pain in children. another study of batra and coworkers 7 showed also the effect of remifentanil especially when give 0.5 g.kg. in our study, incidence of moderate pain was 15 % in tramadol group and 10 % in lidocaine group whereas 35.9 % in the control group and severe pain was found 28.2 % also. no patient in both tramadol and lidocaine group had experience of severe pain. in another study of children, kaabachi 33 compared the effects of ketamine - propofol and lidocaine - propofol mixture in children and they showed that both of them were effective but lidocaine was better than ketamine on propofol injection pain. in our study, we also found both tramadol and lidocaine were effective in reducing pain but clinically lidocaine seems to be better analgesia than tramadol though statistically no significant. we suggested that this clinical difference might be related with the different actions of the studied drugs. pang 14 showed that 50 mg tramadol given as pretreatment following a venous occlusion with a tourniquet for one min significantly reduced propofol injection pain in adults compared with lidocaine 60 mg or placebo group. they found the overall incidence of pain was 23 % in tramadol, 9 % in lidocaine and 69 % in the control group and suggested that tramadol reduced propofol pain due to its peripheral analgesic action which was demonstrated with the use of a tourniquet for one minute. as mentioned before tournique is not used in the current study therefore we could not be explained the analgesic effect of tramadol here with merely peripheral analgesic action of this drug. besides, it was known that tramadol has a dual effect and it does not have a single mechanism on analgesia. we thought that in this study tramadol might be exerted its effect not only with a peripheral action but also with an action on cental monoaminergic system which may be contributed to its analgesic effect. in conclusion, it was shown in this study that pretreatment with iv tramadol to be equally effective in relieving propofol injection pain compared to lidocaine mixed with propofol and it is also useful for intraoperative and postoperative analgesia when relatively such these minor operations are undertaken. further studies comparing tramadol with other opioids that have been shown to reduce propofol injection pain are needed in pediatric population, especially dose ranging studies.	background : the pain on propofol injection is considered to be a common and difficult to eliminate problem in children. in this study, we aimed to compare the efficacy of pretreatment with tramadol 1 mg.kg-1and propofol - lidocaine 20 mg mixture for prevention of propofol induced pain in children.methods : one hundred and twenty asa i - ii patients undergoing orthopedic and otolaryngological surgery were included in this study and were divided into three groups with random table numbers. group c (n=39) received normal saline placebo and group t (n=40) received 1 mg.kg-1 tramadol 60 sec before propofol (180 mg 1% propofol with 2 ml normal saline) whereas group l (n=40) received normal saline placebo before propofol - lidocaine mixture (180 mg 1% propofol with 2 ml % 1 lidocaine). one patient in group c was dropped out from the study because of difficulty in inserting an iv cannula. thus, one hundred and nineteen patients were analyzed for the study. after given the calculated dose of propofol, a blinded observer assessed the pain with a four - point behavioral scale.results : there were no significant differences in patient characteristics and intraoperative variables (p>0.05) except intraoperative fentanyl consumption and analgesic requirement one hr after surgery among the groups (p<0.05). both tramadol 1 mg.kg-1 and lidocaine 20 mg mixture significantly reduced propofol pain when compared with control group. moderate and severe pain were found higher in control group (p<0.05). the incidence of overall pain was 79.4% in the control group, 35% in tramadol group, 25% in lidocaine group respectively (p<0.001).conclusions : pretreatment with tramadol 60 sec before propofol injection and propofol - lidocaine mixture were significantly reduced propofol injection pain when compared to placebo in children.
it is more common in female with female - to - male ratio of 3:1. not all the cases of necrobiosis are associated with diabetes ; hence the terminology necrobiosis lipoidica diabeticorum has been abandoned. the ulcers in nl are quite painful leading to impaired quality of life of these patients. we present a case of ulcerative nl, which failed to respond to traditional treatment modalities but responded quickly to thalidomide. a 58-year - old woman with 15 years history of insulin - dependent diabetes presented with nonhealing ulcers on her shin since 5 years. four months later, she noticed a pinhead sized lesion over the red area, which enlarged and ulcerated. three similar ulcers [figure 1a and b ] appeared on the shin over the next one month. the ulcers were non healing and extremely painful but not associated with any constitutional symptoms. local wound care, topical corticosteroids, topical calcineurin inhibitors were tried along with oral antibiotics, dapsone, pentoxyphylline, and analgesics, without much relief. she had multiple discrete tender ulcers over a waxy, erythematous, atrophic plaque on the anterior aspect of her left leg, variying in size from 3 2 cm to 1 1 cm. the borders of ulcer were undermined, base was indurated, and floor was covered with necrotic debris. her hemogram, renal and liver function tests, and venous doppler test results were normal. pus culture grew staphylococcus aureus sensitive to linezolid, amoxycillin and clavulanic acid, and tazobactam piperacillin. skin biopsy from the edge of the ulcer revealed a hyperplastic, sclerotic epidermis and a dense superficial and deep dermal infiltrate of lymphocyte and plasma cells concentrated around the blood vessels and sweat glands [figure 2a - c ]. we started the patient on oral linezolid 600 mg twice daily for 7 days and chloroquine 250 mg once daily for 4 weeks along with local dressings. topical human recombinant epidermal growth factor, oral clopidrogel and aspirin were added to the above regime and continued for another 2 months, with no improvement. pain reduced dramatically within 2 weeks and the ulcers healed completely after 4 weeks [figures 3a and b ]. (a and b) multiple non healing ulcers over the shin (a - c) hyperplastic, sclerotic epidermis and a dense superficial and deep dermal infiltrate of lymphocyte and plasma cells concentrated around the blood vessels and sweat glands with several granulomas arranged in a horizontal manner in mid and lower dermis. (h and e, 10, 40, 40) (a and b) complete healing and softening of skin after treatment with thalidomide there is no consistently effective therapy, and lack of uniform guidelines make treatment more challenging. many theories were put forward to explain the pathogenesis of nl : diabetic microangiopathy due to deposition of glycoprotein in the blood vessel wall could lead to impaired blood supply to the skin ; greater cross - linking of the collagen fibres in nl could lead to thickening of the basement membrane zone ; immune complex deposition in the dermal blood vessel walls could lead to vasculitis ; recently a role of disturbance in glucose transport by fibroblasts has been postulated. glut-1 is the human erythrocyte glucose transporter, which mediates facilitative transport of glucose across epithelial and endothelial barrier tissues. this protein was expressed in the sclerotic collagen of nl patients, indicating insulin resistance in these tissues. these include cutaneous blood flow enhancers, such as dypyridamol, clopidrogel, aspirin, pentoxyphylline ; topical and intralesional steroids, and topical calcineurin inhibitors ; wound healing enhancers such as epidermal growth factors, platelet - derived growth factors, collagen gel, hyperbaric oxygen ; surgery and pulse dye laser ; and immune modulators such as antimalarials, cyclosporine, and biologics. our patient was treated with various drugs but failed to respond. in several case studies, tnf - alpha inhibitors such as etanercept, adalimumab, and infliximab were shown to improve ulcerated nld. tnf - alpha has been found in high concentrations in the sera and skin of patients with nl thalidomide acts as an anti - inflammatory agent by suppressing tnf - alpha via degradation of its messenger rna and by decreasing the ratio of helper t cells to suppressor t cells. the ulcer healed rapidly and thalidomide was withdrawn over 12 weeks, with no relapse till date.	ulcerative necrobiosis lipoidica (nl) in diabetic patients is a rare, painful condition. it is a difficult - to - treat condition, impairing quality of life of patients. although various drugs have been tried, none of them is consistently effective. biologics in the form of tnf - alpha inhibitors show promising results in the treatment. but because of their high cost we chose thalidomide, which also has tnf - alpha inhibiting properties to successfully treat a long - standing case of ulcerative nl, which was refractory to various treatment modalities.
double dislocations of the thumb joints have only been reported on four previous occasions, in all cases reported to date, the joints have dislocated dorsally. we present the case of a 26-year - old male patient with simultaneous volar dislocations of the carpometacarpal and metacarpophalangeal joints of the thumb. there was delayed operative treatment of this injury with ligament reconstruction and stabilization of the metacarpophalangeal joint. this rare case provides a mechanism to this type of injury, highlights the importance of initial, and repeated clinical and radiographic review, highlights the soft tissue component to this injury, and demonstrates how even delayed treatment can result in a good functional outcome. the thumb joints are vital to the co - ordinated, multidirectional and precise movements of the hand. the thumb is positioned in a perpendicular plane to the other fingers, with the saddle - shaped carpometacarpal (cmc) joint providing six different planes of movement. the metacarpophalangeal (mcp) joint of the thumb is more like an interphalangeal articulation with movements restricted to one plane. dislocations of joints in the hand are relatively common ; however multiple dislocations of the thumb joints have been rarely described [1 - 6 ]. these dislocations involve high - energy axial loading injuries to the thumb, and can also be associated with fractures and ligament disruption. stiffness and pain after injuries to these joints can result in significant impact on activities of daily living. there have been four other reports of double dislocations of the thumb in the literature. our report of simultaneous volar dislocations of both of these joints is the first to be described. a 26-year - old right - handed male horse - trainer injured his right hand while riding, as he raised his arm to protect himself from a low branch. he presented to the accident and emergency department with a painful and obviously deformed right thumb. this was a closed isolated injury, and he had no neurological or vascular deficit of the affected thumb. there was also a fracture of the volar aspect of the base of the first metacarpal, which appeared to be an avulsion - type (fig. 1). ap and lateral radiographs of the initial injury under local anaesthesia block, the two dislocations were reduced with longitudinal traction, and the thumb was immobilized in a bennett s cast. upon review in fracture clinic one week later, the position of the cmc joint was excellent, but there was slight radial translation at the mcp joint (fig. the patient declined operative intervention at this stage, and was maintained in his thumb cast. the patient failed to attend subsequent clinic appointments, but was reviewed again four weeks after the injury. unfortunately in the interim, the patient had attended a minor injuries department and had his cast changed (without a new x - ray) after soiling it while at work. at this stage the cmc joint was again well reduced, but there was approximately 30 degrees of radial angulation of the proximal phalanx at the mcp joint (fig. a dorsal approach was taken over the mcp joint in order to examine both collateral ligaments. the radial collateral ligament was intact but the ulnar collateral ligament was completely ruptured, with disruption of the capsule extending around to the dorsal aspect of the joint. the ulnar collateral was repaired by tying the ligamentous attachments from the metacarpal, to mitek (de puy mitek, warsaw, in, usa) sutures anchored into the proximal phalanx. finally the mcp joint was temporarily immobilized with a kirschner (k) wire (fig. ap and lateral radiographs one week post - operatively post - operatively the patient continued to work with horses, despite advice to the contrary. the kirschner wire was removed three weeks post - operatively due to a superficial pin site infection, which was successfully treated with systemic oral antibiotics (amoxicillin / clavulanic acid). the patient was working and able to perform most of his manual tasks, although he complained of weakened grip strength. he was found to have considerable stiffness at the 1st mcp joint, with range of movement limited from 0 to 30 degrees at 12 months the patient was pain free without restriction of function, and although he could fully extend his mcp joint, he could only flex to 50 degrees only (compared to 90 degrees on the contralateral side). double dislocations of thumb joints are rare, there are only six previous reports of concurrent dislocations of the cmc and mcp of the thumb, and of these only one report also includes an associated fracture at the base of the thumb. interestingly, in the other case reports, both the cmc and mcp dislocated dorsally. the accepted mechanism of this injury is a longitudinally directed force with hyperextension at the mcp, and slight flexion at the cmc joint. when questioned he described gripping the reins with hands together, and then raising both arms above his head for protection just prior to impact. this would cause his wrists to be flexed and radially deviated, with thumb metacarpals extended, and phalanges flexed. the authors feel that a longitudinally directed force with the thumb in this position would account for this unique injury. in all of the previous reports, the cmc joint was unstable and required operative stabilization ; with k - wires in four cases, and ligament reconstruction (eaton s procedure) in two cases [1 - 6 ]. in two of these case reports, the radial collateral ligament of the mcp joint was unstable, and in another the ulnar collateral ligament was unstable. all of these ligaments required repair, with additional k - wire fixation in one of these cases [2 - 6 ]. in their report of concurrent cmc and mcp joint dislocations, drosos categorize mcp joint dislocations as simple (reducible with a closed technique) and complex (irreducible with closed technique, requiring open reduction). it has been postulated that the energy required to produce double dislocations causes more ligamentous damage, and hence greater instability that either injury on its own. four weeks after the injury, the cmc joint of our patient was still in a good position, and although the lateral view of the mcp showed no subluxation / dislocation, the antero - posterior view of the mcp joint showed further radial angulation. even though there was slight translation at the mcp joint at the one - week review, we were forced to opt for conservative management because the patient declined operative intervention. certainly we did no harm by delayed operative fixation until three weeks later. in the case described by ibrahim and noor, the patient had both cmc and mcp joints dislocated for five weeks before operative repair was performed ; the cmc joint was stabilized with a sole k - wire, and at the mcp joint a k - wire plus radial collateral ligament repair. farzan, report a case of a volar dislocated cmc which presented 3 months after the initial injury, due to pain and an inability to pinch and perform opposition. this required open reduction, reconstruction with eaton s procedure, k - wire stabilization, and a thumb spica for six weeks. at four month follow up thumb opposition was possible and pain - free, pinch and grip were near normal compared to the contra - lateral side, and the global reduction in thumb movement was only 10 degrees. this injury can be treated non - operatively if assessed accurately at presentation, with close follow up. marcotte and trzeciak, described a case of cmc and mcp dislocations, which were stable after closed reduction, with only slight laxity of the ulnar collateral ligament. although the option of surgical stabilization was discussed, the joints remained anatomically reduced for the 5 weeks the patient remained in a cast. the patient continued to have clinical and radiological follow up monthly, for 3 months to confirm the joints remained reduced. at 2 year follow up, the thumb was pain free, stable, with a good range of movement and no evidence of arthritis. in our report, the patient was followed up for one year, similar follow up to the other reports. moore followed up their patient for 9 years, with no evidence of arthritis or instability. however, gerard, did report degenerative changes in the cmc and mcp joints in a patient with double dislocations. given the severity of this injury, it is certainly conceivable that these patients would develop symptomatic degenerative changes in the long term. our literature review suggests there are many options for the treating this injury : operative or conservative treatment ; treating both joints as separate injuries or together ; and ligamentous repair and/or k - wire fixation. the key to these decisions seems to rest at the initial clinic review after successful reduction. if instability can be assessed here and operative or nonoperative decisions made, then management can be tailored accordingly. furthermore these injuries need weekly radiographic follow up if conservative treatment is opted for, so that any further subluxation or dislocation is highlighted early. although the patient opted for initial conservative management, and even with delayed operative stabilization of one joint, the patient had a good functional outcome. double dislocation of cmc and mcp joints of the thumb is rare entity ; however it behaves similar to isolated injuries.	introduction : multiple dislocations of joints in the hand are rare. double dislocations of the thumb joints have only been reported on four previous occasions, in all cases reported to date, the joints have dislocated dorsally.case report : we present the case of a 26-year - old male patient with simultaneous volar dislocations of the carpometacarpal and metacarpophalangeal joints of the thumb. there was delayed operative treatment of this injury with ligament reconstruction and stabilization of the metacarpophalangeal joint.conclusions:this rare case provides a mechanism to this type of injury, highlights the importance of initial, and repeated clinical and radiographic review, highlights the soft tissue component to this injury, and demonstrates how even delayed treatment can result in a good functional outcome.

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